International Review of Cytology, Volume 28

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME28

Contributors to Volume 28 E. C. COCKING WILLIAMP.

JACOBS

ROBERTC. KING R. B. MORETON R. L. MOTT MAUREENOWEN CHRISTIAANP. RAVEN R E N ~SIMARD F. C. STEWARD J. E. TREHERNE

INTERNATIONAL

Review of Cytology EDITED BY

J. F. DANIELLI

G. H. BOURNE I’wkes Regional Primate Research Center Emory University Atlanta, Georgia

Center for Theoretical Biology State U n i i w f i f j of N e w Y o r k at Buffalo Buffalo, New YO^

ASSISTANT EDITOR K. W- JEON Center for Theoretical Biology State University of N e w York at BuflaEo Buffalo, New York

VOLUME28

Prepared Under the Auspice! of T h e International Soiiety jor Cell Biolo
ACADEMIC PRESS New York arid London 1970

C O P Y R I G H T @ 1970, BY ACADEMIC PRESS, JNC. ALL RIGHTS RESERVED N O PART OF T H I S BOOK MAY BE REPRODUCED I N A N Y FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, O R ANY O T H E R MEANS, W I T H O U T W R I T T E N PERMISSION FROM T H E PUBLISHERS.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD Berkeley Square House, London W l X 6BA

LIBRARY OF CONGRESS CATALOG CARD

NUMBER: 52-5203

PRINTED I N T H E UNITED STATES O F AMERICA

List of Contributors Numbers in parentheses indicate the pages on which the authors' contributions begin.

E. C. COCKING(SS), Department of Botany, University of Nottingham, Nottingham, England WILLIAMP. JACOBS( 2 3 9 ) , Biology Department, Princeton University, Princeton, N e w Jersey ROBERT C. KING ( 1 2 5 ) , Department of Biological Sciences, Northwestern Universify, Evanston, Illinois R. B. MORETON( 4 5 ) , Depavtment of Zoology, University of Cambridge, Cambridge, England

R. L. MOTT (275), Laboratory for Cell Physiology, Growth, and Development, Cornell University, Ithaca, N e w York MAUREEN OWEN ( 2 1 3 ) , Medical Research Council External Scientific Staff, Bone Research Laboratory, T h e Churchill Hospital, Oxford, England CHRISTIAAN P. RAVEN ( I ) , Zoological Laboratory, University Utrecht, T h e Netherlands

of

Utrecht,

RENB SIMARD(169), Laboratoire de Biologie IMole'cdaire, Faculte' de Me'decine, Universite' de Sherbrooke, Sherbrooke, Canada F. C. STEWARD(275), Laboratory for Cell Physiology, Growth, and Development, Cornell University, Ithaca, N e w York

J. E. TREHERNE ( 4 5 ) , A.R.C. Unit of Invertebrate Chemistry and Physiology, Cambridge, England

V

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Contents

LIST

OF

CONTRIBUTORS . .

V

CONTENTS OF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

The Cortical and Subcortical Cytoplasm of the Lymiiaea Egg CHKISTIAAN P. KAVEN I. 11 . 111. IV . V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Cortical Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Subcortical Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Cortical Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . .....................................

1 3 18 32

37 42

The Environment and Function of Invertebrate Nerve Cells J. 1: . TREHBRNE A N D R . B . MORETON I. I1. I11. IV . V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution and Exchanges of Inorganic Ions and Molecules . . . . . . . . . . . . The Ionic Requirements for Electrical Activity . . . . . . . . . . . . . . . . . . . . . . . . General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45 46 64 70

84 85

Virus Uptake. Cell Wall Regeneration. and Virus Multiplication in Isplated Plant Protoplasts E . C . COCKING I. I 1. 111. IV .

Introduction: The Isolated Protoplast System . . . . . . . . . . . . . . . . . . . . . . . . . Uptake of Viruses by Isolated Protoplasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Wall Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Virus Multiplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

89 92 108 115 122

...

Vlll

CONTENTS

The Meiotic Behavior of the Drosophila Oocyte ROBERTC . KING I. 11. 111. IV .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Morphology of the Drosophila Ovary . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Cytology of the Drosophila Oocyte Nucleus . . . . . . . . . . . . . . . . . . . . . . . Mitotic Crossing-over without Synaptonemal Complexes . . . . . . . . . . . . . . . . . V . The Formulation of a Hypothesis concerning the Origin and Functioning of Synaptonemal Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Nondisjunction in Meiotic Mutants Affecting Crossing-over . . . . . . . . . . . . . . VII . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

125 127 129 134 135 160 164 165

The Nucleus: Action of Chemical and Physical Agents RENB SIMARD I. I1. 111. IV .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Normal Nuclear Fine Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural Support for RNA Synthesis in the Nucleus . . . . . . . . . . . . . . . . . . Agents That Primarily Affect the Nucleolus . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Agents That Primarily Affect the Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Summary and Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

169 170 174 175 195 203 205

The Origin of Bone Cells MAUREENOWEN I. I1. 111. IV .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osteoprogenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other Cells with Potential far Bone Formation . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

213 216 229 234 236

Regeneration and Differentiation of Sieve Tube Elements WILLIAM P. JACOBS I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Regeneration of Sieve Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239 241

CONTENTS

111. Differentiation of Sieve TQbes in Organ or Tissue Culture . . . . . . . . . . . . . . . IV. Normal Differentiation of Sieve Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

264 266 270 271

Cells, Solutes, and Growth : Salt Accumulation in Plants Reexamined F. C. STEWARD AND R. L. MOTT I. Introduction-The Solutes in Cells: Their Composition and Accumulation . . 11. The System Involved: Its Membranes and Fine Structure . . . . . . . . . . . . . . . . rl1. Physiological Studies on Salt Accumulation in Plant Cells: Past and Recent Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Growth Requirement of Cells: Their Implications for Solute IJptake . , . V. Absorption Studies with Cultured Cells and Tissue Explants . . . . . . . . . . . . . VI. Salient Features of Solute Accumulations in Cells: Perspectives and Prospects References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

276 285

294 310 317

355 365

AUTHOR INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

371

SLILIJECTINDEX.

385

........................................................

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Contents of Previous Volumes Ascorbic Acid and Its Intracellular Localization, with Special Reference to Some Historical Features in Cell BiolPlants-J. CHAYI:.N ogy-ARTHUR HUGHES Aspects of Bacteria as Cells and as OrNuclear Reprodution-C. LEONARD HLJSganisms-SmAnT MUDD A N D EDWARD KINS D. DELAMATER Enzymic Capacities and Their Relation to Ion Secretion in Plants-J. F. SUTCLIFFE Cell Nutrition in Animals-GEORGE w . Multienzyme Sequences in Soluble ExKIDDER tracts--HENRY R. MAHLER The Application of Freezing and Drying The Nature and Specificity of the Feulgen Techniques in Cytology-L. G. E. BELL Nucleal Reaction--M. A. LESSLER Enzymatic Processes in Cell Membrane Quantitative Histochemistry of PhosphaPenetration-TH. ROSBNHERG A N D w. taSes-wILLIAM L. DOVLE %rlLBKANDT Alkaline Phosphatase o f the NucleusBacterial Cytology-K. A. BISSET M. C H ~ V K EMOAND N T H. FIRKET Protoplast Surface Enzymes and Absorp- Gustatory and Olfactory Epithelia-A. F. BARADIAND G. H. B O ~ J R N E tion of Sugar-R. BROWN D. Growth and Differentiation of Explanted Reproduction of Bacteriophage-A. Tissues-P. J. GAILLAKD HERSHEY The Folding and Unfolding of Protein Electron Microscopy of Tissue SectionsA. J. DALTON Molecules as a Basis of Osmotic W o r k A Redox Pump for the Biological PerR. J. GOLDACRE formance of Osmotic Work, and Its Nucleo-Cytoplasmic Relations in AmphibRelation to the Kinetics of Free Ion ian Development-G. FANK-HAUSER Diffusion across Membranes-E. J. Structural Agents in Mitosis-M. M. CONWAY SWANN A Critical Survey of Current Approaches Factors Which Control the Staining of in Quantitative Histo- and CytochemTissue Sections with Acid and Basic istry-DAVID GLICK I)yes-hfARcus SINGER Nucleo-cytoplasmic Relationships in the The Behavior of Spermatozoa in the Development of Acetahularia-J. HAMNeighborhood of Eggs-LORD ROTHSVolume 1

MERLING

CHILD

Report of Conference of Tissue Culture The Cytology of Mammalian Epidermis Workers Held at Cooperstown, New and Sebaceous Glands--Wrr.LIAM MONYork--D. C. HIXHERING'I'ON 'SAGNA

AUTHOR INDEX---SURJ

IiCT INDEX

The Electron-Microscopic Investigation of Tissue Sections-L. H. BRETSCHNEIDERVolume 3 The Histochemistry of EsterasesThe Nutrition of Animal Cells-~-CHARITY G. GOMORI WAYMOUTH AUTHOR INDEX-SUB JECT INDEX Caryometric Studies of Tissue Cultures- OTTO BUCHER Volume 2 The Properties of Urethan Considered in Quantitative Aspects of Nuclear NucleoRelation to Its Action on Mitosis-proteins-HswsoN SWIFT IVORCORNMAN

xi

xii

CONTENTS OF PREVIOUS VOLUMES

Composition and Structure of GiantChromosomes-Max ALFERT How Many Chromosomes in Mammalian Somatic Cells?-R. A. BEATTY The Significance of Enzyme Studies on Isolated Cell Nuclei-ALEXANDER L. DOUNCE The Use of Differential Centrifugation in the Study of Tissue Enzymes-CHR. DE DUVEAND J. BERTHET Enzymatic Aspects of Embryonic Differentiation-TRYGGVE GUSTAFSON Azo Dye Methods in Enzyme Histochemistry-A. G. EVERSON PEARSE Microscopic Studies in Living Mammals with Transparent Chamber MethodsROY G. WILLIAMS The Mast Cell-G. ASBOE-HANSEN Elastic Tissu-EDWARD w . DEMPSEY AND ALBERT I. LANSING The Composition of the Nerve Cell Studied with New Methods-SvENOLOPBRATTGARD AND HOLGBR HYDEN

Volume 5

Histochemistry with Labeled AntibodyALBERT H. COONS The Chemical Composition of the Bacterial Cell Wall-C. S. C ~ J M M I N S Theories of Enzyme Adaptation in Microorganisms--J. MANDELSTAM The Cytochondria of Cardiac and Skeletal Muscle-JOHN W. HARMON The Mitochondria of the Neuron-WARREN ANDREW The Results of Cytophotometry in the Study of the Deoxyribonucleic Acid (DNA) Content of the NucleusR. VENDRELY AND c. VENDRELY Protoplasmic Contractility in Relation to Gel Structure : Temperature-Pressure Experiments a n Cytokinesis and Amoeboid Movement-DOUGLAS MARSLAND Intracellular pH-PETER C. CALDWELI. The Activity of Enzymes in Metabolism and Transport in the Red Cell-T. A. J. PRANKERD AUTHOR INDEX-SUB JECT INDEX Uptake and Transfer of Macromolecules by Cells with Special Reference to Volume 4 Growth and Development-A. h.1. Cytochemical Micrurgy-M. J. KOPAC SCHECHTMAN Amoebocytes-L. E. WAGGE Cell Secretion: A Study of Pancreas and Problems of Fixation in Cytology, HistolC. U. JUNQUEIKA Salivary Glands-L. ogy, and Histochemistry-M. WOLMAN AND G. C. HIRSCH Bacterial Cytology-ALFRED MARSHAK The Acrosome Reaction- -JEAN C. DAN Histochemistry of Bacteria-R. VENDRELYCytology of Spermatogenesis-VIsl-IWA Recent Studies on Plant MitochondriaNATH DAVIDP. HACKETT The Ultrastructure of Cells, as Revealed The Structure of Chloroplastsby the Electron Microscope-FRiTroP K. MUHLETHALER S. SJOSTRAND B. AUTHOR INDEX-SUBJECT Histochemistry of Nucleic Acids-N. Ih'DEX KURNICK Volume 6 Structure and Chemistry of Nucleoli-

W. S . VINCENT On Goblet Cells, Especially of the Jntestine of Some Mammalian SpeciesHARALD MOE Localization of Cholinesterases at Neuromuscular Junctions---R. COUTEAUX Evidence for a Redox Pump in the Active Transport of Cations-E. J. CONWAY AUTHOR INDEX-SUB

JECT INDEX

The Antigen System of Pavdmecizcm mVeZiu-G. H. BEALE The Chromosome Cytology of the Ascites Tumors of Rats, with Special Reference to the Concept of the Stemline CellSA-JIROM A K ~ N O The Structure of the Golgi Apparatus-- ARTHUR w. POLI.ISTElr AHD PRISCILIA

F. POLLISTER

CONT E NT S OF PREVIOUS VOLUMES

..A

XI11

An Analysis of the Process of Fertilization Anatomy of Kidney Tubules-JoHANNss and Activation of the Egg-A. MONROY RHODIN The Role of the Electron Microscope in Structure and Innervation of the Inner Virus Research-RoRLEY C. WILLIAMS Ear Sensory Epithelia-HANS ENGThe Histochemistry of PolysaccharidesSIROM AND JAN WERSALL ARTHUR J. HALE The Isolation of Living Cells from Animal Tissues-L. M. J. RINALDINi The Dynamic Cytology of the Thyroid Gland-J. GROSS AUTHOR INDEX-SUB JECT INDEX Recent Histochemical Results of Studies Volume 8 on Embryos of Some Birds and Mammals-ELro BORGHESE The Structure of Cytophm-CHARLEs Carbohydrate Metabolism and Embryonic OBERLING Wall Organization in Plant Cells-R. D. Determination-R. J. OCONNOR PRESTON Enzymatic and Metabolic Studies on IsoSubmicroscopic Morphology of the Synlated Nuclei-G. SIERERT AND R. M. S. SMELLIE apSe-EDUARDO DE ROBERTIS Recent Approaches to the Cytochemical The Cell Surface of Paramecium-C. F. Study of Mammalian Tissues-GEORGE E H R E T A N D E. L. POWERS H. HOGEBOOM, EDWARDL. KUPF, AND The Mammalian Reticulocyte-LEAH MIRWALTER C. SCHNEIDER IAM LOWENSTEIN The Kinetics of the Penetration of Non- The Physiology of Chromatophores-Mrr.electrolytes into the Mammalian ErythTON FINGERMAN rocyte--FREDA BOWYER The Fibrous Components of Connective Tissue with Special Reference to the AU1’HOR INDEX-SUB JECT INDEX Elastic Fiber-DAVID A. HALL CLlhflJLATIVE SUBJECT INDEX (VOLUMES 1-5 ) Experimental Heterotopic OssificationJ. B. BRIDGES Volume 7 A Survey of Metabolic Studies on Isolated Some Biological Aspects of Experimental Mammalian Nuclei-D. B. KOODYN Radiology : A Historical Review-F. G. Trace Elements in Cellular FunctionSPEAR AND FREDERIC L. HOCH BERTL. VALLEE The Effect of Carcinogens, Hormones, Osmotic Properties of Living Cellsand Vitamins on Organ Cultures-ILsE D. A. T. DICK Sodium and Potassium Movements in LASNITZKI Nerve, Muscle, and Red Cells-I. M. Kecent Advances in the Study of the Kinetochore-A. LIMA-DE-FARIA GLYNN Autoradiographic Studies with Sa5-Sulfate Pinocytosis-H. HOLTER D. D. DZIEWIATKOWSKI AUTHOR INDEX-SUBJECT INDEX The Structure of the Mammalian SperVolume 9 matozoon-DON W. FAWCETT The Influence of Cultural Conditions on The Lymphocyte--0. A. TROWBLL Bacterial Cytology-J. F. WII KINSON The Structure and Innervation of LamelAND J. P. DUGUID libranch Muscle-J. BOWDBN Hypothalamo-neurohypophysial Neurose- Organizational Patterns within Chromosomes-BERWIND P. KAUFMANN, HEJ.EN cretion-J. C . SLOPER R. MCDONALD GAY,AND MARGARET Cell Contact-PAUL WEISS BOYD The Ergastoplasm: Its History, Ultrastruc- Enzymic Processes in Celk-JAY BEST ture, and Biochemistry-FRANcoIsE HAThe Adhesion of CellsLEONARD WEISS GUENAU

xiv

CONTENTS OF PREVIOUS VOLUMES

Physiological and Pathological Changes in Mitochondrial Morphology-CH. ROUILLER The Study of Drug Effets at the Cytological Level-G. B. WILSON Histochemistry of Lipids in OogenesisVISHWANATH Cyto-Embryology of Echinoderms and Amphibia-KATsuhrA DAN The Cytochemistry of Nonenzyme Proteins-RONALD R. COWDEN

The Growth-Duplication Cycle o f the Cell D. M. PRESCOTT Histochemistry of Ossification-RoMuLo L. CABRINI Cinematography, Indispensable Tool for Cytology-C. M. POMERAT AUTHOR INDEX-SUBJECT

INDEX

Volume 12

Sex Chromatin and Human Chromosomes JOHN L. HAMERTON AUTHOR INDEX-SUBJECT INDEX Chromosomal Evolution in Cell Populations-T. C. Hsu Volume 10 Chromosome Structure with Special Reference to the Role of Metal Ions--DALE The Chemistry of Schiff's Reagent-FREDM. STEFFENSEN ERICK H. KASTEN Electron Microscopy of Human White Spontaneous and Chemically Induced Blood Cells and Their Stem CellsChromosome Breaks-ARuN KUMAR MARCEL BESSISAND JEAN-PAUL THIERY SHARMA AND ARCHANA SHARMA In V i m Implantation as a Technique in The Ultrastructure of the Nucleus and Skeletal Biology-WILLIAM J. L. FELTS Nucleocytoplasmic Relations-SAUL The Nature and Stability of Nerve Myelin WISCHNITZER J. B. FINEAN The Mechanics and Mechanism of Cleav- Fertilization of Mammalian Eggs in Vitro age-Lswrs WOLPERT C. R. AUSTIN The Growth of the Liver with Special Physiology of Fertilization in Fish Eggs-Reference to Mammals-F. DOLJANSKI TOKI-oYAMAMOTO Cytological Studies on the Affinity of the AUTHOR INDEX-SUBJECl INDEX Carcinogenic Azo Dyes for Cytoplasmic Components-YosHiMr NAGATANI Volume 13 Epidermal Cells in Culture--A. GEDEON The Coding Hypothesis-MARTYNAS YtAs MATOLTSY Chromosome Reproduction--J. HERBERT AUTHOR INDEX-SUB JECT INDEX TAYLOR CUMULATIVE SUBJECT INDEX Sequential Gene Action, Protein Synthesis, (VOLUMES 1-9) and Cellular Diff erentiation-REED A. FLICKINGER Volume 11 The Composition of the Mitochondria1 Membrane in Relation to Its Structure Electron Microscopic Analysis of the Seand Function-ERrc G. BALLAND CLIFFI~ cretion Mechanism-K. KUROSUMI D. JOEL The Fine Structure of Insect Sense Organs ELEANORH. SLIFER Pathways of Metabolism in Nucleate and Anucleate Erythrocytes-H. A. SCHWBICytology of the Developing Eye-ALFRED GER J. COULOMBRE The Photoreceptor Structures-J. J. WOL- Some Recent Developments in the Field of Alkali Cation Transport--W. WILKEN BRANDY Use of Inhibiting Agents in Studies on Fertilization Mechanisms-CHARLES B. Chromosome Aberrations Induced by Ionizing Radiations-H. J. EVANS METZ

C O N T E N T S O F PREVIOUS VOLUMES

xv

Cytochemistry of Protozoa, with Particu- Plant Tissue Culture in Relation to Development cytology-CARL R. PARTANEN lar Reference to the Golgi Apparatus and the Mitochondria-VrsHwA NATH Regeneration of Mammalian LiverNANCYL. R. BUCHER AND G. P. DUTTA Collagen Formation and Fibrogenesis Cell Renewal-FELIX BERTALANFFY AND with Special Reference to the Role of CHOSEN LAU Ascorbic Acid-BERNARD S. GOULD AUTHOR INDEX-SUBJECT INDEX The Behavior of Mast Cells in Anaphylaxis-IVAN MOTA Volume 14 Lipid Absorption-ROBERT M. WOTTON lnhibition of Cell Division: A Critical AUTHOR INDEX-SUBJECT INDEX and Experimental Analysis-SEYMOUR GELFANT Volume 16 Electron Microsopy of Plant Protoplasm Ribosomal Functions Related to Protein R. BUVAT Synthesis-TORE HLJLTIN Cytophysiology and Cytochemistry of the Physiology and Cytology of Chloroplast Organ of Corti: A Cytochemical TheFormation and “Loss” in Euglenaory of Hearing-J. A. VINNIKOV AND M. GRENSON L. K. TITOVA Cell Structures and Their Significance for Connective Tissue and Serum ProteinsAmeboid Movement-K. E. WOHLR. E. MANCINI FARTH-BOTTERMANN The Biology and Chemistry of the Cell Microbeam and Partial Cell IrradiationWalls of Higher Plants, Algae, and C. L. SMITH Fungi-D. H. NORTHCOTE Nuclear-Cytoplasmic Interaction with IonIlevelopment of Drug Resistance by Staphizing Radiation-M. A. LESSLER ylococci in Vitro and in vivo-MARY In Vivo Studies of Myelinated Nerve BARBER Fibers-CARL CASKEY SPEIDEL Cytological and Cytochemical Effects of Respiratory Tissue: Structure, HistophysiAgents Implicated in Various Pathologology, Cytodynamics. Part 1. Review ical Conditions: The Effect of Viruses and Basic Cytomorphology-FirLrx D. and of Cigarette Smoke on the Cell and BERTALANFFY Its Nucleic Acid-CEcmE LEUCHTEN- AUTHOR INDEX-SUB JECT INDEX RERGER A N D RUDOLF LEUCHTENBERGER The Tissue Mast Wall-DOUGLAS E. Volume 17 SMITH The Growth of Plant Cell Walls--K. AIJTHOR INDEX-SUBJECT INDEX WILSON Reproduction and Heredity in TrypanoVolume 15 somes: A Critical Review Dealing The Nature of Lampbrush Chromosomes Mainly with the African Species in the H. G. CALLAN Mammalian Host-P. J. WALKER The Intracellular Transfer of Genetic In- The Blood Platelet: Electron Microscopic Studies-J. F. DAVID-FERREIRA formation-J. L. SIRLIN Mechanisms of Gametic Approach in The Histochemistry of Mucopolysaccharides-ROBERT Plants-LEONARD MACHLIS AND ERIKA C. CURRAN RAWITSCHER-KUNKEL Respiratory Tissue Structure, HistophysiThe Cellular Basis of Morphogenesis and ology, Cytodynaniics. Part 11. New ApSea Urchin Development-T. GUSTAF- proaches and Interpretations FEI.iX D. BERTALANFFY SON AND L. WOLPERT

xvi

CONTENTS OF PREVIOUS VOLUMES

The Cells of the Adenohypophysis and Their Functional Significance-MARC HERLANT AUTHOR INDEX-SUB

JECT INDEX

Volume 18 The Cell of Langerhans-A.

S. BREATH-

NACH

The Structure of the Mammalian EggROBERT HADEK Cytoplasmic Inclusions in OogenesisM. D. L. SRIVASTAVA The Classification and Partial Tabulation of Enzyme Studies on Subcellular Fractions Isolated by Differential Centrifuging-D. B. ROODYN Histochemical Localization of Enzyme Activities by Substrate Film Methods: Ribonucleases, Deoxyribonucleases, Proteases, Amylase, and HyaluronidaseR. DAOUST Cytoplasmic Deoxyribonucleic AcidP. B. GAHANAND J. CHAYBN Malignant Transformation of Cells in Vitro-KATHERINE K. SANFORD Deuterium Isotope Effects in CytologyE. FLAUMENHAPI, S. BOSE,H. L. CRESPI, AND J. J. KATZ The Use of Heavy Metal Salts as Electron Stains-C. RICHARDZOBEL A N D MICHAEL BEER A U T H O R INDEX-SUBJECT

INDEX

Volume 19 “Metabolic” DNA: A Cytochemial Study H. ROELS The Significance of the Sex ChromatinL. BARR MURRAY Some Functions of the Nucleus-J. M. MITCHISON Synaptic Morphology on the Normal and Degenerating Nervous System-E. G. GRAYAND R. W. GUILLERY Neurosecretion-W. BARGMANN Some Aspects of Muscle RegenerationE. H. BETZ,H. FIRKET,AND M. REZNIK The Gibberellins as Hormones-P. W. BRIAN Phototaxis in PhtS-WOLFGANG HAUPT

Phosphorus Metabolism in Plants-“. ROWAN AUTHOR INDEX-SUB

S.

JECT INDEX

Volume 20 The Chemical Organization of the Plasma Membrane of Animal Cells-A. H. MADDY Subunits of Chloroplast Structure and Quantum Conversion in Photosynthesis RODERICB. PARK Control of Chloroplast Structure by Light LESTERPACKERA N D PAUL-ANDRB SXEGENTHA L E R

The Role of Potassium and Sodium Ions as Studied in Mammalian Brain-H. HILLMAN Triggering of Ovulation by Coitus in the Rat-CLAUDE ARON,GITTAASCH,AND JACQUELINE Roos Cytology and Cytophysiology of NonMelanophore Pigment Cells-JoSEPH T. BAGNARA The Fine Structure and Histochemistry of Prostatic Glands in Relation to Sex Hormones-DAVID BRANDES Cerebellar Enzymology-LuciE ARVY AUTHOR INDEX-SUB.JECT

INDEX

Volume 21 Histochemistry of Lysosomes-P. B. GAHAN L. BRAHMAPhysiological Clocks-R. CHARY

Ciliary Movement and Coordination in Ciliates-BELA PARDUCA Electromyography : Its Structural and Neural Basis-JOHN V. BASMAJIAN Cytochemical Studies with Acridine Orange and the Influence of Dye Contaminants in the Staining Nucleic Acids FREDERICK H. KASTEN Experimental Cytology of the Shoot Apical Cells during Vegetative Growth and Flowering-A. NOUGAREDE Nature and Origin of Perisynaptic Cells of the Motor End Plate-”. R. SHANTHAVEEKAPPA AND G. H. BOLJRNE AUTHOR INDEX-SUBJECT

INDFX

xvii

CONTENTS OF PREVIOUS VOLUMES

Mast Cells in the Nervous SystemYNGVEOLSSON Developmental Phases in Intermitosis and the Preparation for Mitosis of Mammalian Cells in V~~YO-BLAGOJE A. NESKOVIC Antimitotic Substances-Guy DEYSSON The Form and Function of the Sieve Tube : A Problem in ReconciliationP. E. WEATHERLEY AND R. P. C. JOHN-

Volume 22

Current Techniques in Biomedical Electron Microscopy-SAUL WISCHNITZER The Cellular Morphology of Tissue Repair-R. M. H. MCMINN Structural Organization and Embryonic Diff erentiation-GA JANAN v. SHERBET AND M. S. LAKSHMI The Dynamism of Cell Division during Early Cleavage Stages of the EggSON N. FAUTREZ-FIRLEFYN AND 5. FAUTREZ Analysis of Antibody Staining Patterns Lymphopoiesis in the Thymus and Other Obtained with Striated Myofibrils in Tissues: Functional Implications-N. B. Fluorescence Microscopy and Electron EVERETT AND RUTH W. TYLER(CAFMicroscopy-FRANK A. PEPE FREY) Cytology of Intestinal Epithelial CellsStructure and Organization of the MyoPETERG. TONER neural Junction-C. COERS Liquid Junction Potentials and Their The Ecdysial Glands of ArthropodsEffects on Potential Measurements in WILLIAMS. HERMAN Biology Systems-P. C. CALDWELL Cytokinins in Plants-B. I. SAHAISRIVASAUTHOR INDEX-SUB

TAVA AUTHOR INDEX-SUB

JECT INDEX

CUMULATIVE SUBJECT INDEX

JECT INDEX

Volume 25

Cytoplasmic Control over the Nuclear Events of Cell Reproduction-NosI. DE TERRA Volume 23 Coordination of the Rhythm of Beat in Transformationlike Phenomena in Somatic Some Ciliary Systems-M. A. SLEIGH Cells-J. M. OLENOV The Significance of the Structural and Recent Developments in the Theory of Functional Similarities of Bacteria and Control and Regulation of Cellular Mitochondria-SYLVAN NASS Processes-ROBERT ROSEN The Effects of Steroid Hormones on Contractile Properties of Protein Threads Macrophage Activity-B. VERNONfrom Sea 1Jrchin Eggs in Relation to ROBERTS Cell Division-HIKorcHI SAKAI The Fine Structure of Malaria Parasites Electron Microscopic Morphology of MARIAA. RUDZINSKA OogefIesis-ARNE N0RREVANG The Growth of Liver Parenchymal Nuclei Dynamic Aspects of Phospholipids during and Its Endocrine Regulation-RITA E. HOKIN Protein Secretion-LOWELL CARRIERE The Golgi Apparatus: Structure and Func- Strandedness of Chromosofnes--~SHEI.DON tion-H. W. BEAMSAND R. G. KESSEL WOLFF The Chromosomal Basis of Sex Deter- Isozymes: Classification, Frequency, and minatiOn-KENNETH R. LEWIS AND Significance-CHARLES R. SHAW BERNARD JOHN The Enzymes of the Embryonic Nephron AUTHOR INDEX-SUBJECT INDEX LUCIEARVV Protein Metabolism in Nerve Cells-B. Volume 24 DROZ Freeze-Etching-HANS MOOR Synchronous Cell Differentiation-GEORGE AUTHOR INDEX-SUB JECT INDEX M. PADILLA AND IVANL. CAMERON (VOLUMES

1-21)

xviii

CONTENTS OF PREVIOUS VOLUMES

Volume 26

Volume 27

A New Model for the Living Cell: A Summary of the Theory and Recent Experimental Evidence in Its SupportGILBERT N. LING The Cell Periphery-LEONARD WEISS Mitochondria1 D N A : Physicochemical Properties, Replication, and Genetic Function-P. BORSTAND A. M. KROON Metabolism of Enucleated Cells-KONRAD KECK Stereological Principles for Morphometry in Electron Microscopic CytologyEWALDR. WEIBEL Some Possible Roles for Isozymic Substitutions during Cold Hardening in Plants D. W. A. ROBERTS

Wound-Healing in Higher PlantsJACQUES LIPETZ Chloroplasts as Symbiotic Organelles--. DENNISL. TAYLOR The Annulate Lamella-SAuI. MISCH-

AUTHOR INDEX---SIJB JECT INDEX

AUTHOR INDEX-SIB

NITZER

Gametogenesis and Egg Fertilization in Planarians-G. BENAZZILENTATI Ultrastructure of the Mammalian Adrenal CorteX-sIMON IDELMAN The Fine Structure of the Mammalian Lymphoreticular SyStem--rAN CARR Immunoenzyme Technique: Enzymes as Markers for the Localization o f Antigens and Antibodies--STRA?’rS AVRAMEAS JECT INDEX

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME28

This Page Intentionally Left Blank

The Cortical and Subcortical Cytoplasm of the Lymnaea Egg CHRTSTIAAN P. RAVEN Zoological Ltzboratory, Univefsity of Utrecht, the Netherldndl

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Cortical Cytoplasm . . . . . . . . . . . . . . . . . A. Physiological Properties of the Egg Cortex in LyflZnded B. Composition, Nature, and Behavior of the Egg Cortex C. The Formation of the Egg Cortex . . . . . . . . . . . . . . . 111. The Subcortical Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Vegetative Pole Plasm . . . . . . . . . . . . . . . . . . . . . . . B. The Animal Pole Plasm . . . . . . . . . . . . . . . . . . . . . . . . . C. Subcortical Accumulations . . . . . . . . . . . . . . . . . . . . . . . IV. The Cortical Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

3 3 11 12 18

1s 21

24 32 37

42

1. Introduction The first indications pointing to a morphogenetic significance of the cortex of the Lymnaea egg were obtained in centrifugation experiments by Raven and Bretschneider (1942). After centrifuging unsegmented eggs of Lymtzaea stagn a h for 5 minutes at 1860 x g, producing a clear-cut stratification of the egg contents, most of the centrifuged eggs developed into normal little snails. It appeared that the egg substances displaced by centrifugation did not remain in their new positions, but were redistributed through the egg, tending to a restoration of their normal arrangement. In this readjustment of the egg substances, two different processes could be distinguished : first, the redispersion of the substances accumulated during centrifuging, leading to a more nearly uniform distribution throughout the egg cytoplasm; second, and on top of this, a selective accumulation of special components of the cytoplasm in certain regions of the egg and embryo, corresponding to the “ooplasmic segregation” of normal eggs. The latter process was studied especially with respect to the redistribution of the lipoid substances in embryos developed from centrifuged eggs. It was concluded that “the factors, which are responsible for the localization of substances in normal eggs (and which . . . are located, probably, in the relatively immovable egg cortex, not displaced by the action of centrifugal force), continue to work in centrifuged eggs till a rather late state of development. ’ ’ This view was corroborated by further observations on centrifuged eggs. When centrifugation takes place before the beginning of the maturation divi1

2

CHRISTIAAN P . RAVEN

sions, the animal pole plasm of the L. stagna2i.r egg forms at the normal time, about 1 hour after the second maturation division, at the animal pole (Raven, 1945; Raven and Brunnekreeft, 1951). If the eggs are centrifuged just prior to first cleavage, the mitochondria, heaped up in the middle zone of the stratified egg, soon disperse through the cytoplasm and accumulate beneath the egg cortex in the neighborhood of the animal pole (Raven, 1946b). A second group of observations pointing to cortical morphogenetic factors concerned the influence of chemical agents, especially lithium, on the development of Lymnaea. Eggs treated with solutions of LiCl at early stages exhibited two kinds of developmental disturbances: exogastrulation, on the one hand, and cyclocephalic malformations, on the other (Raven, 1942), These results were compared to similar nialformations obtained with the same agent in sea urchins and amphibians, respectively, and it was concluded that “the Li effects observed in the eggs of Lymnaea give support to the hypothesis that the development of the Spiralia is controlled by the interaction of gradient-systems, like those discovered in echinoids and Amphibia. The action of Li could be explained, then, by an inhibition of one of these gradients, localised, probably, in the egg cortex.” From a study of the structure of the head region in cyclocephalic embryos produced by lithium (Raven, 1949), it was concluded that the effects of lithium treatment are most pronounced at the animal pole and diminish in intensity with increasing distance from this pole. This was interpreted by assuming that a gradient field with a maximum at the animal pole plays an important part in the development of the head region in Lymnaea. That this animal gradient field is located in the cortex was deduced from the fact that it is not influenced by moderate centrifuging; the pattern of head organs in embryos from centrifuged eggs is relatively undisturbed (Paris, 1953; Raven and Beenakkers, 1955). The centrifugation experiments mentioned above indicated that the cortical factors act, first, by directing the displacement of cytoplasmic substances during ooplasmic segregation. Other observations seemed to show that the localization of nuclei and spindles is likewise dependent on the cortex. LiCl may cause deviations in the direction of both maturation spindles (de Groot, 1948) and cleavage spindles (Raven and Roborgh, 1949). Sometimes, the egg karyotneres appearing after the extrusion of the second polar body are displaced in these eggs toward the egg center, and the migration of the sperm nucleus is suppressed or delayed. Treatment of the eggs with LiCl solutions during third cleavage provokes a reduction in size of the micromeres, presumably owing to a displacement of the spindles toward the animal pole (Raven et al., 1952a). In eggs treated with LiCl at the 24-cell stage, the nuclei in animal cells are displaced toward the surface (Raven and Dudok de Wit, 1949).

CORTEX AND SUBCORTEX OF

Lymnaea

EGG

3

11. T h e Cortical Cytoplasm

A. PHYSIOLOGICAL PROPERTIES OF

THE

EGG CORTEXIN Lymnaea

If we interpret the results of centrifugation experiments showing that the animal-vegetative polarity, the localization of substances during ooplasmic segregation, and the direction of cleavage divisions remain relatively unaffected after moderate centrifuging, by the assumption that the factors controlling ooplasmic segregation and cleavage are bound to the egg cortex, this presupposes that the cortical layer of the egg has a marked rigidity, in contrast to the more fluid internal cytoplasm and, therefore, is not displaced by centrifugal force. The observation that even small lesions of the egg surface lead to a rapid outflow of cytoplasm points to a greater rigidity of the cortex. Direct measurements of the strength of the egg cortex have not been made in Lyvznueu, however. Fluctuations in cortical rigidity during the uncleaved stage were deduced from experiments in which egg samples were centrifuged at different intervals between oviposition and first cleavage (Raven, 1945). The elongation of the eggs by centrifugal force can be used for the evaluation of cortical rigidity. The results indicated that this rigidity undergoes cyclic changes during the uncleaved stage, being low during the extrusion of the first polar body, shortly after the extrusion of the second polar body, and immediately before first cleavage, and higher prior to the beginning of maturation and in the intervals between the minima. The first two minima coincide with periods of ameboid mobility. de Vries (1947) showed by the same method that hypertonic solutions of LiCl and CaC1, cause an increase in cortical rigidity, but that it decreases in a slightly hypotonic (0.024 M ) LiCl solution. Ameboid mobility is increased both in hypotonic (0.0240.048 M ) and hypertonic (0.048-0.144 M ) LiCl solutions, however (de Groot, 1948; Grasveld, 1949; Raven and Roborgh, 1949; Blaauw-Jansen, 1950). This seems to be a rather specific lithium effect, as no increase in ameboid activity was observed in solutions of other chlorides. The number of eggs exhibiting ameboid mobility is strongly decreased at higher temperatures (29-33" C) (Blaauw-Jansen, 1950). The egg cell of Lymnaea is tightly surrounded by a vitelline membrane which is thin and hardly visible in recently laid eggs. When the first polar body is formed, the membrane folds smoothly around it, so that the polar body gives the impression of lying outside the membrane. Shortly afterward, the consistency of the vitelline membrane apparently changes. With the extrusion of the second polar body, the membrane is lifted from the egg surface at the animal pole, and the polar body is distinctly flattened beneath it (Raven, 1945; Hudig, 1946; Grasveld, 1949). During cleavage, the membrane first bends inward with the cleavage furrows, but soon becomes detached from the egg surface in the furrow region (Hudig, 1946). It surrounds the embryo until a

4

CHRISTIAAN P . RAVEN

late gastrula stage; then it is thrown off (Hudig, 1946; Verdonk, 1965). In the electron microscope, the vitelline membrane shows a feltlike structure (Elbers, 1959). It contains star-shaped inclusions which may be remnants of detached microvilli (Bluemink, 1967). The membrane shows a strong metachromatic staining with azure A at alkaline pH and a less intense metachromatic staining with toluidine blue (Ubbels, 1968). The eggs of L. stagnalis show an increase in volume, on an average, of about 45% in the 4-5 hours between oviposition and first cleavage (Raven, 1945). This swelling apparently results from the uptake of substances, mainly water, from the surrounding egg capsule fluid. In decapsulated eggs transferred to distilled water, the rate of swelling is considerably increased. This swelling is an osmotic process. Recently laid eggs are about isotonic with an 0.093 M solution of a nonelectrolyte. The permeability constant for water was found to lie near 2 x 10-7 (expressed in gram-molecules/sec/cm2/gram-molecule/liter) (Raven and Klomp, 1946). Water permeability of the egg surface appears to be increased in 0.02 M and, to a less extent, in 0.01 M solutions of LiCl (Raven et al., 195213). Uncleaved eggs chilled to 3" C in 0.1 M urea solution show a significant increase in volume, on an average, of about 55% over eggs kept at room temperature. A similar, although not fully significant, increase in volume was observed in eggs treated with 0.001 M KCN. It is doubtful, however, that these observations can be interpreted as a proof that the normal egg volume is maintained as a steady state; at best, the active extrusion of water through the egg surface plays only a minor part during the uncleaved stage although it becomes important at cleavage stages (Raven et al., 1953). Elbers (1959), in an electron microscope investigation of Lymnueu eggs, found that LiCl in concentrations of from 1.2 to 4.8 10-2 M has no visible influence on the submicroscopic structure of the cytoplasm and cell membrane. In higher concentrations cytolysis occurs, beginning with disruption of the plasma membrane, followed by a degradation of the cytoplasm and of the cytoplasmic organelles, spreading in a radial direction through the egg from the surface inward. He concluded that lithium ions in subcytolytic concentrations do not penetrate or hardly penetrate into the egg cell. This agrees with the observation of Raven et ul. (195213) that Lymnuea eggs in hypertonic LiCl solutions shrink by dehydration until an equilibrium volume is reached, which then may be maintained for some hours provided no cytolysis occurs. Elbers (1966) showed by means of a microconductometric method that ions leak out only very slowly from the intact egg cell in distilled water. After cytolysis, ions begin to diffuse out of the egg. The salt content of the living egg corresponds to a solution of about 0.042 M NaCl, if the nonsolvent part of the egg is not taken into account. The ion permeability of the intact cell membrane is not increased by treat-

x

CORTEX AND SUBCORTEX OF

Lymizaea

EGG

5

ment with polyene antibiotics (C. E. Hulstaert, unpublished observations). The fact that these substances cause rapid cytolysis of the eggs probably indicates the presence of cholesterol as a constituent of the membrane (Demel et al., 1965). A further argument for a low ion permeability of the surface membrane of the egg is provided by its high direct current resistance. Egg cells were exposed to a direct current electric field in a shallow Perspex electrophoresis cell (Raven, 1948). Decapsulated eggs in distilled water show no electrophoretic movement, nor d o they orient with respect to the direction of the electric field. As long as they remain intact, they pursue their development in a normal way synchronously with the controls; no abnormalities in the position of polar bodies or the direction of cleavages occur. The study of eggs fixed immediately after treatment showed that such eggs exhibit no abnormalities in structure or displacement of egg components, even in a field of 80 V/cm. In eggs previously stained with vital stains (neutral red, Nile blue hydrochloride), no color differences related to the direction of the electric field were observed. In strong electric fields (e.g., 70 V/cm, current density 8-10 x 10-5 A/cm2), the eggs soon cytolyze. Cytolysis as a rule occurs after a certain latency period; this period is shorter, on an average, the stronger the field is. During the latency period, no visible changes of the egg can be observed. Once initiated, cytolysis takes a rapid and characteristic course. A hydration of the central part of the egg, combined with a disintegration of cytoplasmic structure both at the anodal and cathodal poles of the egg occurs. This leads to total destruction of a circumscribed part of the egg cortex on the side of the anode. The cytoplasm flows out, spreading beneath the vitelline membrane which in most cases soon bursts. The contents of the whole egg then show a distinct electrophoretic stratification. Immediately after the bursting of the vitelline membrane, the whole cytoplasmic mass begins to move toward the anode rather quickly. When Lyrnnaea eggs are treated with hypertonic solutions, vesicles and blebs appear on the egg surface beneath the vitelline membrane (Raven and Mighorst, 1946; Grasveld, 1949). They often fuse with each other to form larger vesicles, and may become entirely separated from the egg. These vesicles have a clear citrine fluid content and are separated from the outer medium by a thin film, probably composed of phospholipids. The Lymnaed egg lies singly in a capsule, bathed in a capsule fluid containing about 14.3% dry matter; half of this is galactogen and the other half mainly protein (W. F. Jansen, unpublished observations). The capsule fluid contains diffusible calcium. Egg capsules take up calcium ions from a CaCl, solution and lose calcium ions upon treatment with distilled water or diluted alkali chloride solutions. In the latter case, the amount of calcium ions diffusing

6

CHRISTIAAN P . RAVEN

out of the capsule is dependent upon the concentration of the alkali chloride solution but independent of the kind of alkali chloride. Apparently, the capsule membrane is freely permeable to small ions, but calcium ions cannot diffuse freely from the capsule fluid (Geilenkirchen, 1961). Lyrnnaea eggs in their capsules develop normally when they are kept for 24 hours in a certain amount of distilled water, starting at first cleavage. Treatment for the same period with running distilled water causes a considerable swelling of the eggs and a high death rate; treatment with running tap water (calcium concentration 7.3 x M ) is harmless (Geilenkirchen, 1961). The development of decapsulated eggs in distilled water is soon arrested (Raven and Klomp, 1946; Raven and van Zeist, 1750), but in tap water or in a CaCl, M (Grasveld, 1747) development proceeds. Apparently, the solution 4 x lower concentration of calcium ions that permits development is of the order of 5 x 10-4 M . When Lymnaea eggs are decapsulated and put in calcium-free solutions, cleavage becomes abnormal. The blastomeres remain spherical and do not flatten against each other as they do during normal cleavage. The cells lose their coherence at later stages and fall apart into a loose aggregate of spherical cells loosely surrounded by the vitelline membrane. Apparently, the adhesivity of the cells is greatly diminished. Addition of CaCl, at a concentration of 5 x 10-4 M or more gives normal cleavage (Raven and Klomp, 1946; Hudig, 1946; Grasveld, 1949). If the eggs are treated in their capsules with sodium oxalate or sodium citrate, similar aberrations of cleavage are produced (Stalfoort, 1952). Eggs pretreated for 10-30 minutes with a hypertonic (0.05-0.1 M ) CaC1, solution and then transferred to distilled water may show normal cleavage in this medium (Raven and Mighorst, 1946). It is evident that calcium is essential for preserving the normal adhesivity of the egg surface. Its role can be taken over by magnesium, however, and, to a lesser extent, by lithium (de Groot, 1948; Grasveld, 1949). Cleavage in NaCl solutions, on the other hand, is always abnormal; in KCI it is still more abnormal. It has further been shown that CaCI, protects the eggs against the injurious effects of a heat shock treatment. Both the total risk of abnormality and the severity of the disturbances is reduced in CaCI, solutions (Raven and van Erkel, 1955). On the other hand, an excess of calcium in the capsule fluid is harmful too. If eggs in their capsules are put in CaC1, solutions, the death rate increases according to an S-shaped curve at concentrations of from 2.5 to 3.5 x 10-2 llf (Raven et a/., 1956; Geilenkirchen, 1961). Further information on the properties of the surface layer was gained from experiments with various alkali metal ions. If eggs in their capsules are treated at early stages for 24 hours with solutions of alkali chlorides, in each case with

CORTEX AND SUBCORTEX OF

Lymnaea

EGG

7

increasing Concentration, an S-shaped mortality curve is obtained. Lethal coilM for centrations are very different, however, varying from about 7 x lithium to 2.1 x 1 0 - 2 M for sodium. They increase in the order Li < Cs < Rb < K < Na (Fig. 1) (Raven et al., 1956).

L

0

E + 0 m

._ L

n

-3 log

c

FIG. 1. The relation between mortality and concentrations of LiCl, NaCI, KC1, RbCI, and CsC1. Abscissa, log concentration (in equivalents per liter). Ordinate, percentage mortality before gastrulation.

These relationships were compared with the investigations by Bungenberg de Jong (1949) on the influence of salts on certain dicomplex colloid systems. It was found that the water content and stability of these colloid systems are dependent on the salt concentrations in the surrounding medium. Cations are fixed on the ionized groups of negatively charged colloids. With increasing salt concentration, the charge of the colloid particles decreases until charge reversal takes place. The salt concentration at which this occurs differs for different cations. If the reversal of charge concentrations of several cations are arranged in a diagram, a cation spectrum is obtained which characterizes the ionized group of the colloid. For sulfate and carboxyl colloids, cation sequences always correspond to decreasing atomic weight. This does not hold for phosphate colloids, for which characteristic irregular ion sequences are found. This is explained by differences in the polarizability of the ionized groups of the colloid anion with respect to water. The reversal of charge concentrations can be considered as an inverse measure of cation affinity; for cations with greater affinity for the ionized group of the colloid, reversal of charge will be reached at a lower concentration. A sequence such as we found for the lethal action of alkali metal ions on the Lymnaea egg agrees with the “ion spectrum” of reversal of charge concentrations for phosphate colloids, but differs from that for carboxyl and sulfate

8

CHRISTIAAN P. RAVEN

colloids. To be sure, the actual reversal of charge concentrations found in Bungenberg de Jong’s experiments lie far above the lethal concentrations for Lynzizuea eggs. Therefore, it is unlikely that death of egg cells by alkali metal ions is a direct consequence of reversal of charge of a phosphate colloid. At these low concentrations, no significant change of charge can be expected. The similar action spectra of the two phenomena may, however, point to a similarity of the underlying cause: the differences in cation affinity. Therefore, it was concluded that the effects of the various cations on the egg were connected with their action on a phosphate colloid. In view of the low ion permeability of the egg surface, this phosphate colloid is probably located in the surface membrane of the egg. It was, therefore, assumed that the cations of the outer medium, by being fixed on the ionized groups of the phospholipid component of the egg membrane change the latter’s properties. At a certain concentration of the cation, the physiological properties of the membrane are altered to such an extent that normal development is no longer possible (Raven et d l . , 1956). It was further found that calcium has a marked antagonistic effect with respect to the action of alkali cations (de Vries, 1953; Raven et d., 1956; Geilenkirchen, 1964). When the chlorides of lithium, sodium, potassium, or cesium are combined with CaCI,, the combinations giving 50Yo mortality plotted against the concentrations of the two salts give a distinct antagonism curve (Fig. 2). As the phenomenon of ion antagonism in model experiments with colloids is characteristic of systems containing phosphatides, this is a further argument for the importance of phospholipids in these effects. The shape of the antagonism curves is different in the two cases, however. With respect to charge phenomena of phosphate colloids in vitro, alkali cations antagonize the effects of calcium, but the converse is not true. With respect to egg mortality, however, the antagonism is reciprocal; small amounts of alkali chlorides relieve the effects of lethal concentrations of calcium but, conversely, small amounts of CaC1, diminish the lethal effects of alkali metal cations (Raven et al., 1956; Geilenkirchen, 1964). While these relationships were, in the first instance, established with respect to egg mortality, it must be stressed that the morphogenetic abnormalities (eg., exogastrulation and head malformations after lithium treatment) fit into the same scheme; they occur in special areas of the concentration field (Geilenkirchen, 1964) (Fig. 2 ) . This seems to show that the cortical system important for morphogenesis is identical with the one that is physiologically active. These results have led to the following interpretation (Raven, 1957; Geilenkirchen, 1961, 1964). It is evident that a certain amount of calcium ions in the egg cortex is necessary for its stability and physiological integrity. With increasing calcium concentrations in the medium, more calcium ions are bound to the cell membrane. This causes a decrease in charge and thereby an increase

CORTEX AND SUBCORTEX OF

Lynnaea EGG

3

in density of the cortex until the upper limit of the physiological range is exceeded and mortality occurs. It is important to note that the lethal concentration of CaC1, for Lymnaea eggs is of the same ocder of magnitude as the calcium concentration for reversal of charge of phosphate colloids in vitro in BungenLiCl

4.0 x 10-2M

7 3.0 x 10‘.

28 x

10-2

2 . 0 x l O F GW 0

i)

1.0x 10-2

0.5

1.0

D

2.0

3.0

4.0

5.0

1.0~10-~ 6.0

LiCl

FIG. 2. Interaction of CaCI, and LiCl with regard to “direct” mortality (death before gastrulation). Abscissae: concentrations of LiCl expressed in proportion to concentration giving 50% death when acting alone. The corresponding concentrations in moles are at the top. Ordinates: concentrations of CaCI, expressed in the same way; concentrations in moles at right. Dots: combinations causing 50% death. The curve ABCD is the “isothanate 50” for lithium-calcium mixtures. Combinations within this curve give less than 50% death before gastrulation, combinations outside the curve more than 50% death. In the case of mere additivity of CaCI, and LiCl effects, the straight line AD would have been found as the isothanate 50. Combinations in the lightly shaded area enclosed by the isothanate give more than 50% exogastrulation; those in the darkly shaded area more than SOTo normal development. Other malformations are mostly found in the unshaded area between the two. PQ: concentrations about isosmotic to the eggs. After Geilenkirchen ( 1 9 6 4 ) .

berg de Jong’s experiments. Addition of alkali salts in low concentrations counteracts the lethal effects of calcium as a consequence of ion antagonism which is mainly the result of a lowering of the activity coefficient of calcium in the medium by the added anions. O n the other hand, when the eggs are treated with alkali chloride solutions alone, some calcium diffuses from the capsule fluid into the medium and, presumably, calcium ions are lost from the cell membrane of the egg. This effect does not depend greatly on the kind of alkali metal. Further, the activity coefficient of calcium in the surrrounding egg

10

CHRISTIAAN P. RAVEN

capsule fluid is lowered by the added anions; this effect is also equal for all alkali chlorides at equal concentrations. Moreover, alkali metal ions are fixed on the negative groups of the phosphate colloid in the cell membrane in competition with the calcium present. This effect is greatly dependent on the nature of the ions. The readiness with which a certain ion is fixed on the colloid is inversely related to its reversal of charge concentration. Addition of calcium salts to the medium now counteracts both the loss of calcium ions from the egg membrane and the fixation of alkali metal ions to it. 100

. 0

I

-4

MgCI, CaCI2

-

-3

-2

log

SrCI2

BaCI,

-1

c

FIG. 3 . The relation between mortality and concentrations of MgCI,, CaCI2, SKI,, and BaCI2. Abscissa: log concentration (in equivalents per liter). Ordinate: percentage mortality before gastrulation. After Geilenkirchen (1961).

This interpretation has been further tested by Geilenkirchen (1961) in a comparative study of the effect of the alkaline earth ions magnesium, calcium, strontium, and barium. The concentrations of MgC1, and SrC1, provoking 50% mortality of Lymiraeu eggs were found to be close to that for CaCl,, in the order Mg < Ca < Sr. This fits the hypothesis regarding their action on the charge of a phosphate colloid. MgCl, and SrCI2 also caused considerable mortality at lower concentrations, however, while BaC1, caused an increasing death rate up to 100% at still lower concentrations (Fig. 3 ) . These latter effects of alkaline earth ions are assumed to be the result of their competition with calcium for fixation on the electronegative groups of the cortical phospholipid. To a certain extent, magnesium and strontium are supposed to be able to replace calcium in its effects on the stability of the membrane, so that their mortality curves, after an initial rise, exhibit a plateau or even a drop with increasing concentrations of the salt before rising once more in the region of reversal

CORTEX AND SUBCORTEX OF

Lymizaea

EGG

11

of charge concentrations. Mortality by barium at low concentrations, according to this interpretation, is entirely the result of its fixation on the membrane in competition with calcium, which it cannot replace in preserving the integrity of the membrane. Thus, mortality occurs at concentrations far below those causing reversal of charge. Summarizing, we may conclude that the investigations point to the existence of a surface layer of the Lymnaea egg consisting of a colloid complex in which, in addition to proteins, phospholipids and probably cholesterol take part, while calcium ions are essential for its stability and physiological integrity.

NATURE, AND BEHAVIOR OF B. COMPOSITION,

THE

EGG CORTEX

Cytochemical observations have shown that the cortex of the Lymnaed egg is rich in RNA. When the eggs are stained according to the method of UnnaBrachet, the outer boundary of the cytoplasm is visible as a distinct deep-red line. Beneath it is a narrow subcortical layer staining more deeply than the rest of the cytoplasm. In centrifuged eggs, this subcortical layer is only visible in the zone of hyaloplasm, but the deep-red outer lamella also extends over the oil and yolk zones. After ribonuclease treatment, the whole egg remains colorless. Elbers (1959) observed in his electron microscope study of the uncleaved Lymnuea egg that the egg surface is formed by a triple-layered plasma membrane approximately 100 A thick. No especially differentiated cytoplasmic layer beneath this membrane was found. This was the case even in centrifuged eggs, in which the cytoplasmic inclusions displaced by centrifugal force (lipid droplets, yolk granules, vacuoles) accumulated locally immediately beneath the membrane; no “cortical gel layer” resisting displacement by centrifugal force could be observed. Elbers concluded that the plasma membrane probably is the carrier of the “cortical” factors pIaying a part in morphogenesis. This is in agreement with the fact that, for example, lithium ions even in very low concentrations produce characteristic morphogenetic aberrations, while it is probable that they do not penetrate or hardly penetrate into the egg; apparently they exercise their effects by acting on or in the surface membrane. While a distinct cortical layer beneath the plasma membrane is not visible as a rule in uncleaved eggs, such a layer may be present in places at other times. During cleavage divisions, the cell membrane folds inward and forms the walls of the cleavage furrows; when the blastomeres flatten against each other, the fused membranes form the intercellular partitions. First cleavage begins with an indentation of the egg surface at the animal pole. Here a very distinct cortical layer beneath the plasma membrane can be seen. From this point, the plasma membrane rapidly invaginates into the egg as a narrow fold. It i s lined by the cortical layer, which is especially well developed near the

12

CHRISTIAAN P. RAVEN

advancing tip of the furrow. The two cell membranes forming the sides of the furrow then fuse, forming the partition wall between the cells (W. Berendsen, personal communication) . In this partition wall, a cleavage cavity is already formed at the two-cell stage by the fusion of small clefts appearing in it. This recurrent cleavage cavity, which opens periodically to the exterior and apparently plays an important part in the water regulation of the early embryo, will not be further considered here (cf. Raven, 1946a, 1966).

FIG. 4. Stages of pinocytosis in a blastula cell of L. stagnalis, schematic. N, nucleus, After Elbers and Bluemink (1960).

At later cleavage stages (starting at about the 24-cell stage), the cells begin to ingest albumen from the surrounding egg capsule fluid which is laid down as albumen vacuoles in their superficial parts. At the same time, ring-shaped, conical, and hemispherical projections appear on their outer surface (Raven, 1946a). It has been shown by means of electron microscopy that the uptake of albumen takes place by a peculiar process of pinocytosis (Elbers and Bluemink, 1960; Bluemink, 1967). Ringlike elevations of the cell surface appear, which converge from all sides and fuse in the middle, thus enclosing part of the egg capsule fluid as a vacuole (Fig. 4 ) . This is bounded by a triple-layered membrane identical in appearance to the plasmalemma from which it is derived. The vacuole detaches from the surface and migrates into the cell interior. Additional uptake of albumen occurs through niicropinocytosis by membrane vesiculation at the cell surface. After their formation, the albumen vacuoles coalesce; occasionally they pinch off protrusions. The vacuole membrane may rupture within 15 ininutes after vacuole formation, releasing the albumen into the cytoplasm. After rupture, the discontinuous vacuole membrane curls inward at both margins. In addition, coalescence of albumen vacuoles with yolk granules can be demonstrated. Presumably, the capsule fluid is the main nutrient reserve for the developing embryo, whereas the yolk granules supply the hydrolytic enzymes necessary for its digestion (Bluemink, 1967).

C. THEFORMATION OF

THE

EGG CORTEX

Further information about the cortical cytoplasm of the Lymnueu egg has been obtained by a study of its formation during oogenesis.

CORTEX AND SUBCORTEX OF

Lymnaea EGG

13

The hermaphroditic gonad of Lynmaea consists of numerous acini enclosed within the hepatic gland. The acini are arranged in a cluster around the spermoviduct into which they open by means of short vasa efferentia. The wall of the acini and efferent ducts consists of an outer fibrous layer of connective tissue with small nuclei. In the distal two-thirds of the acini this is lined on the side of the lumen with the germinal epithelium consisting of two regions: (1) the oogenetic zone, occupying the most apical part of the acinus and encircled on all sides by ( 2 ) the spermatogenetic zone, in which the variOLIS stages of spermatogenesis follow each other in a proximodistal direction. In the oogenetic zone, younger and older oocytes lie side by side without any apparent regularity. The early oocytes show signs of ameboid mobility (Bretschneider and Raven, 1951). Presumably, they move about, but do not leave the oogenetic zone in which they originate. Then they become sessile and a follicle is formed around them. The wall of the gonad consists of a 1- to ?+-thick homogeneous basal membrane, which is strongly folded. In this membrane, fibrils about 150 A thick are embedded in two crossing directions; they show no cross striation. Fibroblasts and pigment cells lie on the outside of the membrane. Micropodia of oocytes and follicle cells penetrate deeply into the basal membrane (Recourt, 1961). The substance of the basal membrane is strongly PAS-positive (Recourt, 1961). It further gives a strong reaction for tryptophan with p-dimethylaminobenzaldehyde. With toluidine blue and azure A, a metachromatic staining of the basal membrane adjacent to the oocytes occurs. With Alcian blue, fibrillar structures in the basal membrane stained deeply at all pH values tested (pH 2.2-7.2) (Ubbels, 1968). It is evident, therefore, that it contains acid mucopolysaccharides. The sessile growing oocytes lie with one side against the wall of the gonad (Figs. 5 and 6). Originally they are rather flat, and the side lying against the acinus wall takes up about 40% of their surfaces. With further growth they bulge forth into the lumen, and the part applied to the acinus wall i s gradually reduced to about 15-20% of their surfaces. The rest of the oocyte is surrounded by follicle cells. A thin sheet of cells covers the oogenetic zone on the side of the lumen (Bretschneider and Raven, 1951; Ubbels, 1968). The follicle consists more or less distinctly of two layers of cells. Apparently, it is gradually built up by the recruitment of neighboring cells from the germinal epithelium (Raven, 1963; Ubbels, 1968; LJbbels et a/., 1969). During the first phases of slow oocyte growth, the number of follicle cells increases. When rapid growth begins, the inner layer of follicle cells immediately adjoining the oocyte is completed and no further cells are added to it. In L. s[agnulis, this inner layer then consists of six (exceptionally seven) cells; in Lpnaed peregra it consists of six to eight, occasionally nine, cells.

14

CHRISTJAAN P. RAVEN

The inner follicle cells arc more or less wedge-shaped in cross section (Figs. 5 and 6). They are attached with their wider basal parts to the basal membrane of the acinus wall around the oocyte by means of micropodia penetrating into this layer (Recourt, 1961). The rest of the cell curves smoothly around the side of the oocyte toward its inner pole. The nucleus is large and flat; as a rule it lies in the basal half of the cell close to the surface applied to the oocyte. The peripheral parts of the follicle cells are very thin and membranous. Electron microscope observations have shown that the borders of adjacent follicle cells meet at the lateral and apical side of the oocyte and, by mutuai

FIG. 5 . Position of oocyte against the wall of the gonad, enveloped bjr two layers of follicle cells.

interpenetration of folds, form imbricated complexes of triple-layered membranes (Recourt, 1961; J. E. Rigby, in preparation). The outer follicle cells are less characteristic. Their shape and structure is more or less intermediate between that of the undifferentiated germinal epithelium cells and of the inner follicle cells. Originally, the plasma membranes of the oocyte and of the inner follicle cells are smooth and closely applied against each other (Fig. 6). In older follicles, however, a cleft appears between the oocyte and follicle cells, first at the apex, then gradually extending toward the base of the oocyte (Fig. 7). At the same time, sparse, rather short, blunt microvilli are formed on the surface of the oocyte (Recourt, 1961; J. E. Rigby, in preparation). They are embedded in a layer of a iibrillar or very finely grained substance which now forms on the surface of the oocyte and which probably represents a precursor of the vitelline membrane. The follicular cavity does not for some time extend along the basal surface of the oocyte where this surface lies against the connective tissue wall (Fig. 7 ) and, according to Recourt (1961), penetrates into it with micropodia. Only immediately before ovulation is this surface of attachment rapidly reduced

CORTEX AND SUBCORTEX OF

Lymmed

EGG

15

FIG. 6. Growing oocyte of L. .rtaRnalir in the gonad. AW, Connective tissue wall of iiciiius.

IFC, inner follicle cell. Staining: azan.

x 660.

until the full-grown oocyte becomes entirely free and, after autolysis of the follicle cells and the overlying acinus epithelium has taken place, passes into the lumen of the gonad. The follicle cells contain fat droplets and a special kind of lamcllary bodies mnsisting of concentric triple-layered membranes between which vacuoles or

FIG. 7. Full-grown oocyte in the gonad. AW, Acinus wall; FC, folliculdr cavity; ZK. zona radiata. Staining: azan. x 600.

16

CHRISTIAAN P. RAVEN

globular inclusions may be present. These bodies are rich in lipids and resemble the “yolk nuclei” described in oocytes of various animals (Recourt, 1961). With the Nadi reaction, numerous indophenol blue granules appear in the follicle cells (Bretschneider and Raven, 1951). A study of the follicle cells with various cytochemical methods (Ubbels, 1968) revealed the striking fact that often the reaction of individual follicle cells was variable, ranging from strongly positive in some to almost negative in other cells, even in the same follicle. This was observed with the Hg-BPB reaction for proteins, with reactions for tryptophan, arginine, and histidine, the DDD-reaction for sulfhydryl proteins, and with the Unna-Brachet reaction for RNA. It is not known whether these differences reflect different phases of cyclical metabolic processes or more stable inequalities in the synthetic capacities of individual follicle cells. The cytoplasm of the follicle cells contains small phospholipid granules and metachromatic granules. The latter were found especially along the border between oocyte and follicle cells and, moreover, rows of such granules were observed in the basal part of the follicle cells parallel to this cell border (Ubbels, 1968). Bretschrieider and Raven (1951) observed that during the growth phase of the oocyte, hyaline cytoplasm is accumulated at its apex; it is connected with the perinuclear cytoplasm. In the full-grown oocyte, only a thin layer of hyaline cytoplasm at the periphery, free of granular inclusions, remains; it is bounded peripherally by the oolemina and toward the endoplasm by a layer of fine granules. It has a finely vacuolar appearance after several fixations. Often these vacuoles show a radial arrangement which gives a radially striped appearance to this cortical cytoplasmic layer. This structure has been further studied by Ubbels (1968). A strongly basophilic apical cap of cytoplasm arises in young sessile oocytes. It lies somewhat asymmetrically with regard to the apicobasal axis of the oocyte and contains granular and fibrillar structures rich in protein-bound sulfhydryl groups. The basol2hilic staining is apparently attributable to the presence of large amounts of ribonucleoprotein. The presence of basic protein in it is indicated by an intense histidine reaction. When vitellogenesis begins, this region remains free of yolk. Its connection with the perinuclear cytoplasm becomes progressively narrower, while its peripheral part gradually extends along the surface. At the same time, many small metachromatic granules appear in it. Finally, the connection with the nucleus is broken and a layer of yolk-free cytoplasm spreads along the surface of the oocyte in apicobasal direction, but only as far as the follicular cavity reaches; it does not extend along the basal surface of the oocyte applied against the basal membrane. This layer becomes gradually thinner and

CORTEX AND SUBCORTEX OF

Lymizaea

EGG

17

is about 1 p thick in full-grown oocytes. It often has a radially striated appearance and is, therefore, referred to as the “zona radiata” (Fig. 7 ) . Some observations suggest that synthetic processes in the nucleus or the perinuclear cytoplasm contribute to the formation of the apical cytoplasm and the zona radiata. The zona radiata shows weak to moderate staining reactions for proteins and basic proteins, protein-bound sulfhydryl groups, and RNA. It contains many small metachromatic granules and stains moderately with Alcian blue at pH 5 and above. N o protein-bound SS, phospholipids, or iron could be demonstrated in it. Its inner boundary with the rest of the cytoplasm exhibits somewhat stronger protein and basic protein reactions. A row of small granules lying at the boundary, giving strong RNA and sulfhydryl reactions, may be mitoc-hondria. A thin outer lamella of the zona radiata differs in various respects from its remaining part. It is strongly PAS-positive and gives somewhat stronger protein, basic protein, sulfhydryl, and RNA reactions than the main part of the zona radiata. It shows a strong metachromatic staining with toluidine blue and azure A above pH 2.5-3. Staining with Alcian blue is strong above pH 5 , diminishes at lower pH values to a minimum at pH 3, and then becomes stronger again to pH 2.2, at which point this lamella is the only staining structure in the oocyte. A layer with similar staining properties is found on the inner side of the follicle cells bordering the follicle cavity. It is lacking, however, along the basal surface of the oocyte. The structure of the zona radiata has been further studied by means of electron microscopy. Contrary to expectation, no structural components oriented in a radial direction could be observed in it. It is a rather homogeneous layer of cytoplasm containing free ribosomes and small vesicles, but no mitochondria, yolk granules, or fat droplets (J. E. Rigby, in preparation). The appearance of a radial structure in light microscope sections is probably attributable to the presence of rather large vacuoles which are found in it in places, in agreement with a view already expressed by Bretschneider and Raven (1951 ) . After ovulation, the zona radiata is no longer visible in most cases. It is replaced in light-optical sections by a single dark line, staining rather intensely with the reactions for proteins, protein-bound sulfhydryl groups, and RNA. Further changes seem to occur during the passage of the egg cells through the oviduct, most of the reactions showing a further decrease in intensity. Only with toluidine blue does a peripheral yolk-free layer containing small metachromatic granules, lying beneath the intensely metachromatically staining vitelline membrane, remain visible after oviposition, while the RNA reaction of the outer layer also remains strong in such eggs.

18

CHRISTIAAN P. RAVEN

It is not easy to interpret these observations in terms of the structure of the egg cortex of oviposited eggs. Presumably, the outer lamella of the zona radiata, which apparently is very rich in acid inucopolysaccharides, represents the precursor of the vitelline membrane. The remaining part of the zona radiata must give rise to the definitive egg cortex. Further study of the changes occurring in this region during ovulation, fertilization, and egg maturation is needed to elucidate the details of its formation.

111. The Subcortical Cytoplasm Properly speaking, the term “subcortical cytoplasm” refers to the whole of the cytoplasm inside the cortex. It will be used here, however, in a restricted sense, to denote certain special cytoplasmic differentiations which have the following characteristics in common : they have a definite localization in the egg, they are situated immediately beneath the cortex (or the plasmalemma, respectively), and there is reason to believe that their localization is partly or wholly determined by directing factors located in the cortex. Three different cytoplasmic differentiations of the Lymnded egg answering this definition will be considered here: ( 1 ) the vegetative pole plasm, ( 2 ) the animal pole plasm, and ( 3 ) the “subcortical accumulations.”

A. THEVBGETATTVI~ POLEPLASM The vegetative pole plasm was first observed (Raven, 1945) in sections of eggs of L. stugnulis fixed in Bouin immediately after laying and stained with azan; it was called “ectoplasm” at that time. While most of the cytoplasm stained orange-red with this method, there was a special plasm around the vegetative pole that stained blue. In a meridional section, it covered a sector of about l l O o having its apex near the center of the egg (Fig. 8). It contained a dense mass of proteid yolk granules, staining deep indigo blue with azan. Scattered between them were some larger deep-red y granules. At the vegetative pole there was a small cap of finer granules, staining light greyishblue. When the deep aster of the first maturation spindle was situated at the boundary between the vegetative pole plasm and the remaining part of the cytoplasm, it showed a sectorial coloration; the part of the aster situated within the vegetative pole plasm was blue and the rest was orange-red; the line of demarcation between the differently colored parts within the aster was as sharp as it was elsewhere. The position of the vegetative pole plasni was not strictIy symmetric with respect to the longitudinal direction of the first maturation spindle. Therefore, either the vegetative pole plasm or the maturation spindle at this stage is situ-

s

CORTEX AND

suncORmx

OF

Lynzmeu

EGG

19

ated somewhat obliquely with respect to the main animal-vegetative axis of the egg (Raven, 1963). The position of the vegetative pole plasm, as depicted in Fig. 8, apparently is only of short duration. Very soon after oviposition it begins to spread beneath the egg cortex toward the animal side of the egg. Figure 9 shows the

MSP

FIG, 8. E,gg of L. .r&zgnalis fixed immediately after oviposition. MSP, First maturation spindle; VPP, vegetative pole plasm. Staining: atan. x 800.

situation as it is shortly before the extrusion of the first polar body. The vegetative pole plasm now occupies a crescent-shaped region beneath the egg cortex, leaving free the animal pole and its immediate surroundings. At the vegetative side, it forms a rather thick layer, toward the animal pole it becomes narrower and ends with a sharp edge. The gap at the animal pole remains until after the completion of the second maturation division. Then the pole plasm substance extends over this region also (Raven, 1945). During the first two cleavage divisions of the egg, the vegetative pole plasm substance, forming a layer of nearly uniform thickness beneath the cortex all ;wound the egg, is distributed about equally ainong the blastomeres. At the four-cell stage, however, it concentrates markedly in the animal side of the cells, and unites with the animal pole plasm lying there. A great deal of this common pole plasm substance passes at the next division into the first micromeres,

20

CHRISTIAAN P. RAVEN

which for the greater part consist of it, whereas the macromeres consist mainly of vacuolated cytoplasm derived from the remainder of the egg. This differential distribution of the pole plasm substance is repeated at the following divisions, so that the relative amount of pole plasm substance in the cells of the blastula decreases from the animal toward the vegetative pole (Raven, 1946a). Meanwhile, it has retained its subcortical position so that it lies in the

FIG. 9. Egg fixed about 1 hour after oviposition, shortly before extrusion of first polar body. Vegetative pole plasm has spread beneath egg cortex toward the animal pole. Staining: azan. x 800.

superficial region in all cells. It is more basophilic than the rest of the cytoplasm; this basophily is attributable to RNA. In a full-grown oocyte in the gonad, ready to ovulate, no vegetative pole plasm is visible. Its cytoplasm is filled with numerous granules of different size and stainability. In azan-stained sections, blue, red, and orange-red granules can be distinguished. As they are distributed almost at random throughout the cell, a uniform violet-red staining is observed at low magnifications (Fig. 7). We may conclude that the ooplasmic segregation giving rise to the formation of the vegetative pole plasm of the recently laid eggs takes place during the passage of the eggs through the genital duct of the parent, which takes about

CORTEX AND SUBCORTEX OF

Lymmen

EGG

21

3-5 hours (Bretschneider, 1948), by a local accumulation of cytoplasmic constituents which were almost uniformly distributed in the oocyte. This has been confirmed by the study of some egg samples taken from various parts of the genital tract. They indicate that a gradual separation of the granular inclusions of different staining takes place as the egg passes down the genital tract. The boundaries between regions of different staining become progressively more distinct. A well-formed vegetative pole plasm was observed in some eggs that had reached the pars nidamentaria of the oviduct. de Groot (1948) found that in eggs treated at early stages with LiCl the localization of the vegetative pole plasm is often abnormal. It does not spread evenly beneath the egg cortex but is piled up locally in considerable amounts, whereby no preference for a certain region seems to exist. At other places, it may be lacking altogether. With further development the distribution becomes more regular, and at the two-cell stage it is only slightly abnormal. The abiiormality was most pronounced in an isotonic LiCl solution; to a lesser degree it also occurred in slightly hypertonic solutions. This observation, taken in conjunction with the above-mentioned facts (Section 11, B) leading to the conclusion that lithium probably exercises its effects by acting on the surface membrane of the egg, suggests that the localization of the vegetative pole plasm is controlled by cortical factors. Unfortunately, further analysis of the manner of forination and the developmental significance of this pole plasm has been hampered by technical dificulties. The azan method, which in the first years of our work produced a brilliant coloration of the egg sections with exceptionally clear demarcation of the various plasms, since 1948 has given unsatisfactory results with this material. Attempts to remedy this by alterations in fixation and staining procedures have made no improvement. Presumably, the root of the trouble is some unknown change in the composition or manufacture methods of the dyestuffs. We did not succeed in finding some other staining method or cytochemical reaction producing an equally clear differentiation of this plasm. Therefore, for the time being we had to abandon any further attempts to obtain inore information on the nature of the vegetative pole plasm and the factors controlling its distribution.

B. THEANIMALPOLE PLASM The animal pole plasm has not yet been formed at the time of oviposition, but it appears during or shortly after the maturation divisions by the accumu-

lation of a special plasm beneath the egg cortex surrounding the animal pole. This occurs at various times in different Lymnaeidae. In L. peregra and Lymm e u ouata, the animal pole plasm is already visible at the time of formation of the first polar body, in Lymndea palzlstris, a short time afterward; in MJJX~ J

22

CHRISTIAAN P. RAVEN

~ ~ l z ~ t i n oits aappears , during the second maturation division, while it beconies visible in L. stagnalis about 1 hour after the extrusion of the second polar body (Raven, 1964b). The animal pole plasm is a layer of dense protoplasm lying immediately beneath the egg cortex in the animal hemisphere and staining dark violet-blue with iron hematoxylin (Fig. lo). As opposed to the rest of the cytoplasm at that time, it contains no vacuoles, but a great many mitochondria accumulate in it (Raven, 1945). When the first cleavage furrow indents the animal pole of the egg, it divides the animal pole plasm into two equal parts, each belonging to one blastomere. When the spindles of the second cleavage division are formed, the perinuclear layer of protoplasm fuses temporarily with the animal pole plasm in each of the two blastomeres. At the four-cell stage, the animal pole plasm unites with the greater part of the vegetative pole plasm substance in the animal region of each blastomere, and the common mass of pole plasm substance is at further cleavage unequally distributed among the cells, as described above (Section 111, A) (Raven, 1946a). In eggs of L. stagnalis centrifuged before the first maturation division, the animal pole plasm formed at the normal time beneath the cortex at the original animal side of the egg, irrespective of the direction of stratification of the egg substances brought about by centrifuging (Raven, 1945) (Fig. 11). This was explained by assuming that its formation is the result of an attraction exerted by a particular region of the egg cortex upon certain cytoplasmic components. Raven and Brunnekreeft (1951) studied the manner in which the formation of the animal pole plasm in centrifuged eggs depends on the moment of centrifuging and on the resulting differences in stratification. They found that a distinct animal pole plasm had formed at about the time of first cleavage in eggs centrifuged either before or immediately after the extrusion of the first polar body, but not in eggs centrifuged some time after the extrusion of the second polar body. This was explained by assuming that in the latter eggs the time interval between centrifuging and fixation was too short for the necessary rearrangement of egg substances and segregation of pole plasm substance to take place. Moreover, the possibility was taken into account that the frothy structure of the centripetal parts of eggs centrifuged during the later part of the uncleaved stage might have inhibited or retarded the processes of ooplasmic segregation. Raven and van der Wal (1964) have adduced evidence that the latter explanation is the more likely one. Raven and Brunnekreeft (1951) further found that in eggs centrifuged during the first part of the uncleaved stage, in addition to the animal pole plasm, another accumulation of deeply stained protoplasm beneath the cortex showing

CORTEX AND SUBCORTEX OF

Lymizaea

EGG

23

FIG. 10. Egg of L. stagnulis shortly after second maturation division. Begiiiiiiiig forination of animal pole plasm (APP) . 2PB, Second polar body. Staining: iron licmatoxylin-eosin. x 540.

FIG. 11. Egg centrifuged immediately after oviposition, fixed ;It stage of pronuclei (PR). Egg axis and stratification axis make angle of about 70’; centripetal end at right. Distinct animal pole plasm (APP) and “spurious pole plasm” (SPP). 2PB, Second polar body. Staining: iron hematoxylin-eosin. x 540.

24

CHRISTIAAN 1’. R A V E N

some resemblance to the animal pole plasm might occur. In further experiments, Raven and van der Wal (1964) analyzed the formation of this “spurious pole plasm.” They showed that two components of the animal pole plasm, which are inseparable in normal development, have to be distinguished : the ground substance (“matrix”) of the animal pole plasm, on the one hand, and the mitochondria, on the other. The conclusion that formation of the animal pole plasm is the result of attractive actions exerted specifically by the egg cortex in the neighborhood of the animal pole applies only to the matrix. The mitochondria, however, appear to be attracted in an unspecific way by all parts of the cortex. They tend to accumulate beneath the cortex at all places where their density is high enough. In centrifuged eggs, this occurs in the mitochondria zone, giving rise to a spurious pole plasm (Fig. 11). In normal develolmient, most mitochondria of the egg are conveyed toward the animal pole region by way of the maturation spindles and asters. After extrusion of the second polar body and the breakdown of the spindle and aster remnants, they then accumulate beneath the cortex in the area of the animal pole plasm. The view that cortical factors play a part in the formation of the animal pole plasm finds support in the results of lithium experiments. No aniiiial pole plasm is formed in uncleaved eggs treated with isotonic or hypertonic LiCl solutions (de Groot, 1948). When the eggs are treated for 1 hour with lithium and subsequently transferred to tap water, recovery may take place and a pole plasm is formed after some delay (van den Broek and Raven, 1951). Lithium does not suppress the animal pole plasm once it has been formed (Raven and van Zeist, 1950). C. SUBCORTICAL ACCUMULATIONS

In sections of eggs of L. stapdlis fixed immediately after ovipositioii and stained with azan, in addition to the vegetative pole plasm, some dispersed patches of blue cytoplasm were found in the equatorial region iniinediately beneath the surface (Raven, 1945) (Fig. 8 ) . In a reinvestigatioii of the eggs, these structures were studied more closely (Raven, 1963). These patches [hereafter called “subcortical accumulations” (SCA) ] are more or less lenticular regions of cytoplasm having about the same staining properties as the vegetative pole plasm (Fig. 1 2 ) . The granules they contain are, on an average, somewhat smaller than the p granules in the pole plasm. In all eggs there are six SCA. They are distributed unevenly, four of them lying on one side and two on the other side of the egg; moreover, their distances from the poles of the egg differ. Together with the obliquity of the position of the vegetative pole plasm, the distribution of the SCA indicates a certain dorsoventrality of the egg structure (Raven, 1963). In the full-grown oocyte in the gonad, ready to ovulate, the SCA are not yet

CORTEX AND SUBCORTEX OF

Ly7?z?zdea EGG

25

visible (Fig. 7). Apparently they arise, just as the vegetative pole plasm does, during the passage of the eggs through the genital duct of the parent by a local accumulation of certain cytoplasmic constituents beneath definite parts of the cortex.

.c.A.

FIG. 12. Egg of L. stugnulis fixed immediately after oviposition. ILISP, maturdtion spindle. SCA, subcortical accumulation. VPP, vegetative pole plasm. Staining: a m i .

x

1250.

Cytologically, the SCA consist of a dense cytoplasmic matrix with a great affinity for cytoplasmic stains. The special granules in them apparently are neither fi granules nor mitochondria. Originally, these granules make up the bulk of the granular contents of the SCA, but during early cleavage stages more and more fl granules appear among them. At certain stages of the cell cycle in uncleaved eggs, the granules seem to be replaced by rather coarse fibrils or lamellae, either arranged parallel to the surface, or forming more-or-less lensshaped bodies of coiled threads; whether they arise by a transformation from

26

CHRISTIAAN P. R A V F N

the granules, as seems possible, remaiiis to be established. The SCA are rather rich in RNA and in lipids (Raven, 1967). Further study of the SCA (Raven, 1967) confirmed that at the uncleaved stage they form a regular pattern which is dorsoventral and nearly symmetrical (Fig. 1 3 ) . Its plane of bilateral symmetry coincides with the median plane of

270'-

900

f

180°

FIG. 13. Average position of SCA in uncleaved eggs of L. siagnalir. The surface of the vegetal hemisphere of the egg is represented in horizontal projection. The vegetative pole is in the center; the SCA (stippled) are arranged in a subequatorial ring. The broken line indicates the approximate plane of symmetry of the SCA pattern.

the future embryo. Just before first cleavage, the SCA exhibit a latitudinal extension and fusion. The plane of first cleavage makes an angle of about 50' with their plane of symmetry. The SCA are distributed according to a definite program among the two, then four, blastomeres. Their positions in the four quadrants of the egg exhibit characteristic differences (Fig. 1 4 ) . In each quadrant, they show a gradual shift toward the vegetative pole. During these displacements, they retain their localization immediately beneath the plasma menibrane of the egg. At the third cleavage, all of the SCA substance passes into the macromeres where it is concentrated in the most vegetative part of each cell (Pig. 15). It is very rich in RNA (Minganti, 1950). At the beginning of the fourth cleavage, when the macromeres elongate in a sinistral direction in preparation for formation of the second micromeres, the SCA are drawn out beneath the surface in the same direction. All second rnicro-

27

‘D’

‘C’

‘5‘ FIG. 14. Average position of SCA in early eight-cell stage of L. stugnulis, as viewed from the vegetative side.

FIG. 15. Eight-cell stage of L. Jtugnulis. SCA in vegetative part of ~iiacmiiercs.Staining: iron hematoxylin-eosin. x 580.

28

CHRISTIAAN P. RAVEN

meres 2a-2d acquire a small part of this substance when they are split off from the macromeres. It remains visible for some time in these cells; when they next divide (after about 75 minutes), it goes in about equal parts to their daughter cells, but then soon becomes indistinguishable. The main mass of the SCA plasm has passed at fourth cleavage into the macromeres 2A-2D. In each of these cells, it divides before long into two parts. One portion remains concentrated in the most vegetative region of the cells, occupying the space on either side of the vegetative cross furrow and the angles between adjacent furrows at its extremities. This material, in which a new kind of coarse dark granules is formed at this time, tends to extend into the depth along the interblastomeric planes. The other portion of the original SCA substance passes at the next division for the greater part into the third micromeres 3a-3d, a smaller portion of it remaining in the macromeres along the furrow with the third micromeres. With the 24-cell stage that has now been reached, a break in the progress of cleavage occurs. While the preceding divisions followed each other at intervals of about 1 hour, on an average, cleavage is now interrupted for 3 hours (Verdonk, 1965). During this period, important processes take place. The cleavage cavity, which was already formed at the two-cell stage and showed a sequence of expansion and contraction concurrently with the rhythm of cleavage divisions, disappears altogether at this stage. This is mainly because of a change in shape of the macromere 3D, which partly withdraws from the surface, bulges with the greater part of its mass into the cleavage cavity, and applies itself against the inner side of the animal cells (Figs. 1 6 and 17). Thereby its contact relationships with other blastomeres are greatly enhanced. While most cells are in touch with, on an average, about seven other blastomeres, the cell 3D now has common surfaces with about 14 to 16 of the other cells. Specifically, it is in contact with at least six cells of the first quartet viz. the cells la1-] dl, 1a2, and 13.

Wherever these animal cells apply themselves with their inner ends against the bulging part of the macromere 3D, a special area of cytoplasm appears in them, which is free of yolk but filled with a mass of small granules staining grey-blue with iron hematoxylin. According to their staining properties, they are probably not mitochondria. As this region contains red granules after staining with Best’s carmine, they might be glycogen granules. In meridional sections, it forms a crescentic area surrounding the inner part of the D macromere. At the same time, the blastomeres begin to show pinocytotic activity at their outer surf aces. The coarse dark granules in the SCA plasm at the vegetative pole, which are very rich in RNA, now move along the interblastomeric walls toward the central ends of the macromeres where they fuse into irregular coinplexes (Figs, 16 and 17).

CORTEX AND SUBCORTEX OF

Lymnaed

EGG

29

FIG. 16. Egg of L. stagnaljs, 24-cell stage. Cell 3D (outlined) prulecting into central part of egg, in contact with animal cells. Coarsc “SCA-granules” migrating toward central ends of macromeres in 3D and 3C. Staining: iron hematoxylin-eosin. x 720.

FIG. 17. Same stage. Cell 3D outlined. Coarse “SCA-granules” migrating inward in jA (left), 3C (right) ; have already reached central end of 3D. Fine granules in central ends of micromeres adjacent to 3D. Staining: iron hematoxylin-eosin. x 720.

30

CHRISTIAAN P. RAVEN

After a resting stage of 3 hours, cleavage is resumed, beginning with the division of the macromere 3D. By an oblique and very unequal leotropic division, it is split into the small and superficially located macromere 4D and the primary mesoblast 4d (or M ) , which is much larger and comprises the whole interior part of the cell. By this division, the dorsoventrality of the embryo first becomes visible in the cell mosaic. It is soon followed by divisions of all micromeres. The division of the macromeres 3A, 3B, and 3C occurs about 2 hours after that of 3D. Half an hour later the cell 4d divides into its daughter cells M, and M,, by which a 49-cell stage is reached (Verdonk, 1965). Meanwhile, the complexes of coarse RNA-containing granules at the inner ends of the macromeres have condensed into compact oblong dark bodies which are very rich in RNA. These bodies, which are probably comparable to the “ectosomes” described by Wierzejski (1905) in Physa, pass into the cells of the fourth quartet, including the M blastomere. They lie in the central part of these cells, adjacent to the cleavage cavity which has reappeared with the resumption of cleavage divisions. They remain visible up to a stage of about 60 to 64 cells, then gradually disappear; but basophilic granules are still found in this region in embryos of about 120 cells (Raven, 1946a; Minganti, 1950). In later stages, the cleavage cavity may be bridged by cytoplasmic connections between animal and vegetative blastomeres which are free of granules and particularly rich in RNA. Minganti supposes that a transfer of RNA-containing substances from vegetative to animal blastomeres takes place during cleavage. Verdonlc (1965) discusses the possibility that this is important for the determination of bilateral symmetry in the head region of Lymnaea. This is as far as the history of the SCA and their derivatives has been studied in L. stagrza1i.r. Distinct SCA have also been found in L. p e w p a (Ubbels et al., 1969). They are similar in appearance, localization, and composition to L. r t u p a l i s . Apart from eggs with six SCA, however, eggs with seven SCA were also found in this species. In both groups, the SCA are arranged according to a definite pattern, which is symmetric in the eggs with six SCA (Fig. I S ) but asymmetric in those with seven. Their development has not yet been followed beyond the uncleaved stage in this species. The facts that the SCA are not present in the full-grown oocyte, but are probably formed by ooplasmic segregation during the passage of the egg cells through the genital tract of the parent, and that they retain their close apposition to the plasmalemma of the egg during cleavage divisions up to the 24ceIl stage, are most easily explained by the assumption of a specific attraction exerted upon certain components of the cytoplasm by specific areas of the plasma membrane. This attraction, initiated after ovulation, possibly by the change in environmental relationships of the egg cell, continues up to advanced cleavage stages. This

CORTEX AND SUBCORTEX OF

Lynznueri

31

EGG

FIG. 18. Average position of SCA in uncleaved eggs of L. fieregfa having six SCA. For explanation, cf. Fig. 13; a-f, the six SCA; arrows 1-6, corresponding positions of centers of follicle cells i n six-celled follicles.

supposition is corroborated by observations on centrifuged eggs. They show that the SCA are indistinguishable immediately after centrifuging, but are re-formed in subsequent hours in their typical localization, corresponding to the stage of development reached by the eggs in the meantime (Raven, 1967) (Fig. 19). Table I gives a summary of these experiments. TABLE I SCA Time after centrifugation (hours) ~-

Number of eggs

0-1 1-3

25 18 16

3-4

IN CENTRiFUGED EGGS

SCA Lacking

Doubtful

21

3

1

6

6

1

1

6 14

Prcsent -.

32

CHRISTIAAN P. RAVEN

FIG. 19. Egg centrifuged at uncleaved stage; fixed 4 hours later at four-cell stage. Distinct SCA in both blastomeres. Staining: iron hematoxylin-eosin. x 460.

IV. The Cortical Field If one assumes that the processes of ooplasmic segregation giving rise to the subcortical cytoplasmic differentiations are controlled by directing influences originating in the cortex, it follows that the latter is the carrier of a system of morphogenetic factors exhibiting a certain spatial configuration. This system will be indicated by the term “cortical morphogenetic field.” The use of this term does not imply, to be sure, that only continuous variations of its properties along the surface are allowed; there are several arguments pointing to the existence of rather discontinuous, more-or-less stepwise variations in it (Raven, 1964a). W e have seen above (Section 11, B) that there are good reasons to assume that the cortical factors important for morphogenesis are primarily bound to the triple-layered plasma membrane visible in electron micrographs. In this connection, it is important to note that no local differences either in thickness or structure of this membrane can be observed; as far as can be made out, it has the same appearance throughout. This means that possible differences in its structure must lie below the resolution of the electron microscope. If the plasma mernbrane carries the cortical field, indeed, this must be the expression of local differences in molecular configuration and resulting physiological properties of the membrane. These variations may be continuous, according to a gradient, or discontinuous, in a mosaic fashion, or a combination of the two. In addition to variations in space along the surface, variations in time in the course of early development have to be taken into account (cf. Raven, 1967). Each point within the field is characterized at any moment by a certain constellation of properties

CORTEX AND SUBCORTEX OF

Lymnaea

EGG

33

and may influence the underlying cytoplasm in a variety of ways: either by differential permeability, by exerting local attractions or repulsions (e.g., of an electrostatic nature) upon certain cytoplasmic components, or by any other kind of “resonance relationship” between surface membrane and cytoplasm. It seems that present models of the cell membrane can fulfill the demands to be made on an organelle having a general coordinating and integrating function in the development of the egg (cf. Raven, 1961). If the cortical field is bound to the plasma membrane, indeed, this makes a direct study of its pattern hardly practicable at present. Indirectly, however, some conclusions may be drawn from the observations on the subcortical cytoplasmic differentiations. If their formation is attributable to directing factors emanating from the cortex, their localization in the uncleaved egg cell must represent a more-or-less faithful reflection of the prevailing pattern of the invisible molecular structure of the egg cortex. Starting from these considerations, we may conjecture that there are at least three areas with different properties in the morphogenetic cortical field of L. .rtagnalis: ( 1 ) a vegetative area, occupying about 20% of the egg surface, corresponding in its extension to the vegetative pole plasm in the recently laid egg whose formation it determines; ( 2 ) an animal region, giving rise sooner or later to the formation of the animal pole plasm by the accumulation of its particular “matrix” substance; and (3) an equatorial zone, exhibiting a dorsoventral and nearly bilaterally symmetric mosaic pattern of six patches differing in their properties from the intervening regions. Apparently, a cortical pattern more or less corresponding to this description must be present at the very beginning of embryonic development, i.e., at the moment of oviposition. This raises the question as to its origin. Apparently, it must have been established at some earlier period, most likely during the forination of the egg cell in the gonad. If the structure of the recently laid egg cell is to be compared with that of the oocyte in the gonad, a first question that must be answered is whether or not the animal-vegetative polarity of the former corresponds to the original aiiicobasal polarity of the growing oocyte. Although this generally seems to be the case in molluscs (Raven, 1966), it is difficult to prove this point with certainty in Lymzaea. Ovulation here is followed by pronounced ameboid activity while the eggs are in the genital tract of the parent. Only after the eggs are surrounded by egg capsule fluid in the pars contorta of the oviduct do they round off again. During the ameboid phase, in consequence of the strong distortions the egg undergoes, nuclear polarity is as a rule unrecognizable (Bretschneider and Raven, 1951). Ubbels (1968) recently observed, however, that ovulated oocytes occasionally still showed a zona radiata after staining with pyronin. This was lacking, however, in the region opposite the excentrically situ-

34

CHRISTIAAN P . RAVEN

ated nucleus. This observation supports the assumption that the vegetative pole of the egg arises at the side of the oocyte situated against the bas,d membrane, since a zona radiata never develops in this region. If we accept this, it is obvious to homologize that part of the egg surface beneath which the vegetative pole plasm accumulates with the region formerly applied to the wall of the acinus. The extension of the two regions is about the same, both comprising about 20y0 of the surface of the egg cell. Apparently, this part of the plasmalemma is endowed, by its close association with the coiliiective tissue wall and basal membrane of the acinus, with special properties differing from those of the remaining part of the surface.

A

FIG. 20.

B

Diagram of the arrangement of the inner follicle cells around oocyce of

L. .rtagnnli.r. (A) From inner pole of oocyte. ( B ) From the side. Crosshatchin:,.: area of contact with gonad wall. 1-6, Inner follic!e cells.

In a similar way, we may assume that the animal region of the cortical field, giving rise to the formation of the animal pole plasm, corresponds to the apical side of the oocyte, originally covered by the thin membranous upper parts of the follicle cells, which here form the characteristic imbricated membrane complexes but are soon lifted from the oocyte surface by the formation o f thc follicular cavity. Finally, the six “patches” of the equatorial zone of the cortical field in L. ,i/~i~y/~ul.is giving rise to the SCA of the oviposited egg can be related to tlic perikarya of the six inner follicle cells. The patterns of the follicle cells, 011 the one hand, and of the SCA, on the other, are strictly superimposablc (Raven, 1963, 1967) (Fig. 2 0 ) . This can most easily be explained by the assumption that the plasma membrane of the egg cell facing the perikarya of the follicle cells has acquired special properties by virtue of which it attracts or captures particular cytoplasmic constituents giving rise to the SCA. This explanation has received further support from the fact that it also applies

CORTEX AND SUBCORTEX OF

Lymnaea

EL

35

to the related species L. peregru (Ubbels et ul., 1969). As a matter of fact, in this species the number of inner follicle cells is somewhat less constant; it varies between six and eight, and occasionally is nine. The follicle cells are arranged according to a definite pattern, which is dorsoventral and either symmetric or asymmetric depending on the cell number. In the uncleaved eggs, either six or seven SCA were found. Their patterns conformed to those of six- and sevencelled follicles, respectively (Fig. 18). The fact that no egg cells with eight or nine SCA were found could be explained by the observation that higher follicle cell numbers probably occur only after the formation of the follicle cavity has begun. The pattern of SCA apparently reflects the arrangement of the follicle cells at the moment the follicular cavity begins to form. It is concluded that the cortical pattern in Lymizdea probably arises during oogenesis by interactions between the oocyte and the surrounding structures of the gonad. The structure of the egg follicle is, so to speak, “imprinted” upon the surface membrane of the oocyte. The hypothesis was put forward that this is one of the ways in which information (“blue-print information,” cf. Raven, 1958) is transmitted from the parent to the offspring (Raven, 1961, 1963, 1967). It has been mentioned (Section I) that the results of centrifugation and lithium experiments indicate that not only the directing factors of ooplasmic segregation, but also those controlling the direction of cleavage divisions, are bound to the cortex. The question thus arises whether the structure of the cortical morphogenetic field outlined above also could account for the localization and direction of cleavage spindles. It is evident that this need not be so. The structure of the cortical field, which is not accessible to direct study, has been deduced from our observations on ooplasmic segregation. Only those particulars of the field that influence ooplasniic segregation are brought to light in this way. Since, however, the typical course of development is dependent on the normal distribution of substances among the cleavage cells and, therefore, on an orderly conjunction of the two lxocesses of ooplasmic segregation and cell division, one may expect that the two groups of factors are more or less closely connected into a unified pattern. The positions and directions of cleavage spindles and cleavage planes are related to three perpendicular axes : animal-vegetative, dorsoventral, and sinistrod extra1. It is evident that the structure of the cortical morphogenetic field as described above exhibits a clear animal-vegetative polarity and dorsoventrality, both in L. stagnulis and L. pevegra (Raven, 1967; Ubbels et al., 1969) (cf. Figs. 1 3 and 18). The polarity is expressed in the opposition of the vegetative and animal regions, respectively, matching the pole plasms forming in these areas. It corresponds to the polarity of maturation and cleavage, and becomes the anterioposterior axis

36

CHKISTIAAN P. RAVEN

of the future embryo. The dorsoventrality is mainly expressed in the arrangement of the equatorial “patches” corresponding to the SCA, although a certain obliquity in the vegetal pole plasm of L. stugizulis points in the same direction. The dorsoventrality of the cortical pattern in L. .rtugnulis agrees with that of the future embryo (Raven, 1967). Moreover, the first cleavage spindle has a fixed position with respect to the cortical pattern, and this is intersected by the successive cleavage planes in a determinate way (Raven, 1967). It is evident, therefore, that the factors of ooplasmic segregation and cell division are bound together into a single pattern. However, there is one remarkable exception to this. It has long been known that the asymmetry of coiling of the shell in gastropods is correlated with the asymmetry of spiral cleavage, the cleavage of sinistral species of snails being the mirror image of that of dextral snails. The same regularity holds within a species (Ubbels, 1966). In L. peregru there are two races, one showing dextral coiling and the other sinistral coiling of the shell (Boycott and Diver, 1923). The cleavage pattern of eggs from which dextrals arise is the mirror image of that shown by sinistrals. According to Sturtevant’s (1923) hypothesis, the direction of coiling of the shell is determined by one pair of Mendelian factors, the allele for dextral coiling being dominant. The direction of coiling does not depend on the animal’s own genotype, but on the genotype of the female parent. Therefore, the asymmetry of the future embryo must be laid down in the egg during oogenesis. Since we have seen that the factors determining cleavage are apparently bound to the cortex, one might expect that the cortical patterns of eggs derived from genetically dextral and sinistral L. p e w g m would mirror each other. As the cortical patterns in their turn are related to the topography of the elements surrounding the growing oocyte, the follicle cell patterns of the two races might be expected to mirror each other too. It has been found, however, that the patterns of follicle cells in genetically dextral and sinistral L. peregru do not mirror each other, but are identical (Ubbels et ul., 1969). Apparently, therefore, the determination of the asymmetry of the future embryo does not take place by way of the follicle cell pattern but according to some other mechanism. For the moment, it is not clear how this works. One might think that the factors determining the direction of asyrnmetric cleavage are also built in somehow during oogenesis into the structure of the cortex, which then evidently contains two mutually independent factorial systems, one controlling ooplasmic segregation and the other one cleavage. Another, and perhaps more plausible, possibility might be that the cortical pattern is invariant in eggs of genotypical dextrals and sinistrals, that this pattern allows two alternative and mutually symmetric positions of the first cleavage spindle, and that some property of the egg cytoplasm, dependent on the maternal geno-

CORTEX AND SUBCORTEX O F

Lymnaea

EGG

37

type, determines which of the two positions the spindle will take. It is too early to decide between these possibilities.

V. Discussion The view that one or more factors essential for normal development are located in the superficial layer of the fertilized egg arose in the late 1930’s, mainly as a result of centrifugation experiments with eggs of various animals (annelids, molluscs, echinoderms, and amphibians) (Raven, 1938; Pease, 1939; Peltrera, 1940; Pasteels, 1940; Lehmann, 1941; Raven and Bretschneider, 1 9 4 2 ) . The observation that centrifuged eggs, notwithstanding a serious disturbance of the normal structure of their cytoplasm, in many cases developed normally or nearly so; that, moreover, processes of ooplasmic segregation taking place during normal development in centrifuged eggs occurred at the right moment and at the proper place in relation to the original axes of the egg indicated that the factors responsible for the correct orientation and localization of various processes during development reside in some part of the egg that resists the displacing action of centrifugation. Since Motomura (1935) had shown that the cortical cytoplasm of amphibians and echinoderms is not displaced by moderate centrifuging, it was obvious to conclude that il is the cortex that carries this system of developmental factors. The importance of these cortical factors was especially stressed by Dalcq and Pasteels (1937; cf. Dalcq, 1941) in their theory of the physiological basis of morphogenesis. According to this theory, both the topogenetic and the histogenetic potencies of the cells of the marginal zone of the Amphibia are determined by the interaction of a dorsoventral cortical field and an aninial-vegetative yolk gradient. The ensuing gastrulation, with its directed cell displacements and cell interactions, then ushers in the ordered course of further development. In the following years, interest in the cortical factors of embryonic development gradually subsided. Apart from some further investigations on amphibians by the Brussels embryologists, on the one hand, and the work of our own group, striving to elucidate the structure and the properties of the egg cortex and the mode of action, localization, and origin of the cortical factors in Lymnaea, on the other hand, hardly any contributions to the problem were made for nearly two decades. In recent years, however, interest in the egg cortex has been revived. First of all the work of Curtis (1960, 1962) may be mentioned in this connection. While evidence for the importance of cortical factors in development derived froin centrifugation experiments was merely circumstantial, Curtis succeeded for the first time in obtaining direct relevant evidence by grafting experiments, in which portions of cortex were transplanted in Xeizopus eggs. Grafting cor-

38

CMRISTIAAN P. RAVEN

tex from the grey crescent region of uncleaved fertilized eggs to the ventral side of another egg at the same stage resulted in the development of a whole secondary embryonic axis twinned with the normal one. Such grafts from eightcell embryos placed in uncleaved fertilized eggs also induced a secondary embryo. Grafts of grey crescent cortex placed in eight-cell embryos had no effect on morphogenesis. Excisions of cortex from the grey crescent of uncleaved eggs prevented morphogenesis, although cleavage and mitosis were unimpeded. Excisions from eight-cell stages were without effect on morphogenesis. Neither excisions nor grafts of subcortical cytoplasm from the grey crescent region of eight-cell embryos do affect development. Removal or grafting of large pieces of cortex from regions other than the grey crescent of uncleaved eggs has no effect on the determination of gastrulation, although more localized effects mdy occur. Grafts of cortex taken from outside the grey crescent region and placed in the crescent of uncleaved fertilized eggs may give rise to a splitting of the embryonic axis. Grafts of cortex from unfertilized eggs may block cleavage, even after it has started, in a fertilized one. It was concluded from these results that the cortex carries a great deal of the spatial information required for building an amphibian embryo, “mapping out,” as it were, each part of the embryo. The main features of the cortical field have been laid down at the eight-cell stage. The cortex in the uncleaved fertilized egg may be pictured as containing only a morphogenetic factor centered in one place, the grey crescent. At some time between the first and third cleavages, this acts as a center which initiates and activates the establishment of a cortical field (Curtis, 1962, 1963). Evidence has been growing that the cortex plays a significant role in the early development of insects. In many insects, cleavage nuclei have been shown to be totipotent, their developmental fates being determined only some time after their contact with the cortex. In determinate eggs, the cortex is assumed to be highly organized from an early stage. It is involved with the activation of the nuclei into their varying roles in differentiation (Counce, 1961; Krause and Sander, 1962). Additional information on the significance of the cortex for early development has been obtained in various molluscs by the application of a variety of methods. Jura (1967) inflicted local damage to the egg cortex of uncleaved eggs of Szlccineu by means of ultraviolet microbeam irradiation. With irradiation before extrusion of the first polar body, several abnormalities occurred. The animal pole plasm showed an abnormal distribution. Depolarization of the second maturation division occurred, leading to the formation of giant or abnormally situated second polar bodies. Development either stopped before gastrulation or produced malformed embryos. Irradiation after extrusion of the first polar

CORTEX AND SUBCORTEX OF

Lymizaetl

EGG

39

body had similar effects, although a certain proportion of treated eggs showed normal development. Verdonk (1968) studied the effect of removal of the polar lobe in centrifuged eggs of Dentdhmz. Centrifuged eggs behaved just as uncentrifuged eggs did in Wilson’s (1904) classic experiments; removing the polar lobe at first cleavage produces larvae without apical tuft and posttrochal region; removal of the lobe at second cleavage gives larvae with apical tuft present and a reduced posttrochal region. Apparently, the morphogenetic significance of the lobe does not depend on the nature of the cytoplasmic components it contains. This argues in favor of the view that the morphogenetic factors of the polar lobe are located in the cortex. Clement (1968) centrifuged Zlyanassa eggs in reverse until the yolk had accumulated in the original animal half; then the eggs were centrifuged into two fragments. The nucleated yolk-poor vegetative halves at further development formed polar lobes and cleaved unequally as normal eggs do. Many produced lobe-dependent structures, such as eye, shell, or foot; some developed into small well-proportioned veligers. The animal fragments underwent equal cleavage without polar lobes and developed to defective larvae without lobedependent structures. It is concluded that the morphogenetic factors of the polar lobe region are independent of the yolk, and not movable by a centrifugal force of 2000 x g. It again seems obvious to locate them in the cortex. Finally, it appears that the cortex plays an important part also in the development of cephalopods. Constriction and ultraviolet microbeam experiments indicate that there is a morphogenetic pattern located in the superficial layer, at least from first cleavage up to an early blastoderm stage. Excision or injury of the cortex leads to specific localized organ deficiencies. Regulation is possible only within an organ field, not between fields. Centrifugation experiments show that the pattern is located in the nonmovable part of the cortical layer. The pattern determines which organs are formed in a certain region after cellulation has taken place (Arnold, 1968). Apparently, these relationships show a great simiilarity to those in insects. These new results provide fresh evidence for the importance of cortical factors in development, at least in certain groups of animals. The question whether or not this is a general feature of animal development cannot yet be answered, although the diversity of the animals in which it has been found points to its generality. There are other questions that need further study. One of them concerns the structure and composition of the cortex in various groups, in connection with the question which of its components is the seat of the cortical morphogenetic factors. The structure of the superficial layers of the egg cell varies a great deal among different animals. Sometimes it is rather thick and massive, consist-

40

CHRISTIAAN P. RAVEN

ing of several layers, as in the Amphibia; in other cases, it appears to be composed of a plasmalemma alone, as in Lynzmea. As far as we know, the plasmalemma is the only component generally present. This makes it the most likely candidate as a carrier of the cortical morphogenetic field, but further study is necessary before this can be accepted. If this point has been elucidated, the question of the physical nature of the cortical pattern arises. It has been argued above (Section IV) that it is obvious to think in terms of local differences in molecular configuration of the cell membrane. Starting from the classic Harvey--Danielli model (Harvey and Danielli, 1938) of such a membrane, several possible variations can be imagined. The detailed molecular architecture of the membrane, however, is still rather unknown (Maddy, 1966), and it seems a far cry to the establishment of local variations in the structure of a single cell membrane, relating them to localized differences in its physiological properties and morphogenetic activities. On the other hand, it might be useful to be aware of this possibility in the design of further itivestigations of the cell membrane. A further question concerns the mode of action of the cortical field. As mentioned above, a direct action of the cortex on the nuclei, assigning them their roles in cell differentiation, is indicated in insects. One is tempted here to think in terms of a derepression of genes, but convincing evidence for such a relationship is still lacking. O n the other hand, in groups such as annelids and molluscs the primary action of the cortex seems to be on the cytoplasm, bringing about ooplasmic segregation during early stages of development. This, together with the more-or-less cortex-dependent course of the cleavage divisions, gives rise to regional differences in cytoplasmic composition of the cells (chemodifferentiation), which in its turn is thought to be the prerequisite for differential gene derepression at later stages. The physical nature of the forces playing a part in ooplasmic segregation is still unknown. W e do not know what causes certain components of the cytoplasm to accumulate beneath some part of the cortex, or to move in a certain direction and become shunted into special cells of the embryo. One might think of electrostatic forces of attraction and repulsion, but apart from some uncertain indications there is no proof of it. Further questions concern the origin and evolution of the cortical morphogenetic field. There are clear indications that the cortex is not merely a mosaic of areas of different molecular structure, given once and for all, but that the cortical pattern shows alterations of a dynamic nature during early development (Raven, 1967; cf. also Curtis, 1962). As for its origin, three possibilities have been considered. The first is that the spatially structured system of the cortical field is synthesized under gene control during egg formation. The greatest difficulty of such a view seems to me that it can hardly be imagined how gene

CORTEX AND SUBCORTEX OF

Lymizaeu

EGG

41

products can be dispatched from the nucleus (or from some cytoplasmic site of synthesis) to discrete points of the egg surface unless the latter have an “address,” so to speak, which means that the ordered spatial multiplicity of the cortex that is to be explained must be somehow preexistent. A second possibility to consider is that the cortical pattern is self-replicating. Curtis (1963) has defended this possibility with respect to the Amphibia, on the argumentation that the grey crescent region ultimately gives rise to the germ cells, and that the cell surface is parceled out among the cells during division. In a later paper, Curtis (1965) has given experimental evidence for such a cortical inheritance. It must be said that, in my opinion, the view that all future germ cells acquire a part of the original cortex of the parent egg cell is hard to defend, even in the Amphibia, and can hardly be generalized to other groups. The possibility that developmental information in the egg cortex is bound to certain carriers of highly specific molecular configuration, just as genotypical information in the chromosomes is, and that these specific building blocks are transferred from the parent to the offspring, so that a direct genetic continuity exists between these information carriers in the cortex of an egg cell and in the egg cells of the next generation, cannot be excluded. One must of course assume in that case that these carriers remain in existence during the life of the animal that develops from the egg cell, have the ability to reproduce, e.g., by division or replication, and are built in anew in the egg cells formed by the mature animal. For the moment it seems to me that the results of Curtis’ experiments allow other explanations as well, and give no indubitable proof of the existence of cortical inheritance by a transfer of specific information carriers. For several years (cf. Raven, 1958), I have advocated a third possibility, viz. that the cortical pattern is imprinted on the egg from the outside during oogenesis, and have adduced evidence that such a mechanism plays a part both in L. stugndlis (Raven, 1963, 1967) and in L. peregrd (Ubbels et al., 1969). According to this view, the cortical field somehow is a copy of the structure of the egg follicle, and this copying process represents the manner in which the cortical information is encoded in the egg cell. Evidently, it is too early to decide whether these relationships hold only within a restricted group of animals or represent a more generally valid mechanism of transfer of information from parent to offspring. Only future research into this question can provide us with an answer. A last question concerns the importance of the cortical information for embryonic development. I have argued in a previous paper (Raven, 1755) that the importance of the information carried by the cortical field (dso called “blue-print information”) lies especially in the circumstance that, in contrast to the “executive information” in nucleus and cytoplasm, it has a direct bearing 011 the development of spatial multipldty. The two-dimensional pattern of the

42

CHRISTIAAN P. RAVEN

cortical field provides the egg with a system of coordinates to which all developmental processes are related. It is hard to visualize how ordered spatial multiplicity could come into being without such a preexisting frame of reference. One can calculate that the plasma membrane of the egg, considered as a device for the storage of information, theoretically might carry an immense amount of selective information. For an egg such as that of Lymnaea, the informational capability of the cortex surpasses that of the genome probably by a factor of about 103 (Raven, 1961). This does not mean, of course, that this capacity is fully utilized by the organism. On the contrary, it s e e m probable that the actual information content of the cortex generally is rather low. The eggs of different animals may show great differences in this respect, according as the spatial pattern of the future embryo is “mapped out” with greater or lesser precision in the cortex. It is conceivable that the former distinction between “mosaic eggs” and “regulation eggs” had one of its roots in such differences. However this may be, one must reckon with the possibility that the information content of the egg cortex may differ a great deal among various animals. Information theory provides us with a theoretical possibility for making meaningful estimates and comparisons of the information content of various structures, but a great deal of the factual knowledge necessary for such a program is still lacking. Altogether, it appears that a concerted attack on the problems of the egg cortex in coming years might greatly improve our understanding of animal development.

REFERENCES Arnold, J. M. (1968). Develop. Biol. 18, 180. Blaauw-Jansen, G. (1950). KoninRl. Ned. Akad. Wetenschap., Proc. 53, 910. Bluemink, J. G. (1967). Ph.D. Thesis, Univ. of Utrecht. Boycott, A. E., and Diver, C. (1923). Proc. Roy. Soc. ( L o n d o n ) B219, 51. Bretschneider, L. H. (1948). Koninkl. Ned. Akad. Vetenschap., Proc. 51, 358. Bretschneider, L. H., and Raven, C. P. (1951). Arch. Need. Zool. 10, 1. Bungenberg d e Jong, H. G. (1949). In “Colloid Science” (H. R. Kruyt, ed.), Vol. 2, p. 259. Elsevier, New York. Clement, A. C . (1968). Develop. Biol. 17, 165. Counce, S. J. (1961). A n n , Reu. Entomol. 6, 295. Curtis, A. S. G. (1960). J . Embryol. E x i d . Mor/jl,hol. 8, 163. Curtis, A. S. G. (1962). J. Embryol. Exptl. MoriJhol. 10, 410. Curtis, A. S. G. (1963). Endeavozlr 22, 134. Curtis, A. S. G. (1965). Arch. Biol. (Liege) 76, 523. Dalcq, A. (1941). “L‘oeuf et son dynamisrne organisateur.” Albiti Michel, Paris. Dalcq, A,, and Pasteels, J. (1937). Arch. Biol. (Liege) 48, 669. de Groot, A. P. (1948). Koninkl. Ned. Ahad. Wetenschap., Proc. 51, 588. Demel, R. A., van Deenen, L. L. M., and Kinsky, S. C. (1965). J . B i d . Chem. 240, 2749.

~ l eVries, G. A. (19,$7). Korziitbl. Ned. L4kaJ. IVcterticha/~.. Pvoc. 50, 1 3 3 5 . d~ Vries. L. G. ( 1 9 5 ; ) . Korikkl. Ncd. Akad. l V e t c ~ ~ i c h ~ Pruc. i / ~ . , C56, 5x4. Elbers, P. I;. (1959). P1i.D. Thesis, Uni\. of Utrecht. Elbers, P. F. (1966). Bjochiiu. Biophyr. Acta 112, 31s. Elbers, P. F., and Blueinink, J. G. (1960). Lxxptl. Cell Re.r. 21, 619.

Geilenkirchen, W. L. M. (1961). Ph.D. Thesis, Univ. of Utrrcht. Geilenkirchen, W. L. hf. ( 1 9 6 4 ) . Exptl. Cell Res. 34, 463. Grasveld, M. S. ( 1 9 4 9 ) . Konjizkl. Ned. Akad. Il~etenrchup.,I’roc. 52, 2 S 4 . Harvey, E. N., and Danielli, J. 1;. (1938). Biol. Rev. Cambiidge Phil. Soc. 13, 119. Hudig, 0. ( J9,[6). Konrnkl. Ned. Aknd. Wetentrhap., Pi.or. 49, 554. Jura, C. (1967). Acta Biol. Cyacov., Ser. Zool. 10, 89. Krause, G., and Sander, K. (1962). Advan. Moiphogenesir 2, 259. Lehmann, F. E. (1941). Natul.udrsenschaften 29, 101. Maddy, A. H. (1966). Intevn. Rev. Cytol. 20, 1. hfinganti, A. (1950). Riv. Biol. (Peiugiu) 42, 295. Motomura, I. (1935). Sci. Rept. l’ohoku I m p . Univ., ljouith Ser. 10, 211. Paris, A. 5. ( 1 9 5 3 ) . Konirzkl. Ned. ./lkuil. lVeten.ichap., I’roc. C56, 406. Pasteels, J. ( 1 9 4 0 ) . Aich. Biol. (Liege) 51, 3 3 5 . Pease, D. C. (1939). J. ExXptl. Zool. 80, 225. Peltrera, A. ( 1 9 4 0 ) . Pubbl. Staz. Zool. Napoli 18, 20. Raven, C. P. (1938). Acta N e e d . Morphol. 1, 337. Raven, C . P. ( 1 9 4 2 ) . KoniiikI. Ned. Akad. 1Yeienscbap., Proc. 45, 856. Raven, C. P. (1945). Arch. h’eevl. 2001.7, 91. Raven, C. P. (1946;t). Atch. Neevl. Zool. 7, 3 5 3 . Raven, C. P. ( 1 9 4 6 b ) . Aich. N e e d . Zool. 7, 496. Raven, C. P. ( 1 9 4 8 ) . Koitinkl. N e d . Akad. IV’eterzrchap., Proc. 51, 1077. Raven, C. P. ( 1 9 4 9 ) . Arch. N e e d . Zool. 8, 323. R a e n , C. P. ( 1957). Kuniizhl. Neil. Ahad. IVeterz.rchup., Iferrlag Geuwne Vergadel-. Aldel. Nut. 66, 7 6 . Raven, C. P. (195s). Ajch. Nee1.l. ZOO/.13, Suppl. 1, ISS. Ra\en, C. P. ( 1961 ) . “Oogenesis: ‘lhe Storage of Developmental Information.” Macniillan (Perpimoil), New Y o r k . Raven, C. P. ( I 963). Dewlop. Bzol. 7, 130. R;iven, C. P. ( 1 9 6 4 a ) . ildtuzn. Morl,hogerre.ris 3, 1. R;iven, C. P. (1964b). J. Ettjbvyol. Exptl. Morphol. 12, 805. Raven, C. P. ( 1966). “Morphngenesis. The Analysis of Rfolluscan Dcvrlopmcnt,” 2 n d ed. hlacniillan (Perganioii), New York. R;n en, C. P. (1 967). Det’elop. B b l . 16, 407. Kaven, C. P., and Beenakkers, A. &I. ’r. (1955). J . Embryo/. Exi)tl. Morphol. 3, 2x6. Raven, <;. P., and Bretschneider, L. H. ( 1 9 4 2 ) . Arch. N e e d . Zo o / . 6, 2 5 5 . Raven, C. P., and Brunnrkreeft, F. (1951). Kottivrkl. Ned. Akad. IY/eten.rchrip., l’tor. c54, 440. Raven, C. P., a n d Dudok de Wit, S. (1949). Koninkl. Ned. Akad. TVetertcchap, I’i.uc. 52, 28. Raven, C . P., and Kloinp, H. (1946). Koniuii. Ned. Akarl. Wetenichap., PTOC.49, 101. Raven, C. P., and Mighorst, J. C. A. (1946). Koninkl. N e d . Akad. Wetemchap., P w c . 49, 1003. Raven, C . P., and Rohorgb, J. R. (1949). Ko?zinkl. Ned. Akad. Welenrchap., Proc. 52, 614, 773.

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Raven, C. P., and van der Wal, U. P. (1964). J . Embryol. Exptl. Morphol. 12, 123. Raven, C. P., and van Erkel, G. A. (1955). Exptl. Cell Rer. Su[ipl. 3, 294. Raven, C. P., and van Zeist, \xi. (1950). Koninkl. Ned. Ahad. lYeten.rchap., Proc. 53, 601. Raven, C. P., Bezem, J. J., and kings, J. (1952a). Koninkl. Ned. Akad. Weten(rhap., Proc. c55, 248. Raven, C. P., Bezem, J. J., and van Loo, R. P. (195213). Koninkl. N e d . AKad. Weten.rchap., Proc. C55, 7. Raven, C. P., Bezem, J. J., and Geelen, J. F. M. (1953). Kuniiibl. Ned. Ahad. Wetert.rchap., Proc. C56, 409. Raven, C. P. (in collaboration with Drinkwaard, A. C., Haeck, J., Verdonk, N. H., and Verhoeven, L. A , ) (1956). Pubbl. S t a . Zool. Napoh 28, 136. Recourt, A. (1961). Ph.D. Thesis, Univ. of Utrecht. Stalfoort, T. G. J. (1952). Koninkl. Ned. Akad. V e t e m c h a p . , Proc. C55, 184. Sturtevant, A. H. (1923). Scieiice 58, 269. Ubbels, G. A. (1966). Arch. N e e d . 2001.16, 544. Ubbels, G. A. (1968). Ph.D. Thesis, Univ. of Utrecht. IJbbels, G. A,, Bezem, J. J., and Raven, C. P. (1969). J . Embryol. C x p f l . Morphol. 21. 445. van den Broek, E., and Raven, C. P. (1951). Koninhl. Ned. Akad. Ivetensrhap., P w c . c54, 226. Verdonk, N. H. (1965). Ph.D. Thesis, Univ. of Utrecht. Verdonk, N. H. (1968). J . Embyyol. Exptl. Morphol. 19, 33. Wierzejski, A. (1905). Z. Wiss. Zool. 83, 502. Wilson, E. B. (1904). J . Exptl. 2001.1, 1.

he Environment and Function of Invertebrate Nerve Cells J. E. TREHERNE AND R. B. MORBTON A.R.C. Unit of Invertebrate Chemistry and Physiology and Department of Zoology, University of Cambridge, Cambridge, England I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Structural Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Extraneural Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Nerve Sheath . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Glial Cells and Their Relation to Neuronal Elements D. The Extracellular System . . . . . . . . . . . . . . . . . . . . . . . . 111. Distribution and Exchanges of Inorganic Ions and Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Ionic Requirements for Electrical Activity . . . . . . . . . . V. General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45 46 46 41 53 59 64 70

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I. Introduction ie intensive research of recent decades has contributed to the elucidation veral aspects of the mechanism of excitation in nerve cells. Most of this has been carried out using severely isolated preparations in which it is -ally assumed that the ionic concentrations of the fluid bathing the neuronal ces are similar to those in the bathing media. These investigations have, bver, contributed relatively little to the understanding of the nature of the ma1 environment in intact nervous tissues. In particular, we are still igit of many of the effects of the chemical composition of the extraneuronal on conduction processes in intact central nervous systems. This paucity of dedge can be largely attributed to the experimental difficulties involved orking with complex central nervous tissues, especially those of vertebrate als. For this reason, the relatively simple central nervous systems of some tebrate animals seem to be particularly suited for the study of neuronal -ion in intact preparations. In this review, therefore, an account will be 1 of the current state of our knowledge concerning the nature of the extrama1 environment in invertebrate nervous systems, especially in relation to onic basis of excitation of neurons. This account will be largely confined ie relevant processes taking place in nervous tissues of various annelid, uscan, and arthropod species, which have formed the principal objects of T in recent years. These processes will be interpreted in relation to the tecture of the central nervous tissues, and for this reason an account will iven of some relevant features of the ultrastructure of invertebrate nervous 5.

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11. Structural Considerations Description of the structural features of invertebrate nervous tissues will be largely confined to those aspects that are relevant to the functional interpretations contained in succeeding sections of this review. Accordingly, emphasis will be given to descriptions of the nervous systems of those animals that have been the subject of experimental investigation. A. EXTRANEURAL STRUCTURES In many invertebrate species, the external surfaces of the nervous system are bathed directly by the general body fluids. There are, however, examples in each of the major metazoan invertebrate groups of species in which substantial portions of the nervous system are overlaid by various types of tissue layers. In some species, such as the gnathobdellid leeches, a superficial endothelium may be closely applied to the outer surfaces of the ganglia and connectives (Coggeshall and Fawcett, 1964), while in others, such as lamellibranch molluscs (Gupta et al., 1969) and some insect species (Maddrell and Treherne, 1966), the overlying tissues can interpose an additional fluid compartment at the periphery of the nervous system. The endothelium that encloses the central nervous system of the leech Hirudo medicinalis consists of a continuous layer of squamous cells which represents the lining of the longitudinal ventral sinus (Coggeshall and Fawcett, 1964). These endothelial cells exhibit a number of cytological features, including the presence of apparent pinocytotic vesicles, which suggest the possibility that the endothelium might play an active role in the exchanges of ions and molecules between the blood and the underlying nervous tissues. It is, therefore, coilceivable that such endothelial secretory activity could modify the concentrations of inorganic ions in the extracellular fluid of the nervous tissues and so affect the excitability of the neurons. The cerebrovisceral connectives of the lamellibranch mollusc Anodontd cygnea are situated in a blood space flanked by the epithelial walls of the proximal limb of the kidney (Gupta et al., 1969). This region of the kidney has been shown to be involved in the reabsorption of inorganic ions discharged into the fluid space surrounding the connectives (Picken, 1937; Potts, 1967). The ultrastructure of the epithelial cells also indicates a high degree of specialization for ion transport. Under these circumstances, it is not unreasonable to suppose that this secretory epithelium might be involved in regulating the ionic concentration in the fluid bathing the outer surfaces of the central nervous system. A regulated local ionic environment at the surface of the central nervous system might in turn affect the concentrations of inorganic ions in the extracellular fluid bathing the neurons in the underlying nervous tissues. It is possible to

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correlate such a physiological mechanism with the extremely dilute nature of the blood of freshwater lamellibranchs (Potts, 1954). In some insect species, the surface of the abdominal nerve cord is covered with patches of fat-body cells. In the cockroach Periplaneta americana, for example, these fat-body cells are confined to small areas on the surface of the ganglia and are absent from the connectives. In view of the very close relation of the fat-body cells to the relatively permeable neural lamella, it has been suggested (Smith and Treherne, 1963) that the metabolic processes of the fatbody cells might be linked with those of the perineurium which is thought to be involved in the mobilization of metabolic reserves by the central nervous system (Wigglesworth, 1960). In other insect species, on the other hand, the fat-body cell may be modified to form a continuous sheath which completely encloses both the ganglia and connectives of the ventral nerve cord. This state of affairs is encountered in the stick insect Caramius morosus (Maddrell and Treherne, 1966). Unlike the situation in the cockroach, the fat-body cells in the latter species are not closely applied to the surface of the central nervous system, so that an additional fluid compartment is formed between the blood and central nervous tissues. It appears that there is a potential gradient, of 1520 mV, across the neural fat-body sheath, the inner surface being positive with respect to the exterior (Treherne and Maddrell, 1967a). It was suggested that this positive potential may result from a chloride diffusion potential across the neural fat-body sheath. The blood of C. mo~o.rushas an extremely specialized ionic composition, with a relatively low sodium level, so that it could be postulated that the neural fat-body sheath might be involved in regulating the sodium concentration in the fluid bathing the exterior of the nervous system.

B. THENERVE SHEATH

A common feature of the nervous systems of higher invertebrates is the presence of a superficial envelope or sheath of connective tissue, usually referred to as the neural lamella or capsule. In structure and composition, this layer resembles the basement membrane associated with some non-neural tissues. For present purposes, however, the term basement membrane will be used only for the condition in which there is a relatively thin amorphous layer overlying cell membranes. The superficial noncellular layer of the nerve sheath is, in some groups, associated with a specialized underlying cellular layer. The terminology used to describe the latter layer is confused, the most commonly used terms being “perilemma” or “perineurium.” In most invertebrates, the neural lamella appears to consist of two elements: an amorphous or finely fibrillar one together with a component consisting of coarser striated fibers (cf. Smith and Treherne, 1963; Bullock, 1965; Ashhurst, 1968). The available information seems to indicate that the first element is

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J. E. TREHERNE AND R. 8. MORETON

probably a mucopolysaccharide, the latter being collagenous in nature. In insects, which have been the invertebrates most intensively studied, the data obtained using electron microscope, chemical, histochemical, X-ray, and birefringence techniques clearly indicate that the neural lamella is composed of collagen fibrils embedded in a matrix of neutral mucopolysaccharide (see Ashhurst, 1968, for references). In pulmonate molluscs, such as Lymnaea (Boer et al., 1968) and H e l i x (Newman et ul., 1968), the capsule consists of two layers, the outer thick and loosely constructed, the inner more compact, with partly oriented collagen fibers. Both layers contain fibroblasts; those in the inner layer are highly vesicular, have a few large mitochondria, and show distinct “pores” in the cell membrane facing inward. The capsule surrounding peripheral nerves of H e l i x also contains some smooth muscle cells (Schlote, 1957). In the opisthobranch Aplysia californica (Coggeshall, 1967) , the capsule contains muscle fibers and small blood vessels. The latter often end blindly or empty into large, thin-walled lacunae within the capsule, and India ink injected into the vascular system was found to penetrate into the capsular ground substance, showing that the blood vessels are permeable to rather large particles. The material of the capsule is thus readily accessible from the hemocoel, although the same is not true of the “subcapsular sinus” of Rosenbluth (1963) (see Section 11, C), since the ink was not observed to penetrate beyond the base of the capsule. There is also a system of small capsular nerves, consisting of axons 0.1-2.5 p in diameter having associated satellite cells. Some of these axons presumably innervate the muscle fibers but others, which are highly granular, end in direct contact with the capsular ground substance and are presumed to be neurosecretory in function. The physical and chemical features of the neural lamella that are relevant in the present context can be summarized as follows. First, the appreciable collagen content of the neural lamella suggests the possibility that this peripheral layer could function as a cation reservoir. This supposition is based on the observation that collagen contains 77.2 free anion equivalents per 105 gin protein (Tristram, 1953). Such an effect might be expected to be maximal in the nervous system of a lepidopteran such Galleria mellonella, in which the neural lamella and the associated dorsal connective tissue mass exceeds the volume of the underlying nervous tissues (Ashhurst and Richards, 1964). Second, the neural lamella might reasonably be expected to impose some degree of restriction on the free diffusion of ions and molecules between the blood and the underlying tissues and extracellular fluid. Finally, the arrangement of the collagen fibrils might equip the neural lamella to resist the positive hydrostatic pressure resulting from any excess in osmotic concentration of the

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extracellular fluid arising from the establishment of a Donnan equilibrium with the blood (Smith and Treherne, 1963). The peripheral layer of cells underlying the neural lamella is of obvious potential importance in the regulation of the ionic environment of invertebrate neurons. Most invertebrate ganglia and connectives are avascular organs, so all necessary exchanges of ions and molecules must take place through the cells underlying the neural lamella. The only exceptions to this generalization seem to be the exchanges occurring through the well-developed vascular system described in the crustacean central nervous system (Sandeman, 1967) and the respiratory exchanges mediated by the tracheal system that penetrates the nervous tissues of insect species. The potential role of the peripheral cellular layer in regulating the extracellular concentrations of inorganic ions in the underlying nervous tissues is perhaps most obvious in the case of those invertebrates that possess blood of specialized ionic composition, such as that of some freshwater molluscs (Potts and Parry, 1964) or phytophagous insects (Wyatt, 1961; Shaw and Stobbart, 1963), in which the level of sodium ions may be exceedingly low and the concentrations of potassium or divalent cations correspondingly high. There is considerable variation in the degree of specialiKation 01 the peripheral cells situated beneath the neural lamella in the various invertebrate groups. In some arthropod species, such as those of insects (cf. Smith and Treherne, 1963), the perineurium consists of a continuous cellular layer covering the central nervous system and situated externally to the glial cells associated with the cell bodies of the neurons. The cells of the perineurium in insect species are characterized by the presence of numerous mitochondria, large quantities of glycogen, and lipid droplets (Wigglesworth, 1960; Ashhurst, 1961). In two insect species, P . arnericana and C. mowsus, the perineurium possesses an extensive system of tortuous channels between the lateral cell walls (Maddrell and Treherne, 1967). These channels are open at the outer margins adjacent to the fibrous connective tissue sheath but appear to be closed at the inner margin by regions of septate desmosomes and/or “tight” junctions. There is an increased surface area at the inner margin of the perineurial cells produced by the presence of long inwardly directed flanges (Fig. 1). The organization of the perineurium in these insect species is, in fact, strikingly similar to that observed in a variety of fluid-secreting epithelia. In the smaller peripheral nerves, there is no distinction between glial cells and perineurium (Edwards et dl., 1958; Wigglesworth, 1959a). In the larger peripheral nerves of Rhodnius, the superficial cellular layer is rich in mitochondria but still contributes to the interaxonal sheaths. In the latter species, there is a progressive development of the perineurium as the nerves enlarge and join the ganglia (Wiggles-

FIG. 1. ( A ) Electron micrograph showing a transverse section of the perineuriurn of an interganglionic connective of the stick insect C. ~ Z O I O S U S showing the tortuous path of the lateral walls of the perineurial cells. The arrow (at upper right) indicates the opening of the intercellular cleft (ic) beneath the fibrous sheath. The intercellular cleft can be followed to the channel separating the perineurium from the underlying glial 01

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worth, 1959b). In another arthropod, the arachnid Xiphosara polyphemus, the cellular layer equivalent to the insect perineurim is almost absent and only occurs around ganglia and as occasional patches along the ventral nerve cord (Dumont et al., 1965). The situation in a molluscan ganglion, such as that of A. californica, appears to be essentially similar to that in the peripheral nerves of arthropods in that the peripheral cells enwrap small neuronal processes and come into direct contact with nerve cell bodies (Rosenbluth, 1963). In a terrestrial mollusc such as Helix,however (Newman et al., l96S), fibroblasts in the inner capsule appear to be specialized for active transport, although there is no direct physiological evidence of this; the situation in peripheral nerves is complicated by the existence of extensive neurosecretory areas (e.g., see Simpson et ul., 1966). In lamellibranch nervous systems, there is no cellular layer comparable to the perineuriuin of arthropods (Fahrmann, 1961; Nakajima, 1961; Gupta et al., 1969). In A. cygnea, for example, the inner surface of the neural lamella is bounded by thin glial processes which do not reveal any junctional complexes between adjacent membranes (Gupta et al., 1969). Thus, open channels appear to exist between glial processes so as to give direct access to the outer region of the neuropile. In the annelid H . medicinalis, there is also no periph-

Schwann cell (sc) (arrow at lower left). The lateral walls of the perineurium are held together near the bases of the cells by septate desmosomes (sd) and by a tight junction (tj). Note the presence of microtubules (mt) in the type-I1 cells (11) and the darkly staining granules ( g ) together with the cluster of possible Golgi vesicles (ves) in the type-I cell (I). The extracellular spaces beneath the perineurium contain an electron-dense material (asterisks). Also visible are axons (ax), glial or Schwann cells (sc), and the fibrous nerve sheath (ns) . x 34,000. (B) Micrograph at higher magnification showing the lateral cell walls of the perineurium held together at their bases by septate desmosomes and a tight junction. x 81,000 (from Maddrell and Treherne, 1967).

52 J. E. TREHERNE AND R. B . MORETON

a

c

0

0

24

I

a

d 4 0

FUNCTION OF INVERTEBRATE NERVE CELLS

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era1 cellular layer equivalent to the arthropod perineurium surrounding the central nervous system (Gray and Guillery, 1963; Coggeshall and Fawcett, 1964). Thus, the inner surface of the outer capsule makes direct contact with areas of the outer margin of the giant packet glial cells, which encapsulate the ganglion cells and associated axon processes, the remaining areas of the inner surface of the capsule being lined by thin glial cells (Fig. 2 ) .

C. GLIALCELLSAND THEIR RELATIONTO NEURONAL ELEMENTS

A degree of confusion exists in the terminology employed for the supporting cells in invertebrate nervous tissues. Since, with a few exceptions, there is nothing comparable to the highly developed Schwann cell sheath of vertebrates, the term glia will be employed to describe the non-neural cells in both peripheral and central nervous systems of the invertebrates considered in this review. There is considerable variation in the extent and location of the glial cell elements in the nervous systems of the representative invertebrates considered here. An extreme condition is that encountered in the central nervous system of some lamellibranch molluscs. In both Cr.istariu plicatu (Nakajirna, 1961) and A. cygnea (Gupta et a]., 1969), for example, electron microscope investigation has revealed a paucity of glial elements in the central nervous system. In these species, the glial elements are present as narrow processes which appear to be scattered at random among the axons. N o complete sheaths of glia were observed around the larger axons or around the fasicles of smaller axons which thus occur as naked structures within the connectives (Fig. 3 ) . The larger axons (2-4 p in diameter) do, however, show a more frequent association with glial elements (Treherne et al., 1969b). This association takes the form of a close apposition, to within 100-200 A, of a glial membrane with a portion of the axon surface. The remainder of the axon surface is surrounded by the closely applied membranes of the other axons which also delimit a 100to 200-A space around the axon surfaces (Fig. 3). In other invertebrates that have been investigated, the nervous eleinents are usually separated by more extensive volumes of the cytoplasm of glial cells and their membranes. Several attempts have been made to classify the types of relationships that exist between these supportive cells and the axons (cf. Bullock, 1965; Wiggelsworth, 1959b; Coggeshall and Fawcett, 1964; Hodgkin, 1964). For present purposes, these relationships can be conveniently summarized as follows: (1) those in which individual axons are enveloped by a single glial cell, ( 2 ) those in which a number of axons are surrounded by one glial cell, and (3) those in which individual or a number of axons are surrounded by several glial cells. An interesting example of the second type of relationship listed above is that encountered in the leech ( H . rnedicindis) . Electron micrographs indicate, in general, that gaps of only about 150 A separate the gial and neuronal niem-

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FUNCTION OF INVERTEBRATE NERVE CELLS

55

branes. The area of contact between the glial and neuronal membranes is increased at the surfaces of the neuronal cell bodies where there are numerous glial invaginations which deeply indent the surfaces of the cell bodies (Fig. 2). The surfaces of the larger axons are also deeply infolded so as to increase the area of contact with the processes of the satellite cells. The cytoplasm of the packet glial cells contains numerous mitochondria and an extensive endoplasmic reticulum, together with appreciable quantities of glycogen and scattered lipid deposits. Despite the demonstration of low-resistance connections between glial cells (Kuffler and Potter, 1964), no specialized contacts or tight junctions were observed between adjacent glial membranes in electron micrographs of the leech central nervous system (Coggeshall and Fawcett, 1964). The possibility exists, however, that such regions of specialized membrane contact might not have been recognized because of the extreme complexity of the interdigitating glial processes. The stellar nerves of various tropical squids (Dorytheutis, Sepiotezlthis, and Dosidicus) (Villegas and Villegas, 1960, 1968) show both the first and second types of relationship; larger axons (1.5 ~pupward) are enveloped in a single layer of glial cells, which may be from 0.2 to 6 p thick, and consists of one or several cells, according to the size of the fiber. Of the smaller fibers, several may be enveloped in the same glial cell, singly or in bundles. The glial cells are packed closely together and make direct contact with the axolemma and the capsular basement membrane; access to the axolenima is via tortuous, extremely narrow (70-A) mesaxon gaps, which are filled with what appears to be mucopolysaccharide material. Diffusion through these channels is possible, although restricted. There is some evidence for the presence of pinocytotic vesicles in the glial cells. The structure of peripheral nerve in Octopus (Dilly et ul., 1963) is essentially similar, with bundles of small axons embedded in channels in the cytoplasm of a chain of glial cells. The mesaxon gaps are shorter and less tortuous than those of the squid nerve. The central nervous tissues of cephalopods (Gray, 1969; Stephens and Young, FIG. 3 . Electron micrograph showing a portion of a transverse section of the cerebrovisceral connective of the lamellibranch A. rypzea. The field includes part of one of the sparsely distributed glial cells (gc) which produce finer glial processes that penetrate among the axons. Most of the axons OCCUI as naked structures and no complete sheaths of glia are seen. This field illustrates the frequent association that occurs between the larger axons and the glial elements There are extensive extracellular spaces (asterisks), the larger ones being generally confined to the vicinity of glial cell bodies. The surfaces of Sotile of the axons may be exposed to the fluid in the extracellular spaces (arrow). The iiiicrofiraph also illustrates the wide range of axon sizes encountered in this preparation. The individual axons are generally separated from either the proximate axons or glial processes by narrow spaces usually about 150 A in width. x 18,000 (from Gupta, Mellon, md Treherne, unpublished).

56

J. E. TREHERNE AND R. B. MORETON

1969) show varying degrees of glial-neuron association, although in general glial cells are rather less intimately associated with the neurons than is the case in peripheral nerve. Unfortunately, there is too little experimental evidence regarding the nature of the extraneuronal environment in these tissues to warrant any detailed consideration of their structure in this context. In contrast to the relatively simple situations discussed so far, glial-neuron relationships in gastropod molluscs are highly complex and fall almost entirely into the last of the three categories. The ganglia consist of neurons and glia, mixed and tightly packed together. In the marine opisthobranch A . culifornica (Rosenbluth, 1963), three regions of the ganglion can be distinguished; imrnediately beneath the capsule is a layer composed primarily of glial cells having a few neuronal processes, and a system of “subcapsular sinuses,” which contain a flocculent, electron-dense material. The sinuses are bounded by glia, neurons, and basement membrane and are restricted to certain regions, so that over large areas the glial cells make direct contact with the basement membrane. They are continuous with small intercellular clefts between glial processes, which also contain the flocculent material, especially in dilated regions where the intercellular spaces may be up to 1000 A wide. Below the peripheral layer, neurons and glia are tightly packed together, one neuron making contact with many glial cells; the surfaces of the neurons are highly invaginated and interdigitate with glial processes. Dilatations of glial-neuron intercellular spaces are not observed, the typical separation between cells being of the order of 100 A. In the central neuropile, axons and neuronal processes are again mixed together with a tight packing of glial processes, although the proportion of glia is smaller, so that direct contact between neurons is not uncommon. Many glial cells, particularly those in the peripheral layer and the glial processes interdigitating with neurons, are densely packed with glycogen granules. Others in the peripheral layer are glycogen-free and have a highly convoluted profile. There is little evidence of pinocytotic vesicles. The structure of peripheral nerves is essentially similar to that in the ganglia (Batham, 1961; Coggeshall, 1967), except that the proportion of glial cells is smaller. As in the peripheral nerves of the squid, the degree of glial association varies with the size of the axons, small axons being collected together in bundles enveloped by a single stellate glial cell so that axons in the center of the bundle are completely surrounded by other axons. A feature not found in the ganglia, however, is the presence of specialized intercellular junctions between glial processes (Coggeshall, 1967) resembling the desmosomes seen in vertebrate tissues. Local widening of the intercellular cleft to about 450 A is associated with symmetrical regions of electron-dense cytoplasm on either side, and a 3oo-A-thick disc of dense material in the cleft. It seeins likely that these specialized junctions are concerned with mechanical attachment between cells; they are evidently different from the tight junctions seen between glial cells in arthropods (see Section 111).

F UNCT ION OF INVERTEBRATE NERVE CELLS

57

In terrestrial pulmonates, such as Helix apersu (Fig. 4 A ) , the situation in the ganglia is very similar. Invagination of the neuronal surfaces tends to form fingerlike processes running parallel to the main part of the surface, so that the space between neurons is filled with a multiple layer of alternating glial and neuronal processes. Small dilatations of the intercellular clefts occur (Fig. 4B) ; there is no evidence to show whether they occur specifically between neurons and glia or whether they form effective channels to the capsule. There is some evidence of a system of enlarged extracellular spaces immediately under the basement membrane, although there does not appear to be a system of sinuses as organized as that found in Aplysiu. Peripheral nerves of Helix, however, show a much lesser degree of glial investiture (Schlote, 1957; Hanneforth, 1965) more closely resembling that found in Anodonta. Other molluscs for which structural data are available include the freshwater pulmonate Lynznaea stugnulis (Boer et al., 196S), some species of nudibranchs (Schmekel and Wechsler, 1968), and the marine prosobranch Culliostoma (Simpson et al., 1966). All of these show neurons surrounded by multiple layers of glial cells and processes. A variety of relationships between glial cells and neurons is encountered in arthropods. These relationships are well exemplified in the central nervous systems of various insects, which have been the most intensively studied among arthropod species. In the cockroach P. amevicuna, for example, the subperineurial glial system of a ganglion can be approximately divided into two regions: a peripheral layer of cells, which invests the neuron cell bodies, and an underlying layer, which sends a complex system of processes into the neuropile (Wigglesworth, 1960). These two layers are separated by large extracellular spaces, the so-called “glial lacunar system.” The types of relationships that exist between the supporting cells and the axons in the neuropile are illustrated in Fig. 5. The larger axonh are frequently invested with a concentric glial cell sheath. The glial cell membrane is separated from the plasma membrane of the axon by a gap of between 100 and 150 A which is confluent with the spirally oriented mesaxon invagination. An unusual feature of these structures is the presence of appreciable dilatations in the otherwise narrow (i.e., 100-1 50 A ) extracellular channel defined by the axon folds. The smaller axons may be associated with a separate mesaxon invagination or a group of them may be surrounded by a common cell process. In the central nervous system of the phytophagous insect C. moYOSZIS, in which there is a very extensive volume of glial tissue, even the smaller axons tend to be surrounded by separate glial invaginations (Fig. 6) (Treherne and Maddrell, 1967b). The situation in some other arthropods appears to be essentially similar to that outlined above for an insect species. In both a crustacean, Armadilliunz, and a myriapod, Lithobius, it is possible to recognize two rather ill-defined regions in the glial system corresponding to the peripheral layer and inner zone

58

J. E. TREHERNB AND R. B. MORETON

59

FUNCTION O F INVERTEBRATE NERVE CELLS

described in Periplanetd (Trujillo-Cen6z, 1962). In the relatively few species that have been investigated, the majority of the axons are associated with extensive glial processes. Details of glial-neuron relationships in nervous tissues of other crustacean species can be found in papers by Hamori and Horridge (1966), Malzone et d l . (1966), and Abbott (1969).

D. THEEXTRACELLULAR SYSTEM In the nervous systems of the invertebrates that have been examined using conventional electron microscope techniques, large areas appear to exist in which the bulk of the extracellular space consists of restricted extracellular channels only a few hundred angstroms in diameter. This situation is well exemplified in the central nervous system of the leech (Coggeshall and Fawcett, 1964). In the central nervous connectives of this annelid, planimetric measurements have shown that the extensive system of intercommunicating extracellular channels account for approximately 5 % of the volume of the tissue (Kuffler and Potter, 1964). The total volume of extracellular space measured in this preparation was found to be approximately 38%, most of which was accounted for by the extensive connective tissue capsule. This morphologically determined value is in excellent agreement with the figure of 40% (of the total tissue water) calculated from the exchanging fractions of sucrose and inulin in this preparation (Nicholls and Wolfe, 1967). The picture that has emerged from electron microscope studies of very restricted extracellular spaces in large areas of invertebrate nervous tissues is essentially similar to that obtained using conventional techniques with vertebrate central nervous tissues (cf. Horstmann and Meves, 1959). The nervous systems of some invertebrate species differ, however, from those of vertebrates in that, with the electron microscope, relatively large extracellular spaces are observed within the nervous tissues. Examples of such spaces can be cited in the central regions of the connectives of the lamellibranch Anodontu cygizen (Gupta et al., 1969), in the peripheral “subcapsular sinuses” of the opisthobranch Aplysia culifornicu (Rosenbluth, 1963), in the “extracellular cisterns” of the lobster optic lamina (Hamori and Horridge, 1966), and in the peripherally ________

FIG. 4 . ( A ) Electron micrograph from

~~

~

~

~

a section through the right parietal ganglion

of the snail H . aspersa showing part of the cytoplasm of three giant neurons (nc) and

a glial cell (gc), extensively invaginating one of the neurons. The neurons are separated by a complex network of interlocking glial processes (gp) which invaginate their surfaces (arrows). Also shown are the nucleus of the glial cell (gn) and part of the nucleus of one neuron (nn). Osmic fixation. x 4200. ( B ) Micrograph from a similar section, showing at higher magnification the region between two neurons (nc) separated by several layers of glial processes (gp). Particularly prominent are small dilatations of the intercellular clefts (ic) and the invagination of one of the neurons by a glial process (arrow). x 48,000 (Moreton, unpublished).

60

J. E. TREHERNE A N D R. B. MORETON

situated “glial lacunar system” of the cockroach (Wigglesworth, 1960; Smith and Treherne, 1963). The presence of such extensive extracellular spaces could contribute to the relatively large estimates obtained for the chemically deter-

FIG. 5. Diagrammatic representation of the relationship between axons and the ensheathing glial cells in the central nervous system of P . anzefirund. A larger axon (ax) is invested in a concentric glial or Schwann cell sheath. (sc). The mesaxon invagination (mx), which defines a channel 100-150 A in width, follows an approximately spiral course which contains frequent dilatations ( d ) . The dilatations and the larger extracellulai spaces (ECS) contain an electron-dense material (*), most probably acid mucopolysaccharide. The smaller axons may be associated with separate mesaxon invaginations, as between the long and short arrows at axp, or a group of axons may be surrounded by a common glial cell process as at ax3 (from Smith and Treherne, 1963).

FIG. 6. A portion of a transverse section of a connective from the ventral nerve cord of C. ~ O Y O S Z I J , showing axon profiles (ax) and glial cell layers (gc). The extracellular clefts (arrows) occasionally dilate to form larger extracellular spaces (ECS) which are filled with an electron-opaque deposit. x 29,000 (from Treherne and Maddrell, 196713).

62

J. E. TREHERNE AND R. B. MORETON

mined extracellular volume of some invertebrate nervous tissues. In addition, as we have already seen in the leech, the presence of an appreciable volume of connective tissue at the periphery of the nervous system would also contribute to the relatively large extracellular volume determined by chemical means. It seems likely that the value of 36.6% for the extracellular water in the ganglia of Helix pomatia (Sorokina, 1966) could also be accounted for by the volume of the peripheral connective tissue layers, for the central nervous tissues of gastropods do not appear to possess extensive systems of enlarged extracellular spaces (Fig. 4A and B) . Some degree of caution must be exercised in interpreting the validity of the extracellular volume determined by chemical means. It has been shown in the leech central nervous system, for example, that with sucrose, inulin, or dextran, a fraction corresponding to about 5-10% of the total tracer could not be removed by prolonged washing. This would correspond to a volume of about 3% of the total water in the preparation. Autoradiographic evidence indicated that this “bound” fraction was, in fact, located within the cells of the central nervous tissues (Nicholls and Wolfe, 1967). In the central nervous system of A. cygnea, however, the sucrose space did not show a significant increase when the soaking period was increased by a factor of 2, which was interpreted as an indication that intracellular uptake was not a significant factor in this case (Mellon and Treherne, 1969). There is also a reasonable measure of agreement between the measured inulin space and the rapidly exchanging fraction of tritiated water in the cockroach nerve cord (Treherne, 1962a). In some crustacean (Mauchline, 1958; Sandeman, 1967; Malzone et al., 1966) and earthworm (Hama, 1960; Coggeshall, 1965) central nervous tissues, quantitative measurement of extracellular volume is complicated by the presence of well-developed blood vascular systems. Measurements with dextran molecules and labeled red cells indicate that approximately 8-12% of the total water in the cerebral ganglion of Carcinus maenas is contained in the blood vessels (Abbott, 1969). Electron micrographs indicate that the blood vessels are surrounded by mesodermal endothelium, but the majority of vessels possess a naked basement membrane which is invariably surrounded by at least one specialized glial layer. There seem to be few enlarged extracellular spaces in the highly vascular regions of the cerebral ganglion, and preliminary measurements indicate that the extracellular compartment accounts for approximately 10-1 5 % of the total tissue water. Recent electron microscope investigations on invertebrate nervous tissues have revealed the presence of amorphous or granular material of appreciable electron density in the various extracellular spaces. Such material has, for example, been observed in the intercellular clefts and larger extracellular spaces in central nervous tissues of the leech (Coggeshall and Fawcett, 1964), A . californica (Ro-

FUNCTION OF INVERTEBRATE NERVE CELLS

63

senbluth, 1963), C. maenas (Abbott, 1969), P. americaiaa (Smith and Treherne, 1963), and C. mo~osz~.r(Maddrell and Treherne, 1767), as welf as in peripheral nerves of cephalopods (Villegas and Villegas, 1968). The available evidence for the leech (Bradbury, 1958), cephalopods (Villegas and Villegas, 196S), and an insect species (Ashhurst, 1961) seems to indicate that this material is probably acid mucopolysaccharide. The apparent presence of abundant acid mucopolysaccharide in the extracellular channels of invertebrate nervous tissues has two important physiological implications relevant to the present context. First, such material could function to restrict the movements of ions and molecules in the extracellular spaces and, second, it is possible that the anion groups associated with the acid mucopolysaccharide could function as a significant cation reservoir in the region of the neuronal surfaces (cf. Treherne, 1967). Finally, it should be emphasized that the coininon ultrastructural feature of the invertebrate nervous tissues that have been studied is the extremely tortuous nature of the restricted extracellular pathways interposed between the neuronal surfaces and the fluid bathing the surface of the nervous system or contained in the vascular system of some crustaceans and annelids. In general, these extended and restricted pathways are formed by various glial processes, but even in a freshwater lamellibranch such as A . cygnea, in which there is a paucity of glial elements, the extracellular pathways formed by the closely apposed axonal membranes form a relatively tortuous pathway for the axons situated deep within the neuropile although the superficial axons are readily accessible from the blood or bathing fluid (Gupta et al., 1969). In general, electron microscope studies have not revealed the presence of appreciable visible structures in the intercellular clefts of the nervous tissues of the invertebrates discussed above, either in the form of tight junctions or desmosomal contacts, which would be likely to severely restrict intercellular movements of small ions and molecules. Examples of such continuous systems of narrow extracellular channels can be cited in the central nervous tissues of species from each of the major invertebrate groups, for example, in the leech (Coggeshall and Fawcett, 1964), Anodonta cygnea (Gupta et al., 1969), Aplysia cdifornica (Rosenbluth, 1963), and P. americuna (Smith and Treherne, 1963). Exceptions to the above generalization are the well-developed desmosomes observed between glial processes or between cell bodies and glial processes in the lobster optic lamina (Hamori and Horridge, 1966). In the absence of three-dimensional reconstructions it is, however, not possible to speculate as to what extent the desmosomes function in restricting access within extracellular channels and particularly to the extracellular cisterns referred to previously. In the insect species (P. americaiza and C. m o ~ o s a s ) regions , of septate desmosomes and tight junctions were confined to the narrow extracellular channels of the perineurium (Maddrell and Treherne, 1967). There is an urgent need for investigations,

64

J. E. T R E H E R N E A N D R. B. MORETON

using electron-opaque extracellular indicator molecules, to determine the extent to which the apparently open extracellular clefts are in fact accessible. The current picture of the extracellular system summarized in this section has been largely derived from studies using conventional electron microscope techniques. The demonstration of the very restricted extracellular system in large regions of invertebrate nervous systems is, as has already been pointed out, essentially similar to that described in vertebrate central nervous tissues. It should be borne in mind, however, that electron micrographs have been obtained of vertebrate cerebral tissues, using a freeze-substitution technique, which show extracellular spaces appreciably larger than those obtained using conventional methods of fixation (Van Harreveld and Malhotra, 1966). The freeze-substitution technique has not been applied to the representative invertebrate nervous tissues considered in this review, so that an element of caution must be introduced in the interpretation of the extracellular systems described above. In the single case of the leech central nervous system, however, there is a reasonable measure of agreement between the chemically and the morphologically determined extracellular space (Nicholls and Wolfe, 1967), which does lend some support to the validity of the ultrastructure of the extra cellular system as revealed by the electron microscope.

111. Distribution and Exchanges of Inorganic Ions and Molecules There is considerable variation in the composition of body fluids between the various species of higher invertebrates that have been studied. In marine annelids, molluscs, and arthropods, the concentrations of inorganic ions in the blood frequently approximate those of sea water, with relatively high levels of sodium ions and low concentrations of other monovalent and divalent inorganic cations (cf. Prosser and Brown, 1961; Potts and Parry, 1964). Terrestrial and freshwater invertebrates, on the other hand, tend to exhibit blood concentrations in which the sodium levels are variable and generally considerably lower than in their marine counterparts. To choose examples from the representative species considered in this review, the leech ( H . m e d i c i d i s ) possesses blood with sodium and potassium concentrations of 130 and 4. l nimoles/liter, respectively, (Kuffler and Potter, 1964). In various terrestrial pulmonates, the sodium concentrations of the blood lie between 46 and 107 mmoles/liter, with potassium levels of 2.8-9.7 mmoles/liter and concentrations of calcium and magnesium as high as 10.3 and 13.2 mmole/liter, respectively (cf. Robertson, 1964; Sorokina and Zelenskaya, 1967; Burton, 1968b). The lamellibranch A. cygnea exemplifies the extremely dilute nature of the blood that can occur in some freshwater molluscs. The total concentration of the blood in this species is equivalent to

F U N C T I O N O F INVERTEBRATE NERVE CELLS

65

cnly 44.0 milliosmoles with a sodium level of 15.6 mmoles/liter (Potts, 1954). Among arthropod species, the insects exhibit the most extreme variations in blood composition (cf. Treherne, 1966). In omnivorous species such as the cockroach P. umevicund, the blood sodium level averages 157.4 mmoles/liter (Treherne, 1961a), while in a phytophagous insect such as C. morosus, the sodium concentration of the blood has been found to be only 11.0 mmoles/liter (Rainsay, 1953) and in the hymenopteran Ptevonidea ribesii, to be as low as 1.6 mmoles/liter (Duchgteau et al., 1953). Unlike the blood of freshwater molluscs, the osmotic concentration of that of insects, with relatively low concentrations of inorganic ions, is maintained by extremely high concentrations of amino acids (cf. Wyatt, 1961). The various extraneural structures described previously (Section 11, A) appear to offer little restriction to the movements of inorganic ions from the blood, and there is no evidence that they are responsible for maintaining specialized ion concentrations in the fluid bathing the surfaces of the nervous system. Despite the presence of pinocytotic vesicles in the endothelium of the leech central nervous system (Coggeshall and Fawcett, 1964), inorganic ions were found to pass rapidly in and out of the nerve cord even in preparations cooled to 1.3'C (Nicholls and Kuffler, 1964). It was concluded from this that active transport does not play a significant role in ion movements across the endothelium. Similarly, movements of labeled inorganic cations take place rapidly between the bathing medium and the central nervous tissues of the insect C. moromr (Treherne, 1965), despite the presence of the neural fat-body sheath (Maddrell and Treherne, 1966). The apparent chloride diffusion potential measured across the fat-body sheath (Treherne and Maddrell, 1967a) does not appear to be of primary importance in regulating the extracellular sodium level in this insect, for the central axons still continued to function for appreciable periods in preparations bathed in low sodium media (Treherne and Maddrell, 1967b). There is also no evidence that the epithelial walls of the proximal limb of the kidney play a significant part in regulating the concentration of inorganic ions in the fluid bathing the central nervous system, for action potentials were recorded for extended periods in low-sodium solutions (Treherne et al., 1969a). The available evidence seems to indicate that the peripheral connective tissue sheath surrounding ganglia, connectives, and peripheral nerves does not function as an appreciable diffusion barrier as has been suggested in vertebrate nerves (Feng and Liu, 1950). This conclusion is based on observations that the relatively large anionic dye molecules of trypan blue readily passed into the neural lamella of the earthworm nerve cord (Levi et al., 1966) and that of the cockroach (Wigglesworth, 1960), although no staining of the underlying tissues was observed. It also appears that the peripheral cellular layer underlying the connective tissue sheath is relatively permeable to small water-soluble ions atid

66

J. E. TREHERNE AND R. B . MORETON

molecules in the invertebrate species that have been studied. This conclusion is based on observations that alterations in the ionic composition of the bathing medium are quickly reflected in changes in the resting and active membrane responses of neurons in the intact central nervous systems of H . medicinalis (Nicholls and KufAer, 1964) and A. cygneu (Treherne et ul., 1969a), and on the rapid exchanges of radioactive substances observed in the central nervous systems of Hirudo medicindis (Nicholls and Wolfe, 1967), A. cygneu (MelIon and Treherne, 1969), Homdrus americanus (Nevis, 1958), P. umericunu (Treherne, 1961a,b,c,d, 1962a; Eldefrawi and O’Brien, 1966, 1967a,b; Eldefrawi et al., 1968), and C. rno~osus(Treherne, 1965). The endothelium of the capillaries, which channel blood deep into the central nervous system of Carcinus, also appears to be relatively leaky to small ions and molecules, for it has been recently observed that relatively large ferritin molecules were able to pass from the blood into the underlying extracellular spaces and to reach the axonal surfaces (Abbott, 1969). In view of the dramatic physiological changes induced by removal of the nerve sheath in insect species (Holyle, 1953; Twarog and Roeder, 1956; Treherne, 1962b), it is of some relevance to examine some of the effects produced by this procedure. The desheathing procedure involves the removal of the connective tissue layer and also substantial portions of the underlying perineurium (Twarog and Roeder, 1956). It was originally supposed that changes in the response of axons to alterations in the ionic concentration of the bathing medium produced by the desheathing procedure resulted from the removal of a peripheral diffusion barrier associated with the nerve sheath. As is apparent from the results of isotope experiments summarized previously, this hypothesis is no longer tenable. The desheathing procedure does, however, produce a number of dramatic effects in the underlying nervous tissues. In particular, it has been shown to involve disruption of the extracellular Donnan effect with the blood and to result in a fivefold increase in the measured inulin space in the central nervous tissues of the cockroach (Treherne, 1962a). The increase in the measured inulin space does not appear to be correlated with any significant expansion of the extracellular spaces, for electron micrographs of desheathed preparations showed extracellular spaces of dimensions essentially similar to those of intact preparations (Smith and Treherne, unpublished observations; Lane and Treherne, 1969). The use of peroxidase and other extracellular indicator molecules has shown, however, that cytoplasm of the underlying glial cells is invaded by these relatively large molecules in desheathed preparations (Lane and Treherne, 1969). It appears from the latter investigation that molecules are able to pass from the region of the damaged perineurial cells into the adjacent glial cells and thence into more distal glial cells by means of tight junctions between glial membranes (Fig. 7). Apparently, massive additional diffusion channels are 1x0-

FUNCT ION O F INVERTEBRATE NERVE CELLS

67

duced through nervous tissues by the conventional desheathing procedure. The demonstration of the movement of peroxidase molecules between glial cells via tight junctions is also of interest in that it provides the first structural evidence for the low-resistance connections between adjacent glial cells demonstrated electrically by Kuffler and Potter (1964) (Section IV). The exchange of inorganic ions between nervous tissues and the bathing medium appears to take place as a two-stage process in the relatively few invertebrate species investigated. This is particularly apparent in efflux experiments carried out on the cerebrovisceral connectives of A. cygnea (Mellon and Treherne, 1969) and the abdominal nerve cords of P. u m e h m a (Treherne, 196ld, 1962a) and C. MOYOSZLS (Treherne, 1965). In the case of the two insect species, it was found that the slow exponential fraction obtained for the efflux of radiosodium was reduced in the presence of metabolic inhibitors such as cyanide, dinitrophenol, and ouabain. The initial fast fraction was, however, not affected by these substances. The slowly exchanging sodium observed in these experiments was, therefore, identified as the intracellular fraction, and the rapidly exchanging one as the extracellular fraction. An essentially similar interpretation has been applied to the exchanges observed in mammalian central nervous tissues (Ames and Nesbett, 1966). The rapidly exchanging fraction of tritiated water in the peripheral nerve of the lobster (Nevis, 1958) and of some nonelectrolytes in the nerve cord of the leech (Nicholls and Wolfe, 1967) have also been identified as being extracellular. The concentrations of both monovalent and divalent cations in the extracellular fraction, calculated using the measured inulin space or the rapidly exchanging tritiated water fraction (Section 11, D), were found to exceed that of the hemolymph in the nerve cord of Periplaneta (Treherne, 1962a). These elevated cation concentrations and the relatively low level of chloride ions appeared to result from the existence of a Donnan equilibrium between the hemolymph and the extracellular fluid. It has been suggested (Treherne, 1962a) that such a Donnan equilibrium might result from the presence of free anion groups associated with the extracellular acid mucopolysaccharide demonstrated in cockroach ganglia (Ashhurst, 1961). The situation in the freshwater lamellibranch A. cygneu differs in several respects from that described for insect species (Mellon and Treherne, 1969). Although it is possible to separate sodium exchange into two fractions, a rapidly exchanging one (with a half-time of approximately 150 seconds) and a slower one (with a half-time of 980 seconds), the d u x was accelerated and the amount of the rapidly exchanging sodium was reduced at low temperature. It seems clear, therefore, that it is not possible to postulate any simple separation in the exchanges of the intracellular and extracellular sodium fractions in the central nervous tissues of Anodontu. It seems most likely that these observations at low

FIG. 7. Section of neuropile from abdominal ganglia of P. nrnericdmz which have been desheathed and treated with horseradish peroxidase for 30 minutes ( A ) and for 60 minutes (B) . The tissues were subsequently incubated to visualize the sites of protein uptake, and fixed in formaldehyde-glutaraldehyde (A) and buffered glutaraldehyde ( B ) . The thin section in A is unstained, the better to demonstrate the dense reaction product, while in B the tissue has been stained with uranyl acetate and lead citate. N, Axons cut in cross or tangential section; G, sections through glial cells ensheathing the axons and containing dense depositions of reaction product indicating the presence of peroxidase. Arrows in B indicate tight junctions between adjacent glial cells. Ganglia with intact sheaths do not exhibit this glial uptake of peroxidase. ( A ) x 19,500. ( B ) x 50,000. (From Lane and Treherne, 1969).

FUNCTION O F INVERTEBRATE NERVE CELLS

69

temperature resulted from a drastic slowing down of the efflux of some rapidly exchanging intracellular sodium so as to leave a fast fraction largely composed of extracellular sodium ions. It was found that at low temperature the fast fraction amounted to about 3.9 mmoles per kilogram tissue. By using a figure of 0.23 p1 per milligram tissue for the volume of the extracellular fluid (i.e., the measured sucrose space), it was then calculated that the concentration of sodium in such a rapidly exchanging extracellular fraction would amount to approximately 15.6 mmoles/liter, which is very close to the concentration of this cation in the bathing medium (Mellon and Treherne, 1969). It appears, therefore, that there is no elevation of the extracellular sodium resulting from a Donnan equilibrium with the blood, as in the case of the insect species. Despite the very low sodium level in the blood and extracellular fluid of A?zodonta, it was calculated, nevertheless, that there was a gradient of sodium ions between the extracellular fluid and the nerve cells, for the intracellular sodium concentration was found to average the exceptionally low value of 8.6 mmoles/liter. Another unusual feature of the nervous system of Anodoiztd is the presence of a small sodium fraction that does not exchange with that in the bathing medium even after prolonged soaking in a solution containing radiosodium (Carlson and Treherne, 1969). This sequestered sodium fraction, which amounts to 0.51 mmoles per kilogram tissue, was identified in experiments using 2zNa of high specific activity and low concentration in the bathing medium. Stimulation of the cerebrovisceral connective in sodium-free solution resulted in a significant depletion of this residual sodium (Carlson and Treherne, 1969). There is relatively little quantitative inforniation on the movements of inorganic ions in restricted extracellular channels between adjacent cell membranes in invertebrate nervous tissues. The data obtained in radioisotope studies on insect nervous tissues are difficult to interpret in this respect, for the relatively rapid exchanges of inorganic ions between the bathing medium and the extracellular fluid may well be those taking place with the large peripheral glial lacuna spaces, thus masking the movements of inorganic ions within the narrow intercellular spaces in the neuroprofile. A more satisfactory analysis has been made in the leech central nervous system in which changes of the membrane potentials of the neurons have been related to alterations in the ionic comnposition of the bathing medium (Nicholls and Kuffler, 1964). By using the diffusion coefficients for NaCl and for sucrose, the theoretical half-time was calmlated for linear diffusion within an individual intercellular cleft using the following formula: C

-- 1 -

co

~

2 1 , e-Y'dV -

vn

where C, is the initial concentration and y = x/2 -\/nt,.Y being the distance

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J. E. TREHERNE AND R. B. MORETON

(in centimeters), t the time (in seconds), and D the diffusion coefficient (a2 sec-1). By using values of 30 p for the mesaxon length in a connective and 50 p for that in the packet in the ganglion, it was found that there was reasonable agreement between the calculated and the observed half-times for diffusion. For example, in the case of potassium ions diffusing through a ganglion packet, the calculated value for to,5was 1.5 seconds as compared with the observed value of 4 seconds. It is concluded, therefore, that the observations on the rates of change of potentials of neurons in response to changes in the ion content of the bathing medium support the concept that there is little restriction on diffusion through the narrow intercellular clefts visualized in electron micrographs (Nicholls and Kuffler, 1964). The movement of small organic molecules such as glycerol and ethylene glycol through mesaxon channels of the squid giant axon also follows an equation of the above type (Villegas et ul., 1962). Rates of diffusion agree well with those calculated from diffusivities in free solution.

IV. The Ionic Requirements for Electrical Activity The electrical function of nerve cells is profoundly affected by the ionic composition of the fluid bathing their surfaces. Information gained from electrical experiments is thus of the most fundamental kind; unfortunately, it is often also the most dificult to interpret. The responses of a neuron to changes in the ionic concentration of the blood or bathing medium are affected by the extent to which these relatively large-scale changes are reflected in alterations in the cell’s immediate extracellular environment. Some idea of the nature of this environment can be obtained by measurement of the changes in potential in neurons in intact nervous structures in response to alterations in the ionic content of the bathing medium; by experiments involving elimination of the control of the extraneuronal environment, for example, by mechanical disruption of regulating structures; by direct measurement of chemical changes such as those discussed in the preceding section of this review; or by analysis of the behavior of neurons through the use of drugs with known effects on neuronal mechanisms and subsequently inferring the nature of the extracellular changes to which they are responding. This section is devoted to the results obtained from experiments involving the measurement of the resting and active potentials of invertebrate neurons in preparations subjected to varying degrees of surgery and variations in the ionic concentration of the bathing medium. One invertebrate system in which a relatively complete analysis has been possible is the central nerve cord of the leech H . medicimlis (Kuffler and Potter, 1964; Nicholls and Kuffler, 1964, 1965). Here, both neurons and glial cells have resting potentials, of around 50 and 70 mV, respectively, which are deter-

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mined by the extracellular potassium concentration; at concentrations above 20 ininoles/liter the behavior is closely approximated by the Nernst equation, so that it was possible to make estimates of the intracellular potassium concentrations in good agreement with the mean value of 130 mmoles/liter determined by flame photometry (Nicholls and KufAer, 1965). The glia showed no trace of electrical excitability, but many of the neurons gave overshooting action potentials which were rapidly abolished by exposure of the cord to solutions free of sodium ions. As noted above, from the rapidity with which changes in both resting and action potentials occurred following changes in the bathing solution, it was possible to show that diffusion of ions through the intercellular clefts is practically unrestricted, so that the glial cells can play very little part in regulating the ionic environment of the neurons. The ready accessibility of the intercellular spaces from the outside medium was confirmed by the observation that a microelectrode placed in one of the clefts recorded a potential and input resistance indistinguishable from that recorded in the bathing solution (Kuffler and Potter, 1964). Repeated stimulation of either single neurons or the whole cord failed to cause any accumulation of potassium ions in the extracellular spaces, as shown by the fact that the glial resting potentials were unchanged. [Compare the situation in the amphibian Nectarus (Orkand et al., 1966; Kuffler, 1967), in which repetitive stimulation of neurons causes considerable potassium accumulation and associated glial depolarization; compare also the giant axon of the squid (see below).] Neurons and glia are electrically quite independent apart from possible localized interactions, a fact which can again be shown to result from their high membrane resistance as compared with the longitudinal resistance of the unobstructed channels between (KuHer and Potter, 1964). Some direct electrical connections between glial cells were found, which is surprising, in that no physical contacts between cells were seen in the micrographs (Section 11, C). In an insect species, however, there is some structural evidence for the existence of communication channels between adjacent glial cells (Lane and Treherne, 1969) ; as described in Section 111, tight junctions have been observed to exist between glial membranes which appear to be responsible for the movement of peroxidase molecules between adjacent glial cells. In the leech central nervous system, it was possible to remove the glial investiture from some of the neurons without significantly altering their excitability for many hours, again showing that their function does not depend upon the provision of any specialized ionic environment by the glial cells. In molluscs, the situation is in general more complicated than in the leech. The simplest system to have been studied is the familiar giant axon of Loligo, whose properties are so well known that it has come to be regarded as a prototype for all nerve cells; it is convenient when discussing the properties of neu-

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rons to speak of those resembling the squid axon in behavior as “conventional.” Details of the ionic processes underlying the resting and action potentials in the squid axon have been reviewed by Hodgkin (1958) (see also Conference on Newer Properties of Perfused Squid Axons, 1965) and are in any case outside the scope of this article. The volumes of extracellular space and glial cytoplasm surrounding the axon are very small in comparison to that of the axon itself (Villegas et aL, 1965) and could not be expected to contribute significantly to direct measurements of the exchange of ions between the preparation and its surroundings. Electrical nieasurements (Frankenhaeuser and Hodgkin, 1956) have shown, however, that the tortuous mesaxon channels between the glial cells do restrict the diffusion of potassium ions released from the axon during the action potential. Passage of a single impulse was found to raise the extracellular potassium concentration by about 1.6 mmoles/liter the excess ions then diffusing away with a time constant of 30-100 msec. The thickness of the space in which the ions accumulated was calculated to be about 300 A, which is comparable to the size of the intercellular space seen in electron micrographs. The reason why this effect was not observed in the leech may be partly that the area of excitable membrane of the squid axon is relatively enormous compared to the total area of the mesaxon channels available, or may lie in the presence of the amorphous, electron-dense intercellular substance seen by Villegas and Villegas (1968) (Section 11, D). The diffusion of potassium ions away from the surfaces of insect giant axons appears to take place more rapidly than in those of the squid, for the negative after-potential decayed with a mean half-time of only 9.2 msec (Narahashi and Yamasaki, 1760). It is conceivable that the dilatations in the concentric extracellular channels (Fig. 5 ) surrounding the insect axons could function as reservoirs which, by increasing the volume of extracellular fluid in the region of the axon surfaces, could facilitate the dispersal of potassium ions from the axolemma (Smith and Treherne, 1963; Treherne, 1966). The fact that the decay of the negative after-potential does not assume a simple exponential decline following repetitive stimulation, but shows a secondary component roughly equivalent to that encountered in the squid axon (Frankenhaeuser and Hodgkin, 1756), could also result from the reservoir effect of the relatively large extracellular spaces adjacent to the axon surfaces. Thus, it is envisaged that the initial rapid fall in the negative after-potential obtained after rapid stimulation (Narahashi and Yamasaki, 1960) represents the dispersal of potassium ions from the axon surfaces, while the subsequent slower decay results from the relatively slow fall in the potassium concentration of the extraaxonal fluid as the reservoirs of ions in the larger spaces disperse into other parts of the extracellular system (Smith and Treherne, 1963; Treherne, 1966). Glial cells surrounding the squid axon thus appear to play a small passive

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role in regulating the ionic environment of the axolemma. Their own resting potentials are, however, as in the leech, quite independent of electrical activity in the axon (Villegas et al., 1963). Glial resting potentials are comparatively low (33-46 mV) and vary only slowly with potassium concentration in the physiological range (Villegas et al., 1968), so that the effect on them of the potassium ions released from the axon during activity is probably much less than the effect on the resting potential of the axon itself. There is no evidence that glial cells have a significant restricting or regulating effect on the access of any other ions to the squid giant axon, for exposure of the system to sodiumfree solutions abolishes the axonal action potential within 2 1 seconds, as reported by Villegas et d. (1965). On the other hand, the same authors have shown that the glial cells contain an exceptionally high concentration of sodium ions, which they suggest (see also Villegas, 196s) may be largely in bound form, and which can be extruded from the cells by a “conventional” sodium pump, coupled with uptake of potassium ions. It is thus conceivable that the glial cells could, in addition to any passive role, play an active part in regulating the extraaxonal environment by providing a store of sodium ions to be released into the extraaxonal space during activity. It is, however, evident from the rapid and complete abolition of the spike, which follows exposure of the preparation to sodium-free solution, that any such regulation that occurs must be extremely ineffective; the behavior of the action potential overshoot (Hodgkin and Katz, 1949) and membrane conductance (Hodglrin and HUXley, 1952; Chandler and Meves, 1965) as functions of the sodium concentration in the bathing medium can be quite adequately accounted for on the assumption that the extraaxonal sodium concentration closely follows that in the external fluid. Uptake by the glial sodium pump could also assist in the dispersal of potassium ions released from the axon during electrical activity, although again there is very little evidence of any significant effect. The time constant for dispersal of potassium ions (Frankenhaeuser and Hodgkin, J 9 5 6 ) varies only slowly with temperature (Ql0 = 1.3), suggesting that the mechanism is predominantly one of passive diffusion. Electrical studies have been made on the ganglia of a number of gastropod and bivalve species from marine, freshwater, and terrestrial habitats; as might be expected, the results vary widely. Most electrical experiments have been aimed principally at elucidating the mechanism of generation of the nervous action potential and have been conducted on desheathed preparations 111 which the glial system surrounding the neurons has been disrupted to an unknown extent. There is thus, in general, little information on the function of connective tissue or glial cells in the maintenance of electrical activity in intact systems. It is, however, particularly interesting that certain molluscan nervous systems, even after such drastic interference wlth their extraneural structures,

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still show behavior that cannot be explained in terms of a simple, more-or-less rapid exchange of ions with their surroundings. In these cases, it must be assumed that exchange is being affected by the presence of structures or substances close to the neurons themselves. While in an active state, molluscs are characteristically unprotected against gain or loss of water through their body surface resulting from changes in their environment. While in marine species this presents no problem, terrestrial and, to a lesser extent, freshwater molluscs are subject to considerable changes in the composition and osmotic pressure of their body fluids (e.g., Duval, 1930; Arvanitaki and Cardot, 1932; Burton, 1964, 1968a; Sorokina and Zelenskaya, 1967; Little, 1968). Burton (1968b) has tabulated the hemolymph composition of a number of terrestrial and freshwater pulmonates; in marine species, the hemolymph differs little from the surrounding sea water, although there may be some small discrepancies in ionic balance (e.g., Hayes and Pelluet, 1947). The degree of control over the extraneural environment within the central nervous system is, however, not at all well correlated with the degree of variability to be expected in the environment of the animal. Thus, of the marine species examined, the lateral giant neurons of A. californicu (Junge, 1967; Geduldig and Junge, 1968) appear to form part of a simple, unregulated system. Generation of the action potential is “unconventional” in that both sodium and calcium ions appear to be jointly responsible for carrying the inward current; thus, tetrodotoxin and cocaine, which are generally considered to act on current-carrying systems involving sodium ions, cause only partial inhibition of the action potential. Manganous ions, which affect calcium-carrying mechanisms, similarly reduce the action potential overshoot and when applied in conjunction with tetrodotoxin lead to total and reversible abolition of the spike. Moreover, the effect of either tetrodotoxin or manganous ions can be quantitatively reproduced by exposure of the preparation to solutions devoid of either sodium or calcium ions, respectively. It thus appears that during the rising phase of the action potential in Aplysid neurons, both sodium and calcium ions enter the cell by way of two independent mechanisms. Of chief interest in the present context, however, is that the effects of changes in the concentrations of both these ions in the bathing medium, and of the addition of tetrodotoxin, cocaine, or manganous ions, were observed quite rapidly, being generally complete within 5 minutes after changing the bathing solution. It thus appears that inorganic ions and small organic molecules are able to penetrate to the neuronal surface in this preparation with very little hindrance. In the ganglia of the freshwater gastropods Pluuorbis comeids and L, stugt2ali.r (Gerasimov et dl., 1964), the situation is simpler in that spike generation is by a conventional, sodium-dependent mechanism. As in AplysLz, the exchange

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of sodium ions between bathing medium and extracellular spaces is rapid, action potentials being abolished within a few minutes of exposure of the preparation to sodium-free solution. The concentration of sodium ions in the hemolymph of these species is only about 50 mmoles/liter (Sorokina and Zelenskaya, 1767; Burton, 196sb), but varies comparatively little throughout the year. In Planorbis, Sorokina (1966) gives a mean value for the intracellular sodium activity of only 8 mmoles/liter, which corresponds to a sodium activity ratio of 4.4: 1 between cells and hemolymph; the maximum action potential overshoot that could be generated by this gradient is 37 mV, which agrees quite well with the observed value of around 33.2 mV (Maiskii and Gerasimov, 1964), suggesting again that the extracellular sodium activity differs little from that in the hemolymph. The terrestrial species most studied are H. ponzatia and H. aqkvsa, two closely related snails from very similar habitats. In spite of the similarity of the snails, there appear to be some differences in the functioning of their nervous systems which have led to considerable confusion. Giant neurons of both H . pomatia (Gerasimov et al., 1964, 1965; Meves, 1966, 1968) and H . uspersd (Kerkut and Gardner, 1967; Moreton, 1968a) continue to give action potentials after the ganglia have been exposed to sodium-free solution for 1 hour or more. In H. pomatia, this behavior has been shown largely to result from the use of calcium rather than sodium ions to carry the inward current during the action potential (Gerasimov et al., 1965; Meves, 176s); the overshoot in sodium-free solution is linearly related to the logarithm of the calcium concentration, and the action potential is abolished by manganous ions, but not by tetrodotoxin or cocaine. There is little evidence for regulation of the extraneural environment. In H. aspersa, on the other hand, the action potentials of neurons are reversibly abolished by tetrodotoxin, although only in rather high concentrations (Moreton, 196Sa), and most cells are little affected by manganous ions (Moreton, unpublished observation). Although the response to a reduced concentration of calcium was a rapid decline in overshoot, raising the concentration failed to produce any increase (Chamberlain and Kerkut, 1967, 1969). It thus appears that, in H. aJPersa at least, the mechanism of spike generation is sodium-dependent, and that the persistence of the spike in sodiumfree solution is attributable to the retention of sodium ions in the extracellular system. In further support of this, prolonged exposure to sodium-free solution is found to cause a very slow decline in the overshoot, which could be rapidly reversed by restoring the sodium concentration of the bathing fluid (Moreton, 19682~).The regulation evidently applies specifically to sodium ions, since the effects of changes in the concentrations of calcium (Kerkut and Gardner, 1967) and potassium (Kerkut and Meech, 1967; Moreton, 196Sb) on the resting and action potentials of the neurons are complete within a few minutes after ex-

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posure of the ganglia to the new solution. Whether the sodium ions are retained by binding to some specific structure or substance [such as the mucopolysaccharide found in the intercellular clefts around the squid giant axon (Section V, D ) ] , or by local recycling between extracellular spaces and the glial cytoplasm via the sodium pump in the glial cell membranes, is not clear. The highly complex system of glial investiture found in H . aspersa is an obvious candidate for involvement, although it is difficult to see why Aplysiu, which has a system of multiple glial sheaths, apparently shows very little regulation. Regulation of the extraneural environment in H . aspersd is obviously a good solution to the problem of the large variations in composition of hemolymph this species must tolerate (Burton, 1968a). Possibly, the adoption of a calcium mechanism for generating the action potential in H . pomatia could represent an alternative solution to the same problem, although the calcium content of hemolymph in this species may also vary considerably (Sorokina and Zelenskaya, 1967; Burton, 1968b). Systems similar to that in H . asperj-a are also found, somewhat surprisingly, in the two marine gastropods T. diomedid (Magura and Gerasimov, 1966; Veprintsev et al., 1966) and 0. werruczllatvm (Oomura et al., 1961). The neurons are freely accessible to potassium ions, but exposure to sodium-free solution has only a slow effect on Tritonia neurons, and none at all in 012chidiztm. Removal of calcium has a depolarizing and inactivating effect in both species, although rather more slowly than in Helix. There is little information on the structure of the ganglia in these species. A recent study carried out in this laboratory has revealed a mechanism responsible for the regulation of the extraaxonal sodium in the central nervous system of the freshwater bivalve A . cygnea (Treherne et al., 1969a; Carlson and Treherne, 1969). The compound action potential of the cerebrovisceral connective, recorded from external electrodes, consists of fast and slow components, which can be related to groups of large and small axons, respectively. Whereas the small axons show relatively conventional behavior, being “knocked out” by sodium-free solutions within 10 minutes, the large axons in this system were found to be conduct action potentials with very little attenuation for periods of up to 3 hours in isotonic sucrose or dextran solutions. This is in contrast to both Helix species, which require the presence of at least some calcium ions in the bathing medium for the maintenance of neuronal function. LOW concentrations of tetrodotoxin or procaine rapidly abolished the action potential, but manganous ions had only a small effect, suggesting strongly that the actual mechanism of spike generation resembles that in a conventional, sodium-dependent system. This idea is supported by observation of the effects of both dinitrophenol and ouabain, prolonged application of which in Ringer’s solution caused decline and eventual failure of the action potential, which could

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then be rapidly but temporarily restored by exposing the preparation to a sodium-enriched solution; raising the concentration of calcium, on the other hand, did not produce any recovery of the action potential. Substitution of sulfate or acetate ions for chloride in the bathing medium was also without effect on the action potential, which tends to rule out any possibility that efflux of anions might be responsible for the inward current. The rapid recovery in a high-sodium solution also suggests, in coinhillation with the fact that most of the sodium in the connective exchanges quite rapidly with the bathing medium (Section IV) , that the intercellular spaces are readily accessible to small ions and molecules, as is the case in Helix. Activity in sucrose solution must, therefore, he maintained through the existence of a store of sodium ions, in or close to the axon membranes, as was proposed by Chamberlain and Kerkut (1967, 1969) for H . a.rpersa. Direct evidence for this store was provided by two rather surprising observations. First, conduction in the connective could be rapidly abolished by bathing it in distilled water, and then restored by bathing in isotonic dextran, with no further access to inorganic ions. Second, and again in contrast to HeLix, replacement of external sodium by tris or choline, instead of sucrose, resulted in rapid conduction failure, which in this case could be restored by the addition of sodium to the bathing medium. Furthermore, it was also observed that the relation between the conduction velocity and the relative sodium concentration of the bathing medium was similar to that in conventional sodium-dependent axons, such as those of Carcinur (Katz, 1947) and Loligo (Hodgkin and Katz, 1949), when sodium was replaced by tris chloride, but showed no appreciable reduction with decreasing external sodium concentration in the presence of dextran (Fig. 8). It was, therefore, postulated that the connectives contain a store of “bound” sodium, which can exchange with other monovalent cations but which is otherwise only available during passage of an action potential. This idea is supported by the observation (Section 111) that there is a residual sodium fraction in the tissue which exchanges with sodium in the bathing medium only when the connective is stimulated repeatedly; this is correlated with the observation that repeated stimulation in a sucrose solution causes progressive conduction failure over a period of 30 minutes (Carlson and Treherne, 1969), as compared to the 3-hour survival of conduction in connectives oiily occasionally stimulated. Any remarks as to the nature o f the structures responsible for retaining such a reservoir of sodium ions must remain at present highly speculative. It should be noted that most of the experiments on Anodonta were carried out on intact connectives, in contrast to those on Helix, which were made on desheathed ganglia. Experiments on connectives that had been split down the center (Carlson and Treherne, 1969) indicated, however, that the behavior of the Iarge axons was not significantly altered by this treatment, suggesting that, as in

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Helix,regulation is mediated by structures intimately associated with the axons. Glial cells are again the obvious candidates for consideration, particularly since the larger axons, which are the ones whose conduction is maintained for the longest time in sodium-free solution, are also the ones that tend to have the 1.2

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FIG. 8. The relative conduction velocity (6test/6normal) of the fast axons in the cerebrovisceral connective of the lamellibranch A. cygnea plotted as a function of the square root of the relative sodium concentration (Nat,st/Na,,r,al) %, in solutions in which the sodium ions were diluted with dextran and tris chloride, respectively. This graphic form permits a comparison to be made with data for Carcinus (Katz, 1947) and Loligo axons (Hodgkin and Katz, 1949). The data for Loligo axons was calculated from mea~urement~ made on the maximum rate of rise of action potentials in test solutions of varying sodium concentration. These calculations were made on the assumption that in a simplified theoretical system the conduction velocity can be related to the square root of the rate of rise of action potential (Hodgkin and Katz, 1949) (from Carlson and Treherne, 1969).

most complete glial investiture (Treherne et al., 1969b). On the other hand, Anodonta shows a much more marked regulation of the extraneural environment than does Helix,whereas Helix has the more complex glial system. It is conceivable that sodium ions could be sequestered by large, indiffusible anions in the intercellular spaces, so that their thermodynamic activity normally remains very low, and released, possibly as a result of the electrical charges occurring at the onset of an action potential, to take part in the conduction process.

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Such an extracellular sodium store is apparently not the only source of sequestered sodium in the region of the axons surfaces in Anodonta, however, for it was observed that there was a rapid return of axonal function when preparations were removed from tris or choline chloride to isotonic dextran solutions (Carlson and Treherne, 1969). It appears necessary, as indicated above, to postulate an additional intracellular sodium fraction, perhaps associated with the glial processes, to explain the ability of the fast axons of Anodonta to function in sodium-free nonelectrolyte solutions. It is of particular interest in this respect to note there is histochemical evidence for the existence of nonfreely diffusible sodium in the Schwann cells of cephalopod giant axons (Villegas, 1968). The neurons of the arthropod species that have been studied appear to be conventional in the ionic mechanisms responsible for determining the resting and action potentials. Comparison of the measured resting potentials with the calculated potassium equilibrium potentials for a number of crustacean and insect species indicates that this cation is largely responsible for carrying the outward current associated with the resting potential (cf. Treherne, 1966). Departure from the relation predicted by the Nernst equation with varying external potassium concentrations described by various workers (e.g., Dalton, 1958, 1959; Yamasaki and Narahashi, 1959; Julian et al., 1962a,b; Edwards et al., 1963; Treherne and Maddrell, 1967b) indicates, however, that some other ion species are also involved in determining the resting potential. This state of affairs is essentially similar to that in the squid giant axon (Curtis and Cole, 1942) where, allowing for the permeabilities of potassium, sodium, chloride, and the variations in potential, the resting potential can be well accounted for (Baker et al., 1962). As in the conventional squid giant axon preparation, the action potentials of isolated crustacean (Dalton, 1958, 1959) and insect axons (Boistel and Caroboeuf, 1958; Yamasaki and Narahashi, 1959; Treherne and Maddrell, 1967b) show a striking dependence on the external sodium concentration. In crayfish giant axons, the magnitude of the action potential was close to that which would be predicted for an ideal sodium electrode (Dalton, 1959), although in cockroach axons the departure from the predicted Nernst relation makes it necessary to postulate that the conductances of other ions may participate in determining the peak of the action potential (Yamasaki and Narahashi, 1959; Narahashi, 1963). Voltage-clamp experiments also confirm the dominant part played by sodium ions in the inward current of the action potential in lobster (Julian et al., 1962b) and cockroach giant axons (Pichon and Boistel, 1967; Pichon, 1968). The existence of apparently conventional, largely sodium-induced action POtentials in arthropod axons is of some interest in relation to the bizarre ion

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concentrations encountered in the blood of some insect species (Section 111). In particular, the extremely low sodium concentrations, which are frequently exceeded by those of potassium ions, present difficulties in the interpretation of action protential production in terms of the conventional membrane theory. The efficiency of the physiological mechanisms involved in maintaining appropriate extraaxonal ion concentrations in the central nervous system of Curuzisizls is well illustrated in the experiments in which axonal function was maintained for extended periods in preparations bathed in sodium-free solutions, whereas desheathed preparations showed a rapid decline in excitability under these circumstances (Treherne and Maddrell, 1967b). The ability to function for appreciable periods in the absence of external sodium ions does not, however, appear to be confined to the axons of phytophagous insects, for conduction was maintained for several hours in the intact nerve cord of Periplaneta in sodiumdeficient saline (Twarog and Roeder, 1956) and in isotonic glucose (Yamasaki and Narahashi, 1959). The sodium dependency of the axons of Caruusiiis under these conditions is also confirmed by the rapid effect of dilute tetrodotoxin on action potential production in intact preparations maintained under these conditions (Treherne and Maddrell, 1967b). The neural fat-body sheath (Section 11, A ) which surrounds the central nervous system of CurdzoinJ- (Maddrell and Treherne, 1966) does not appear to be involved in regulating the sodium level in the extraaxonal fluid, for the ability of the axons to function in sodiumfree solutions was not impaired by removal of this structure (Treherne and Maddrell, 1967b). It is also apparent from the preceding section that the ability to function in sodium-free solutions does not result from an appreciable peripheral diffusion barrier associated with the nerve sheath in insect species. It also appears unlikely that the perineuriuin is involved in actively pumping sodium ions into the general extracellular system, as was suggested by Shaw and Stobbart (1963), for it has been shown that the magnitude of the rapidly exchanging sodium fraction, which has been postulated to be largely extracellular (Section HI), is related to the concentration of sodium in the bathing medium (Treherne, 1962b). Finally, it seems reasonable to reject the possibility that elevated concentrations of cations might be maintained at the axon surfaces in Cdr~~u.riaiand Peripbneta because of the presence of extracellular indiffusible anion molecules (Section 111). It has been pointed out (Treherne, 1967) that the thermodynamic activity of sodium ions associated with a fixed-charge system, such as the anion groups associated with extracellular acid niucopolysaccharide (Section 111), could only be equivalent to that in the bathing solution. Thus, the activity of the cation would be no higher than that in the bathing medium. The possibility cannot be eliminated, however, that the sodium associated with such fixed-anion groups could form part of a cation reservoir

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which might be mobilized if local circuit current flow altered the configuration of the anion groups as has been tentatively suggested earlier in respect to molluscan nervous systems. The possibility also exists that regulation of the extraaxonal sodium level might be achieved by the activity of the glial cells (Treherne and Maddrell, 1967b; Treherne, 1967). In both Peripbneta and Carausius, the subperineurial glial system consists of a peripheral cell layer with an extensive system of cytoplasmic processes (Section V, C) (Wigglesworth, 1960; Smith and Treherne, 1963; Maddrell and Treherne, 1967; Treherne and Maddrell, 1967b). The axons are surrounded by at least one glial fold, the glial membranes being closely applied to the axon surfaces so as to leave extracellular clefts about 150 A in width (Fig. 6). The mesaxons pursue a tortuous course so as to form an extended pathway between the perineurium and the axon surfaces. In view of the demonstrated movement of molecules between adjacent glial cells by tight junctions (Section 111), it seems possible that there could be a rapid intracellular movement of sodium ions to glial processes which could be associated with an extrusion of the cations into the very restricted extracellular spaces adjacent to the axon surfaces, perhaps by conventional sodium pumping mechanisms. The efficiency of such a glial sodium-regulating system would depend on some degree of restriction on sodium movements away from the axon surfaces. It would not be unreasonable to suppose that the tortuous nature of the intercellular channels in the neuropile might restrict sodium movements away from the axon surfaces relative to intracellular movements of the cation via the glial processes. The above system would be in accord with the observations that even local desheathing results in a loss of function in preparations maintained in sodium-free solutions, for, as already mentioned (Section 111) , this procedure results in an invasion of the glial system by relatively large peroxidase molecules via the damaged perineurium and the tight junctions between adjacent glial membranes (Lane and Treherne, 1969). Accordingly, it would be possible to postulate that the loss of extraaxonal regulation in desheathed preparations results from the disruption of normal glial function. The response of insect neurons to surgical damage of the preparation contrasts strikingly with the situation in molluscan nervous systems. As already emphasized, isolated neurons of Helix, with the associated glial coverings, and the split connectives of Anodonta are able to maintain action potential production for appreciable periods in sodium-free solutions as compared to the extremely rapid loss of axonal function encountered in insect nerves and ganglia that have suffered damage to the perineurium during the desheathing procedure. It seems possible that this difference might be a reflection of the differences in the extraneuronal sodium-regulating systems in the two invertebrate groups.

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Thus, as has been shown in Anodoiztd (Carlson and Treherne, 1969), the sodium utilized in maintaining action potentials in sodium-free nonelectrolyte solutions results from the utilization of a small sequestered sodium source in the region of the axon surfaces, probably in the form of an accessible extracellular fraction together with an intracellular fraction which cannot be readily displaced by organic cations such as tris or choline. In the insect nervous system, on the other hand, the extreme sensitivity of extraaxonal sodium regulation to glial damage suggests a more dynamic system perhaps, as suggested above, involving local recycling of sodium ions in the region of the axon surfaces so as to maintain axonal function in sodium-deficient solutions. An interesting feature of the cockroach central nervous system is the description of a difference in magnitude of the resting potential and overshoot of the action potential between desheathed and intact preparations (Pichon and Boistel, 1967). The amplitude of the action potential was found to be greater in intact preparations (103.0 t 5.4 mV) than in desheathed ones (85.9 2 4.6 mV) with an external bathing solution containing 2 10.2 mmoles/liter sodium. The effect appears to be associated with a positive potential, which in a subsequent paper was found to average as much as 28.2 mV (Pichon and Boistel, 1968), between the extracellular fluid and the Lathing solution or hemolymph, for potentials of this magnitude were recorded when the tip of the microelectrode was withdrawn into an apparently extracellular position following impalement of axons in intact preparations (Pichon and Boistel, 1967). This positive potential, recorded between the indifferent electrode and the recording electrode with the tip in an apparently extracellular position, cannot be wholly attributed to variations in tip potential obtained as a result of differing ion concentrations at the tip of the electrode (cf. Adrian, 1956). These results are somewhat difficult to interpret in view of the possible damage caused to the giant axons in the desheathing procedure (although it should be emphasized that isolated giant axons do maintain steady resting and action potentials for appreciable periods) or to other possible effects such as swelling of the axons in desheathed preparations. If it is assumed, however, that the intracellular concentrations of sodium and potassium remain unaffected by the desheathing procedure, then it seems clear that larger action potentials recorded in the intact preparations could result from higher concentrations of sodium ions in the fluid bathing the axonal surfaces in intact as compared with desheathed preparations. If it is further assumed that the concentration of sodium ions in the extraaxonal fluid in desheathed preparations is similar to that in the bathing medium, then it is possible to calculate the concentration of sodium in the fluid immediately surrounding the axons in intact preparations. In saline containing 210.2 mmoles/ liter sodium, the values for the resting and action potentials, 67.4 and 85.9 mV,

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respectively, give an overshoot of 18.5 mV in desheathed preparations. In the intact preparation, the resting and action potentials were 103.0 and 58.1 mV, respectively, with a positive extracellular potential of 6.7 mV. If the positive potential is added to the measured resting potential of the axon, these figures yield a value of 38.2 mV for the overshoot of the action potential in intact preparations. This would correspond to a difference of 19.7 mV between the measured overshoot in intact and desheathed preparations. From the data of Yamasaki and Narahashi (1959), it can be calculated that the active membrane potential of cockroach giant axons shows a 48.0 mV increase per 10-fold increase in external sodium concentration. A difference of 19.7 mV would thus correspond to a concentration of sodium ions at the axon surfaces which would be greater, by a factor of about 2.3, in intact as compared to desheathed preparations. With a bathing medium of 210.2 mmoles/liter sodium, this would correspond to a concentration of 483.0 mmoles/liter sodium in the fluid bathing the axon surfaces in intact preparations used by Pichon and Boistel (1967). In view of the possible secondary effects produced by the desheathing procedure, it would obviously be unwise to examine the calculated values obtained above too closely. These considerations do show, however, that there is the possibility that the extraaxonal fluid niay well have a socliuiii concentration veiy much higher than that of the hemolymph or bathing medium. It is of some interest to consider the above in relation to the suggested mechanism involving a regulation of the extraaxonal sodium level by the activity of the glial cells (Treherne and Maddrell, 1967b; Treherne, 1967). The polarity of the extracellular potential described by Pichon and Boistel (1967, 1968) is the opposite of that which would be expected if it resulted from the outward diffusion of cations maintained by Donnan forces in the extracellular fluid. A positive extracellular potential would be obtained, however, from the presence of an electrogenic sodium pump extruding the cations into the extracellular system in the vicinity of the axonal surfaces. If such an electrogenic sodium pumping system involved glial cells, and perhaps also the perineurium, then the loss of the extracellular positive potential (which, as we have seen, is also correlated with the loss of ability of the axons to function in sodium-deficient media) could be explained by changes in the glial system produced by the desheathing procedure. The fact that the glial cytoplasm in desheathed preparations becomes accessible to molecules as large as those of peroxidase (Lane and Treherne, 1969) clearly indicates that there must be a rapid leakage of small water-soluble ions and molecules from within the glial cells which would produce a dramatic interference with normal glial function. It is possible, therefore, to relate the electrophysiological findings of Treherne and Maddrell (1967b) and of Pichon and Boistel (1967, 196s) to the ultrastructural evi-

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dence (cf. Smith and Treherne, 1963; Treherne and Maddrell, 1967b) and to recent findings concerning the accessibility of insect central nervous tissues to large extracellular indicator molecules (Lane and Treherne, 1969) .

V. General Conclusions It is apparent from the preceding discussion that there is considerable variation in the degree of regulation of the extraneuronal environment as far as inorganic ions are concerned. In the leech central nervous system, for example, movements of inorganic ions take place rapidly through the narrow intercellular channels between the bathing medium and the extraaxonal fluid, and the glial cells appear to play very little part in regulating the immediate ionic environment of the neurons. An essentially similar state of affairs exists in the cephalopod giant axons. In some molluscan and insect species, however, there is clear evidence of regulation of the content of inorganic ions in the extraneuronal fluid. This generalization does not imply that all neurons in the nervous systems exist in a regulated environment. In the freshwater lamellibranch A . cygzea, for example, the majority of the axons in the cerebrovisceral connective respond to changes in the ionic composition of the bathing medium in much the same way as has been described for the leech neurons, and only relatively few large axons show evidence of functioning in a controlled ionic environment. Despite the extremely specialized nature of the blood and the demonstrated ability of some molluscan and insect neurons to function for appreciable periods in preparations bathed in sodium-free solutions, it appears, nevertheless, that the ionic basis of action potential production is conventional in that the inward current is largely carried by sodium ions. There also appears to be a ready access of inorganic ions from the bathing medium to the neuronal surfaces in at least one such molluscan species. In the case of a gastropod and a lamellibranch species it seems likely that the sodium utilized by these neurons is sequestered in the region of the neuronal surfaces and is released into the extraneuronal fluid, thus sustaining neuronal function in preparations bathed in sodiumdeficient media. The available evidence indicates that this extraneuronal sodium store, which has been shown to be depleted by stimulation of the neurons in sodium-free conditions, may exist in two fractions : an extracellular fraction, one perhaps associated with indiffusible anion molecules, which is accessible to and can be displaced by small organic cations, and an intracellular fraction, most probably situated in glial elements, which cannot be easily displaced by small organic cations but which can be mobilized to maintain an appropriate sodium level in the fluid immediately surrounding the neurons. Extraneuronal sodium regulation in insect species appears to be achieved by

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physiological mechanisms somewhat different from those in the molluscan nervous systems that have been investigated. In particular, the ability of axons to function in sodium-deficient media is dependent upon the integrity of the glial system. Loss of the ability to regulate the sodium concentration in the fluid immediately surrounding the axons, observed in desheathed preparations, does not appear to result, as was originally supposed, from the disruption of a peripheral diffusion barrier associated with the nerve sheath, but from effects on the underlying glial system. Thus, even relatively large molecules can move from the region of the damaged perineurium in desheathed preparations into the glial processes surrounding axons within the neuropile by means of transversely permeable tight junctions between adjacent glial membranes. It is envisaged, therefore, on the basis of the currently available evidence, that there is an active extrusion of sodium ions from the glial cytoplasm into the restricted 150-200 A extracellular channels adjacent to the axon surfaces. It is also suggested that the positive potential shown to exist between the extracellular fluid and the bathing medium could result from the activity of an electrogenic sodium pump situated in the glial membranes. The system of fixed-anion groups, which may be associated with the extracellular acid mucopolysaccharide of insect nerves and ganglia, is unlikely to produce sodium of higher activity in the region of the axon surfaces relative to that in the blood or bathing medium. The possibility cannot be eliminated, however, that the sodium associated with such fixed-anion groups could form part of a cation reservoir which might be utilized if there were a transitory drop in the sodium activity of the extraaxonal fluid or if local circuit current flow altered the configuration of the anion groups.

ACKNOWLEDGMENTS Me are grateful to Drs. B. L. Gupta and N. J. Lane for reading and commenting on some portions of the manuscript for this article and for their help, and that of Dr. R. L. Tapp (Department of Physiology, Cambridge) in preparing some of the electron tnicrographs.

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Virus Uptake, Cell Wall Regeneration, and Virus Multiplication in Isolated Plant Protoplasts E. C. COCKING Department of Botany, University o/ Nottinghnnz, Notzingham, England

I. Introduction: The Isolated Protoplast System . . . . . . . . . . . 11. Uptake of Viruses by Isolated Protoplasts . . . . . . . . . . . . . . A. Entry of Macromolecules into Plant Cells: Evidence for Pinocytosis in Isolated Protoplasts . . . . . . . . . . . . . B. Virus Uptake and the Initiation of Infection . . . . . . . III. Cell Wall Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Virus Multiplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

89 92 92 100 10s 115 122

I. Introduction : The Isolated Protoplast System One of the distinguishing characteristics of most higher plant cells is that they possess a rigid cell wall. The phenomenon of plasmolysis in which it is possible to observe the protoplast of the cell drawing away from the cell wall is dependent in part on the presence of this rigid wall. Yotsuyanagi (1953) observed that plasmolysis of the cells of an Elodea leaf in a solution of CaC1, or Ca(N03) produced protoplasts within the cells which often survived for more than 50 days. He also observed (Fig. 1) small spherical fragments of cytoplasm which had become isolated spontaneously from the protoplast during this prolonged plasmolysis. Similar small spherical fragments of cytoplasm (balls of protoplasm) were produced in the microdissection studies of Plowe (1931) on the cells of the epidermis of bulb scales of Bermuda onions. Strips of epidermis were plasmolyzed in 18% (0.56 M) sucrose for about 2 0 minutes; during this time the protoplasts were reduced to about half their original volume and were well rounded away from the end walls. A strip was then cut with a sharp razor transversely to the long axis of the leaf, and the blade passed between the end walls and protoplasts of many cells, leaving the protoplasts untouched and uninjured. This mechanical method of isolating protoplasts was originally described by Klercker in 1892. Plowe noted that when the solution in which plasmolyzed, sectioned material was mounted was diluted, the protoplasts moved toward the open end of the outer cells as they swelled until they partially protruded or were even set free into the surrounding medium. Frequently, she was able to pinch these protruding protoplasts in two using a needle, and initially a thin strand of plasmalemma connected the two portions (Fig. 2 ) . She noted that one portion was enucleate, yet streaming in it con89

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FIG. I. Cell of Elodea plasmolyzed for a long time in a solution of CaCI, or Ca(N03)2. Small spherical fragments of cytoplasm (subprotoplasts) are present as well as the large subprotoplast (Yotsuyanagi, 1953).

tinued in exactly the same manner as in protoplasts containing a nucleus even after the connecting thread was broken. The strand or connecting thread is apparently self-sealing. Each of the portions of the original protoplast was surrounded by part of the plasmalemma that covered the protoplast initially, and the plasmalemma was unbroken. Somewhat similar division of the protoplast on plasmolysis, particularly in the presence of Ca++ ions, has been observed by Yoshida (1962), Kamiya (1959), and Stadelmann (1956). These portions of the protopIast have been named subprotoplasts by Cocking (1963). Mechanical methods for the isolation of plant protoplasts, although providing suitable material for investigating, for instance, the iiifluence of environmental factors on the osmotic behavior of isolated protoplasts (Vreugdenhil, 1957), have always been limited by the small number of protoplasts that can be

FIG.2. A protoplast protruding from the cut end of a cell is seen dividing into two. The needle (arrow) is pulling one of these subprotoplasts away from the other, but at this stage the two subprotoplasts are still connected by a thin strand of plasinalemina (Plowe, 1931).

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readily isolated. The introduction of a method by Cocking (1960) for the isolation of protoplasts which involved the enzymic degradation of the cell wall by Myrothecium verrucaria cellulase has permitted far greater numbers of protoplasts to be readily isolated from a wide range of different tissues and their

FIG.3. Isolated tobacco leaf protoplasts together with isolated vacuoles (Power and Cocking, unpublished observations).

properties investigated. The use of cellulases for the isolation of root, cotyledon, and fruit protoplasts has been described (Cocking, 1960, 1961a; Gregory and Cocking, 1963) , and Ruesink and Thimann (1965) have also reported the use of M. verrucaria cellulase for the isolation of protoplasts from Avena coleoptiles and from a wide range of higher plant tissues including tissue culture cells (Ruesink and Thimann, 1966). The large-scale isolation of protoplasts from immature tomato fruit using commercially available pectinase to

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degrade the highly pectinaceous cell walls of tomato locule tissue has also been described (Gregory and Cocking, 1965). Recently, Power and Cocking (1968) have reported a simple method for the isolation of very large numbers of leaf protoplasts involving the use of mixtures of commercially available Trichoderma viride cellulase and pectinase to degrade the cell walls, chiefly those of the mesophyll cells of the leaf. Isolated vacuoles are often produced as well, and a typical preparation of such isolated leaf protoplasts and vacuoles is shown in Fig. 3. The ready availability of these enzymically isolated protoplasts has greatly stimulated work particularly in relation to studies on the entry of macroniolecules into protoplasts (see Section II), on cell wall regeneration (see Section HI), and on the ability of such regenerated isolated protoplasts to support the multiplication of plant viruses (Section IV) . Although it will not be discussed further in this review the ability of isolated protoplasts to fuse (Michel, 1937), which has recently been more fully investigated by Binding (1 9 6 6 ) , should be noted. This work was carried out using mechanically isolated protoplasts, and it could well be that far more extensive fusion is possible with the greater range of isolated protoplast types now available. To some extent, the use of enzymes to liberate protoplasts from cells by digestion of the cell wall can be regarded as one of the simplest forms of cell fractionation and, indeed, pectinase and cellulase have been used in more extensive degradation procedures on leaf tissue to obtain readily large quantities of nuclei fractions uncontaminated with other organelles (D’Alessio and Trim, 1968). 11. Uptake of Viruses by Isolated Protoplasts A. ENTRYOF MACROMOLECULES INTO PLANTCELLS:EVIDENCE FOR PINOCYTOSIS IN ISOLATED PROTOPLASTS Several workers, including Whaley et al. ( 1 9 6 4 ) , have commented on the numerous bays and infoldings in the plasmalemma of plant cells, observable by electron microscopy, and have noted that many of these membrane irregularities are to be seen in cells in which there is no conspicuous secretion of a Golgi product. These workers have concluded that it seems quite likely that a process similar to pinocytosis may occur in plant cells despite the presence of the cellulosic wall. Other workers, particularly Bradfute et al. ( 1 9 6 4 ) , have observed that morphological evidence suggestive of vacuole formation by the cell membrane may be found as a result of phenomena other than pinocytosis. The liquid endosperm of Piszm sativum was employed in these studies so that the presence of a rigid highly impermeable cell wall was not a complicating factor in following the uptake of fluorescent-labeled basic proteins by phase and fluorescent microscopy. The isolated protoplast system is one in which marker mole-

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cules can be presented directly at the surface of the plasmalemma to a relatively uniform population of protoplasts which can be readily handled for electron microscope observations, whether by thin-sectioning or by freeze-etching, or for biochemical assays. Since the cell wall is absent, it is not a complicating factor and it is possible to devise experiments to follow readily any pinocytic uptake of ferritin, viruses, or suitably labeled material. The extent, however, to which pinocytosis detected in isolated protoplasts is comparable to that in the cell itself is more difficult to ascertain (Clowes and Junniper, 1968). Isolated protoplasts of higher plants, because of their special osmotic relationships, have to be kept in a suitable plasmolyticum. Usually this is 20% sucrose together with small amounts of various salts and special nutrients. From work with amoebas and mammalian cells, it is known that pinocytosis is induced by a wide range of different substances and inhibited by others (ChapmanAndresen, 1964), and it could well be that on occasion the extent of pinocytosis in isolated protoplasts is greatly in excess of that in the cell itself. The real difficulty in comparing these two systems is that, as previously mentioned, the cell wall acts as a very efficient ultrafilter largely preventing the penetration through it of ferritin (Barton, 1964) or of certain viruses (Cocking and Pojnar, 1969) and, as a result, it is impossible to use marker molecules with cells as it is with protoplasts. It seems likely that all modes of vesicle formation by the plasmalemma may have a basic course. Threadgold (1967) has provided a very lucid account of the various processes involved in this vesicle formation as far as animal cells are concerned. Phagocytosis i s the ingestion of large particles such as bacteria and results in the formation of relatively large vacuoles (Fig. 4 ) . Pinocytosis proper is also regarded as involving the formation of active fringes by the cell surface (plasma membrane), but these occasionally fall back into the cell (Fig. 4). In micropinocytosis no pseudopodia or veils of cytoplasm are formed; in this instance there is invagination of the plasma membrane. Rhopheocytosis involves attachment of macromolecules to the plasma membrane as an essential sequel before uptake takes place (Fig. 5 ) . Cytopemphis involves the formation of vesicles as in rhopheocytosis but these vesicles are then discharged into a main control vacuole system (Fig. 5 ) . As Threadgold has pointed out, pinocytosis in animal cells has come to be used as a term to describe the formation of any small vacuole formed by invagination of the cell surface regardless of whether or not prior attachment to the plasma membrane takes place. Holter (1959) has repeatedly stressed that there seems little effectual difference between pinocytosis and phagocytosis since the main diff ereiice does not appear to be the mechanism of the process but more the dimensions of the vesicles formed and the nature of the material being taken up. Moreover, in relation to pinocytosis itself, Holter (1963) has pointed out that pinocytosis by invagi-

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nation is fairly variable with regard to the shape of the cavities formed and also extremely variable with regard to dimensions. In animal cells, pinocytosis and phagocytosis has also frequently been directly implicated in intracellular digestive processes. Gordon et al. (1965) were able a

C

1

0 t

uf

( a ) Serial drawings illustrating phagocytosis. The opposing evaginated pseudopodia eventually fuse and trap the particle. ( b ) Pinocytosis. (c) Micropinocytosis (Threadgold, 1967).

FIG. 4 .

to show in electron microscope studies the phagocytic uptake of DNA-protein coacervates containing colloidal gold by strain L fibroblasts. From studies of the progressive morphological alterations of these phagocytized gold-inarked coacervates, these workers were able to formulate the structural pathways involved in their degradation. Various forms of lysosomes were found to be in-

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volved, and a schematic scheme of the proposed structural pathways of intracellular digestion in these strain L cells is shown in Fig. 6. The origin and development of a possible lysosomal apparatus in higher plants has been investigated by Matile and Moor (1968) using the freezeetching technique in rootlets of corn. Vacuolation was seen to take place by four main processes (a diagrammatic representation of these processes is shown in Fig. 7 ) : first, formation of provacuoles derived from the endoplasmic reticulum ( 1 ) ; second, fusion of vacuoles ( 2 ) and the expansion of the vacuolar a A

I 1

FIG. 5 .

-

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( a ) The process of rhopheocytosis. (b) Cytopemphis (Threadgold, 1967).

volume; third, invagination of the tonoplast ( 3 ) ; and fourth, encapsulation of the larger Golgi-derived vesicles by invaginations of the tonoplast ( 4 ) . These workers do not comment on possible involvement of the plasmalemma in vesicle or vacuole formation but clearly, as is shown in Fig. 7 , the possibility also exists for vacuole formation by pinocytic vesicle formation. Direct evidence for pinocytosis in isolated protoplasts was provided from studies in which isolated fruit protoplasts were incubated in 5% ("/v) ferritin for several hours prior to fixation and embedding for electron microscope studies. The pH of the incubation mixtures was maintained at 4.5, since in comparable studies in amoebas Nachimias and Marshall (1961) observed that

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(Phagacytotic vacuole)

1

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~rotolysosomes (Golgi ~ e s i t l e s l

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Telolysosome (Dense body1

FIG. 6. Schematic representation of proposed structural pathways of intracellular digestion in strain L cells (Gordon et al., 1965).

the initial bending of ferritin to the cell surface was optimal at a pH near the isoelectric point of ferritin ( 4 . 4 ) . Accumulation of ferritin particles was observed in the cytoplasmic vesicles of these protoplasts. No initial binding of ferritiii to the plasinalemina was, however, observed. It was considered that pinocytosis was taking place in these isolated fruit protoplasts, thereby enabling ferritin molecules to be accumulated in pinocytic vesicles in the cytoplasm. In

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this electron microscope study, particular care was taken to ensure that the finding of ferritin in vesicles in the cytoplasm was not a fixation artifact (Cocking, 1966a). The experimental advantages of this isolated protoplast system for studying pinocytosis were obvious, yet largely because the initial events of this pinocytic uptake remained obscure and also because the relevance of these ob-

FIG. 7. Diagrammatic representation of processes contributing to the formation of vacuoles. For detailed discussion of 1-4 see the text plasmalemma (PL), cell wall (CW), endoplasmic reticulum ( E R ) , dictyosome (D) , dictyosome vesicle (DV) , tonoplast (T) , vescicular body (VB) (Matile and Moor, 1968).

servations on pinocytosis to pinocytosis in plant cells under natural conditions was unclear, botanists were slow to make use of this system. It was stressed (McLaren and Bradfute, 1966) that pinocytosis was a dynamic process and not a static end result and that no one had at that time visually observed the dynamic events with plant systems. Neither ferritin particles nor most viruses, because of their small size, are ideally suited to follow the early stages of pinocytosis in isolated protoplasts. Furthermore, it also seems likely that early stages may differ as between different protoplast systems. The nature of the particles being taken up is also of importance. The ready availability of polystyrene latex particles in high concentrations and in a range of particle sizes suggested their general applicability in these investigations. After incubation, latex particles were readily detected attached to the plasmalemma of protoplasts. Uptake proper was observed to occur in localized regions of the plasmalemma. In these

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regions, several stages of pinocytic uptake were evident and vesicles containing one to several latex particles were seen detached from the plasmalemma within the cytoplasm (Fig. 8) (Mayo and Cocking, 196%). The latex particles employed were usually 0.088 p in diameter, but it was also observed that particles of up to 0.25 p diameter could also be taken into the cytoplasm by pinocytosis. Electron microscope studies were made difficult by the fact that the particles were soluble in the usual embedding media but the use of hydroxypropyl niethacrylate as an embedding medium largely overcame these difficulties. It was observed that the uptake vesicles containing the latex particles were small and in the range 0.1-0.3 p in diameter. The diameter of vesicles of the pinocytic vesicles observed in the cytoplasm of protoplasts after incubation in ferritin or tobacco mosaic virus (TMV) (Cocking, 1966a,b) were, however, much more varied in diameter and often greatly in excess of 0.3 p. Hirsch et al. (1969) have noted that in the case of pinocytic vesicles formed in macrophages from tiny invaginations of the surface membrane these vesicles commonly fuse with one another to form vacuolar structures 0.5-1.0 p in diameter. There thus exists the possibility that during ferritin and virus uptake by fruit protoplasts pinocytic vesicles coalesce, either with themselves or with existing vesicles, shortly after entering the cytoplasm, to form larger vesicles; but the possibility cannot be eliminated that the range of initial size of pinocytic vesicles during ferritin and virus uptake is very much more varied than when latex particles are being taken LIP. The use of phosphotungstic acid (which occasionally stains only regions of the membranes surrounding larger cytoplasmic vesicles) as a selective stain for pinocytic activity (Mayo and Cocking, 1969b) suggests that sometimes coalescence of pinocytic vesicles with existing cytoplasmic vesicles does take place in these protoplasts. Although, as previously mentioned, uptake of ferritin by isolated fruit protoplasts does not appear to involve any binding of ferritin to the plasmalemma, a binding of ferritin to the plasmalemma of isolated leaf protoplasts (Power and Cocking, 1968) has, however, been observed preliminary to pinocytic uptake. Clearly, differences are to be expected in the detailed mechanism of pinocytic uptake depending on the nature of the protoplast surface and the nature of the materials being ingested. What is evident is that the FIG. 8. ( a ) Section of a protoplast following incubation in 2% latex particles for i hours. Material was fixed in glutaraldehyde, postfixed in osmium tetroxide, stained with uranyl acetate during dehydration, and embedded in 95% hydroxypropyl methacrylate. Sections were poststained with 5% phosphotungstic acid for 1 hour. The membranes and latex particles appear in negative contrast. Particles are attached to the plasmalemma and indented into the membrane (Mayo and Cocking, 196%). ( b ) Outer region of a protoplast incubated in 2% latex for 3 hours. Fixation and embedding was as in ( a ) . Sections were poststained with lead citrate. A region of active pinocytosis is shown containing several stages of engulfment of latex particles (arrows) (Mayo and Cocking, 1969a).

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plasmalemma, in relation to pinocytic activity, is not very discriminating with respect to the material being taken in. It is the subsequent fate of pinocytosed material that is of the greatest interest in relation to the overall physiological behavior of the protoplast or cell. Discrimination appears to occur within the pinocytic vesicle. B. VIRUSUPTAKEAND

THE

INITIATION OF INFECTION

Before attempting to survey the available conclusions regarding virus uptake and the initiation of virus infection in the isolated protoplast system, it will be particularly instructive to review briefly the situation in bacteria and in animal cells. Bawden (1964) has called for caution in extrapolating our knowledge of bacteriophages to considerations of plant viruses and plant cell interactions, especially since bacteriophages such as T2 organize their own transmission and spread unaided, whereas plant viruses depend on other organisms to transmit them. Nevertheless, Best (1965) provided strong evidence in favor of the view that in plant virus infections the protein of the virus becomes adsorbed to the cell wall and ejects its RNA which then enters the cell alone and mediates the synthesis of more virus. A diagrammatic representation of Best’s ideas on the sequence of events in the biosynthesis of an RNA virus is shown it1 Fig. 9. Work on isolated plant protoplasts and the detection of pinocytic activity at the plasmalemma in these protoplasts, particularly the pinocytic uptake of virus intdct, has now focused attention on comparisons with the animal cell system in which, in many instances, it has been shown that viruses are taken up intact, become uncoated, and subsequently initiate infection. It appears that in many respects the isolated plant protoplast is similar to the animal tissue culture cell. The forces responsible for holding virus particles on the surface menibrane of animal cells are considered to be electrostatic, and it has been suggested that strongly acidic phosphate groups of the cell surface interact with amino groups in the virus. It is generally held that most animal viruses are taken into cells by pinocytosis after specific interaction of the virus with receptor sites at the cell surface (Dales, 1965). It is interesting to recall that following the demonstration by Hershey and Chase (1952) in which only the infectious component of the T2 bacteriophage was injected into the host, the idea held, in the absence of any direct evidence, that the viral nucleic acid was first released by rupture of particles at the surface followed by direct penetration through the surface membrane into the host cell. The process envisaged was comparable to that represented diagrammatically by Best for the initiation of infection of plant cells (see Fig. 9 ) . As critically discussed by Dales (1965), however, further experiments have indicated that the converse viewpoint, namely that infection occurs after the cell has engulfed the intact virus, is probably the correct one, Vaccinia has been shown to be phagocytized by L cells and fowl pox by chorio-

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allantoic membrane cells. Myxoviruses have also been shown to be taken up into animal cells by phagocytosis, as have adenoviruses, herpesviruses, and reoviruses. The evidence for this comes mainly from electron microscopy, but more recently biochemical techniques applied to the study of viral entry have served to confirm these observations and to indicate that following uptake the virus

R @

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2

Viral protein

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Viral RNA (parent)

Viral RNA (cornplernentary 1 q T r a n s f e r RNA Host chromosomes C 3 Viral RNA replicase N = Nucleus R = Ribosome n = Nucleolus

5

FIG. 9. Diagrammatic representation of the sequence of events in the hiosynthesis of an RNA virus (Best, 1965).

particles become decoated. Thus, Silverstein and Dales (1968) were able to show in a combined electron microscope radioautographic and histochemical approach that reovirus type 3 was phagocytized by L cells and rapidly sequestered inside lysosomes. These workers also showed that hydrolases within these organelles were capable of stripping the viral coat proteins but failed to degrade the double-stranded RNA genome, and Silverstein and Dales concluded that “sojourn of reovirus in lysosomes, when the lytic enzymes uncoat its genome, is an obligatory step in the sequence of infection.” Reovirus labeled in its RNA was obtained by infecting cells and allowing the virus to multiply in a medium containing cytidine3H and uridine-3H (Dales and Gomatos, 1965). Since, as we shall see later, plant tissue culture cell studies are not as yet sufficiently

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developed to enable plant viruses to be labeled in a comparable fashion, such studies have not been carried out using these viruses. Nevertheless, Mayo and Cocking (1968) developed a simple and rapid in vitm labeling technique for preparing T M V labeled with 1251. Only the protein of the virus particle is, however, labeled and although very high specific activity virus can be obtained by this in vitro technique the application of such labeled virus in following the uptake and development of plant viruses is necessarily limited. In the future, infection studies using plant protoplasts should clearly allow plant viruses of high specific activity, labeled in their nucleic acid moiety, to be readily obtained (see Section IV). It has recently been suggested, exclusively on electron microscope evidence, that herpes simplex virus and influenza virus can both become uncoated at the cell surface very rapidly, with rupture of the viral core permitting release of nucleoprotein directly into the cytoplasm (Morgan et d.,1968; Morgan and Rose, 1968). As emphasized by Watson (1968), however, “electron microscopy, by itself, disassociated from allied or complementary techniques, can raise new questions, but it cannot provide the answers.” For the present we can still conclude, as did Dales in 1965, that the preponderance of available evidence favors the conclusion that upon attachment to host cells, which may involve specific membrane receptors, animal viruses are transferred intact, with their genomes still protected, into phagocytic vacuoles by an active and unspecific engulfment. In animal cells, this engulfment is as mentioned quite unspecific. Particles of ferritin and colloidal gold, polystyrene latex spheres, high-molecular-weight D N A and DNA-protein coacervates, as well as viruses, are all capable of being engulfed (Dales, 1965). As far as has been investigated, isolated fruit and leaf protoplasts behave comparably (see Section 11, A ) but, as we have seen, the detailed mechanism of this engulfment seenis to vary depending on the nature and size of the material being taken up. Great interest, therefore, centers on the subsequent fate of engulfed, or pinocytosed material in both the animal cell and in the isolated protoplast system. In both instances, lysosomal enzymes may be involved. Intracellular digestion of phagocytized material is known to occur frequently in animal cells, and the observation that heat-denatured vaccinia is degraded within vacuoles is probably another example of intracellular digestion. In the sense that the internal milieu of a pinocytic vesicle differs from that existing on the cell surface, it has frequently been suggested that virus inside vacuoles is no longer extracellular. The release of the infectious genome of the virus particle that occurs in such a vesicle has been termed the “uncoating process.” This uncoating process involves the activities of the host cell as well as the specific nature of the virus coats. Lysosomal enzymes may, in animal cells, participate in the uncoating process of virus nucleic acid after the intact particle has entered the cell (Allison and

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Sandelin, 1963) . Novikoff ( 1963) has demonstrated an accumulation of lysosomes near pinocytic vesicles. The rate at which this uncoating process takes place varies markedly. In the case of Newcastle disease virus and pox virus, the viral nucleic acid is rapidly released into the cytoplasm as a result of prompt lysis of both the outer viral envelope and the pinocytic (phagocytic) vesicle (Dales, 1963; Silverstein and Marcus, 1964). As previously mentioned in the case of reovirus in L cells, Silverstein and Dales (1968) observed that a sojourn of the virus in lysosomes was an obligatory step in the sequence of infection. When viral nucleic acid itself is used to infect cells, clearly no uncoating process is involved. It is, however, far more difficult and usually impossible to follow by the usual electron microscope methods entry of viral nucleic acid into animal cells or isolated plant protoplasts, since in thin-section studies the free nucleic acid of the virus is not readily detected. Cocking and Pojnar (1968a) and Mayo and Cocking (1969a) have suggested that by selective staining of TMV with uranyl acetate or phosphotungstic acid it may be possible to detect stages in the uncoating process before the nucleic acid of the virus is released (see Section I V ) . A large number of electron microscope and cytochemical studies have demonstrated that protein molecules can be taken up by tumor cells in suspension by pinocytosis. One of the difficulties in investigatiiig the effects of viral nudeic acid is that free viral RNA, particularly single-strandcd, and D N A are quite readily degraded by nucleases present in incubation mixtures. It has been demonstrated, for instance, that infectious RNA, obtained from encephalomyocarditis virus is very rapidly degraded on contact with Krebs mouse ascites tumor cells. It has also been demonstrated that nuclease inhibitors such as bentonite, DEAE-dextran and polyamino acids augment the detectable infectivity of various RNA preparations; recently, Stonehill and Huppert (1 968) have detected an endonuclease associated with cell wall preparations obtained from Krebs ascites cells. These workers suggest that mammalian cell walls may be a source of nuclease activity and may contribute to the inactivation of infectious RNA molecules. It has also been observed that short exposure of cell cultures to hypertonic media enhances the biological effects of nucleic acids on animal cells. Ryser (1967) has suggested that maximum enhancement of RNA infection may be the result of a form of severe, but reversible, cell damage. He points out that cell damage caused by hypertonicity and other conditions may modify the usual digestion process of lysosomal enzymes creating new paths of uptake, the cytopathic uptake vacuoles. These large cytopathic uptake vacuoles could account for an increased or an abnormal penetration of nucleic acids into cells exposed for a short time to adverse conditions. Ledoux (1965) has critically reviewed the phenomenon of uptake of DNA by living cells and has concluded that the physicochemical state of the DNA

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preparations used is a variable of paramount importance; more recently, Olenov (1968) has stressed the importance of the physiological state of recipient cells if transformation phenomena are to be observed in somatic cells. Rogers and Pfuderer (1968) added nucleotide sequences (poly A) to TMV RNA and were able to induce polylysine formation in tobacco plants. It was shown that the poly A must be attached to the virus RNA to be effective. This is an instance of a virus (or more precisely its nucleic acid) being modified to transmit added genetic information. The demonstration by Kovics and Bucz (1967) of the isolation of complete virus from yeast and Tetrahymena experimentally infected with picorna viral particles or their infectious RNA serves also to demonstrate the universality of the RNA code. It raises the possibility of the infection of plant cells by animal viruses. As we shall see later, the isolated plant protoplast system may be very well adapted for experimental work along these lines. In this connection, it is of interest that Sander (1964), working with tobacco leaves, claimed that phage could multiply in the leaves provided they were inoculated with the free nucleic acid of the phage. As discussed by Kassanis (1967), plant tissue cultures cannot be infected without wounding the cells, and this factor, together with the rather low virus concentrations usually present in the infected cells, has resulted in the usefulness of tissue cultures to plant virus research being rather limited. An approach to the use of plant cells in culture that could largely overcome difficulties was suggested from the initial studies of Cocking (1966b) using isolated protoplasts that had been incubated with TMV. No cell wall was present around these isolated fruit protoplasts so that one of the principal barriers to the entry of viruses had been removed. The suspension of protoplasts was incubated with a 1% suspension of TMV for varying periods of time up to 7 hours. Protoplasts were fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in butyl methacrylate, and sections were poststained with lead citrate. These protoplasts showed in thin section (Fig. 10) some deep invaginatiom of the plasmalemma where virus particles appeared to be present, some apparently attached to the plasmalemma. After a few hours, virus particles were detected in vesicles in the cytoplasm, some attached to the periphery of the vesicles, others apparently lying free. It was suggested that the virus entered the protoplast by pinocytosis so that vesicles containing virus in the cytoplasm were pinocytic vesicles. As noted earlier (Section 11, A ) , further studies have indicated a general pinocytic activity in these isolated protoplasts which is not restricted to the uptake of virus particles. As correctly pointed out by Esau (1967), the plasmalemma is not clearly visible in this early work in which methacrylates were employed as embedding media; but there is no difficulty in seeing the plasmalemma when better embedding media are used (see Fig. 8). The use of a high concentration of virus facilitates the detection of pinocytic uptake. At lower virus suspension

FIG. 10. ( a ) Outer region of isolated tomato fruit protoplast incubated in a suspension of TMV. A deep invagination of the plasmalemma (arrow) is visible. ( b ) Higher magnification of deep invagination. Thin particles of TMV are clearly visible and pinocytic activity is also evident (arrows) (Cocking, unpublished observations),

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concentrations (0.1 “/o), fewer virus particles are detected (Fig. 11A) in pinocytic vesicles in the cytoplasm, but when detected the characteristic vesicle with virus particles is present (Fig. 11B) . These findings led Cocking (1966b) to suggest that uptake of virus particles intact by pinocytosis might be an initial stage in the infection of these isolated fruit protoplasts by TMV, just as we have seen is the situation in most instances in the infection of animal cells by viruses. It is of interest that a year earlier Mundry (1965) had concluded that although the mechanism of virus uptake by plant cells was still obscure it was not inconipatible with the hypothesis of virus uptake into plant cells by pinocytosis. One of the difficulties in this work was that at that time protoplasts could only be isolated readily in large quantities from the locule tissue of tomato fruit using a commercially available pectinase (Gregory and Cocking, 1965). Little was known about the rate or extent of infection of the locule tissue of tomato fruit during the systemic infection of tomato plants by TMV. Cocking and Pojnar (1968b) showed, however, that the rate of multiplication of TMV in fruit tissues was comparable to that in the leaves and that there were comparable levels of infection in the various tissues of the fruit. More recently, Power and Cocking (1968) were able to obtain from tobacco leaves very large numbers of isolated protoplasts by using a mixture of commercially available cellulase (from Trichodermu viride) and pectinase so that comparable studies are now possible using isolated leaf protoplasts. Since intact particles of TMV were detected within pinocytic vesicles, it seems likely that infecting virus particles were being taken up intact prior to being uncoated, releasing their nucleic acid and initiating infection. However, in the early studies of Cocking (1966a) protoplasts were incubated continuously in very high concentrations of TMV. Moreover, after about 9 hours the highly vacuolated fruit protoplasts began to burst progressively with time. As a result, a detailed study of the fate of pinocytosed virus was impossible in this early work. It was also evident that high levels of ribonuclease were present in the incubation mixtures (Cocking and Pojnar, 1969). As we shall see later (Section III), it was only with the development of incubation conditions that enabled isolated protoplasts to regenerate a new cell wall and the incubation (“inoculation”) of protoplasts with virus for relatively short periods of time under coilditions of low ribonuclease activity prior to wall regeneration, that it became possible to begin to follow the fate of pinocytosed virus and the establishment of infection. Isolated leaf protoplasts are considerably less highly vacuolated than isolated fruit protoplasts and are inherently more stable, and it may prove possible in the future to follow pinocytic uptake of virus, initiation of infection, and virus multiplication while these are still protoplasts.

FIG. 11. ( a ) Region of isolated tomato fruit protoplast incubated in a suspension of TMV (0.1%) for 6 hours. Note virus in pinocytic vesicle (arrow). ( b ) Higher magnification of pinocytic vesicle. The particles of TMV are clearly visible (Cocking and Pojnar, unpublished observations).

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111. Cell W a l l Regeneration Following this work on the uptake of viruses by isolated protoplasts, the idea arose of allowing the virus to enter the protoplast by pinocytosis and then “triggering off” cell wall regeneration so that the isolated protoplast was converted into a cell containing the virus, which could then be cultured under conditions in which multiplication of the virus would result. It seemed likely from the early work of Townsend (1897) that enzymically isolated protoplasts and sub-

FIG. 12. Drawing of plasmolyzed cell of G. Lnceolata. Only the nucleate subprotoplast within the leaf cell regenerates a new cell wall (redrawn from Townsend, 1897).

protoplasts, provided that they were nucleate, would regenerate a new cell wall. Townsend showed that in cells of Elodeu cunadensis and Gaillardiu lanceolata that had been plasmolyzed the plasmolyzed protoplast, or the nucleate subprotoplast within the leaf cell, regenerated a new wall when maintained in 2070 sucrose (Fig. 1 2 ) . H e also showed that “free pieces of protoplasts” (subprotoplasts in the terminology of Cocking, 1,963) isolated by the mechanical method of Klercker (1892) from protonema of mosses, prothecia of ferns, hairs of stems, and leaves of higher plants could be kept alive for days, and even weeks, during which time they built distinct cell walls; pieces without nuclei (enucleate subprotoplasts) never made cell walls (Fig. 13). More recently, Binding (1966) has reported the regeneration of protoplasts isolated by the method of Klercker (1892) froin the musci Fzmaria hygrometrica, Physcomifrizm ezoystom,zlm, and Pipiforme and Brynm erythrocarpum. Here again, wall formation was only carried out by nucleate protoplasts and most of these isolated protoplasts formed a rigid wall after at least 13 days. Some of these protoplasts (plaslocytes) germinated to protonemata. Yoshida (1961), using leaf cells of Elodea densa, investigated the role of the nucleus in some activities of protoplasm by comparing the behavior of nucleated and enucleated halves of protoplasts which he separated by means of plasmolytic treatment of intact cells. H e observed that the membrane that was reformed on the surfaces of divided protoplasmic halves (subprotoplasts) was more solidified and less elastic in the nucleated halves than in the enucleated halves. H e also noted that by treatment with ribonuclease the properties of nucleated protoplasmic halves could be rendered similar to those of the enucleated halves.

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Up to 1967, although the general properties of enzymically isolated protoplasts had been extensively investigated by Cocking (1961b) and by Ruesink and Thimann (1965, 1966), there was little or no evidence of cell wall regeneration. In 1967, Pojnar et ul. reported that isolated fruit protoplasts, when well washed to remove contaminating enzymes and incubated in suitable media, ra-

FIG. 13. Isolated protoplasts of Bvyum caespititium held in contact for 4 days. The protoplasts possessing a nucleus (a and b) formed a cell wall; the protoplast (subprotoplast) without a nucleus ( c ) did not (redrawn from Townsend, 1897).

pidly began to regenerate new cell walls. It was established from electron niicroscope observations on thin sections of suitably fixed and embedded freshly isolated tomato fruit protoplasts that there was no detectable wall material on the surface of the protoplasts; surface replica studies largely confirmed this conclusion. More recently, freeze-etching studies of glutaraldehyde-fixed freshly isolated fruit and leaf protoplasts have further substantiated this conclusion. Evidence was obtained that essentially the entire wall substance was removed from the surfaces of the protoplasts. It should be noted that in a freeze-etch study of the surface structure of yeast protoplasts Streiblova (1968) obtained evidence that, at least in some cases, the entire wall substance was not removed from the surfaces of the protoplasts. She showed that after treatment of the yeast with snail enzymes an innermost thin wall layer, as well as remnants of the fibrillar middle layer, could sometimes be demonstrated. Perhaps the extensive plasmolysis of fruit and leaf cells prior to protoplast release greatly facilitates the removal of the cell wall from the surface of the protoplasts. A general view of a region of a freeze-etched freshly isolated leaf protoplast is shown in Fig. 14. When isolated tomato locule protoplasts that had been in 20% sucrose were transferred to a culture solution consisting of a modified White’s medium (Lam-

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port, 1964) but without 2,4-dichlorophenoxyaceticacid, together with an OSmotic stabilizer, cell wall regeneration was rapidly initiated. After about 3 hours the plasmalemma, but not the tonoplast, was no longer mainly smooth but possessed numerous infolds. Electron-dense material was massed near the plasmalemma and outgrowths from the plasmalemma were being formed (Fig. 1 5 ) .

FIG. 14. Outer region of a freeze-etched isolated tobacco leaf protoplast. Note absence of the cell wall. Plasmalemma (PL), plastids ( P ) , fat body ( F B ) , tonoplast ( T ) , central vacuole ( V ) , and possible pinocytic vesicles (arrows) (Power and Cocking, unpublished observations).

As regeneration proceeded, an initial multilayered wall was formed and, after 3 days, plasmolysis of the protoplast away from this wall was often evident (Fig. 16). Pojnar and Cocking (1968) observed that if these isolated protoplasts were kept in contact with each other during cell wall regeneration, cell aggregates were formed. A typical cell aggregate is illustrated in Fig. 17. Many of the cells within these cell aggregates are no longer spherical and cells of a variety of shapes and sizes are present; it appears that a single common wall is formed at the contact area of their surfaces. It is of interest that NeEas and Svoboda (1967) observed that similar aggregates of yeast cells are formed during the regeneration of isolated protoplasts of Sacchnroniyces cereuiseae growing contiguously in gelatin. These workers showed in an electron microscope study

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that a single common wall was formed at the contact area of the surface of the regenerating yeast protoplasts. These cell aggregates of higher plant cells could be very useful for following movement of virus from cell to cell. Border bodies (lomasomes), which are aggregates of membranes in a matrix between the plasmalemma and the cell wall, are sometimes detected in thin-section studies of these regenerating fruit protoplasts (Fig. 18). In thin-section electron

FIG. 15. Very early stage in the regeneration of a cell wall by isolated tomato fruit protoplasts. Material was stained during dehydration with uranyl acetate and poststained with lead citrate. Note the convoluted plasmaleinina and dense material massed at its surface (Cocking, unpublished observations).

microscope studies, Bowes and Butcher (1967) examined cell wall inclusions in Androgruphis punicalutu callus. These workers detected complex invaginations of the cell wall and plasmalemma into the cytoplasm which they interpreted as border bodies. They suggested that peripheral regions of the cytoplasm could become incorporated into the cell because of the formation of a new plasmalemma. A typical region showing a freeze-etched border body is shown in Fig. 19. It has frequently been suggested that these border bodies may be concerned in wall synthesis as well as playing a role in extracellular enzyme secretion (Calonge et ul., 1969) and micropinocytosis (Bracker, 1967). The marked layering of this newly formed wall is clearly shown by freeze-

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etching in Fig. 20 and after 5 days’ culture a few fibrils (probably microfibrils of cellulose) are frequently visible, being formed at the outer surface of the initially formed wall (Fig. 21). The finding by freeze-etching (of what are in all probability cellulose fibrils on the outer surface) agrees with the surface replica studies of Pojnar et al. (1967) in which fibrillar material (approximately 100 A thick) was detected at the outer surface of regenerating protoplasts.

FIG. 16. Region of regenerating tomato fruit protoplast after 3 days in the cell wall regenerating medium. Plasmolysis of the plasmalemma away from the newly formed wall is evident. Newly formed wall ( W ) , plasmalemma (PL), and toiioplast (T) (Cocking, unpublished observations).

The process of cell wall regeneration in this protoplast system appears to parallel in certain respects the pattern of cell wall formation in the soil amoeba Acanthamoeba during encystation. This amoeba has cyst walls containing cellulose (Tomlinson and Johnes, 1962). Bauer (1967) showed that at an early stage of wall formation during encystation a lamellae pile (Lumellenstupel) was first formed, and later cellulose fibrils. A somewhat similar sequence of events also appears to occur during cell wall formation in Chlorellu (Staehlin, 1966) ; this has been presented in diagrammatic form by Muhlethaler (1967) and is shown in Fig. 22. There is little evidence in the case of Chlorellu for the initial formation of a lamellae pile prior to the formation of the primary wall. In the regenerating protoplast, reverse micropinocytosis (Pickett-Heaps, 1967) has

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been implicated in the deposition of amorphous material on the outside of the plasmalemma (Pojnar et dl., 1967). It is difficult to draw any conclusions concerning the involvement of plasinalemma particles in protoplast wall regeneration, since prior to freeze-etching protoplast material was fixed in glutaraldehyde and these particles are known to be very labile during fixation (Miihlethaler, 1967). Preston and Goodman (1967) have stressed the need for an

FIG. 17. Characteristic cell aggregate which is formed when isolated protoplasts are kept in contact with each other during cell wall regeneration (Pojnar and Cocking, 1 9 6 s ) .

examination of naked protoplasts during the initiation of wall formation. They observed, by freeze-etching of unfixed material, ordered rows of granules with attached fibrils at the surface of Chlamydomonas protoplasts. The detailed investigation of cell wall regeneration by these fruit protoplasts is, as yet, at too early a stage of development to discuss profitably the implications of any finding of a hydroxyproline-rich protein in these cell walls; but it will be particularly instructive to determine whether the bulk of the hydroxyproline present occurs in the cell wall, as is the case in the callus cells of sycamore and bean (Northcote, 1969), or whether it is present mainly as a structural entity in their groundplasm as suggested by Israel et al. (1968) for cultured carrot explant cells.

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Cocking and Pojnar (1969) have shown that the onset of cell wall regeneration in protoplasts leads to a diminishing pinocytic uptake of virus. This is probably not only associated with the fact that the newly formed wall is highly impermeable to virus, but that Golgi vesicle activity is at this time contributing to cell wall synthesis by reverse pinocytosis. Leaf protoplasts isolated by the

FIG. 18. Outer region of an isolated tomato fruit protoplast after 3-day incubation in cell wall regeneration medium. A border body (BB) (or lomosome) is clearly visible between the plasmalemma and the newly formed wall (W) (Cocking, unpublished observations).

method of Power and Cocking (1968), which involves the use of a concentrated mixture of cellulase and pectinase, do not readily regenerate a new cell wall, yet as previously mentioned, they are stable in suitable culture media for several days. This could permit their use in studies of virus multiplication without their first having to be converted into cells. Svoboda and NeEas (1968) have reported that snail enzyme (which contains cellulase) prevents regeneration of 3. cerevisiue protoplasts, and this observation may explain the difficulty encountered in getting isolated leaf protoplasts to regenerate a wall. Only nucleate fruit protoplasts regenerate a cell wall under the present experimental conditions. As a result, the isolated single cell cultures formed from isolated

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protoplasts that have taken up virus by pinocytosis provide a uniform population of suitable nucleate material for studying virus multiplication.

IV. Virus Multiplication In no instance does a virus divide or increase in size and, therefore, the term “virus replication” would perhaps be better than virus multiplication. Viral nu-

FIG. 19. Freeze-etched outer region of an isolated tomato fruit protoplnst after ?-day incubation in the cell wall regeneration medium. A border body (BB) (or lomosonie) i s clearly visible. Note the suggestion of a multilayered wall which is beginning t o hrenk down (arrows). Plasmalemma (PL) (Willison and Cocking, unpublished observations)

cleic acid and protein are probably synthesized separately and the virus assembled later. It is clear that whatever may be the mechanism of infection of plant cells by viruses, the nucleic acid of the virus must first be freed of its protein coat after entering a susceptible cell. In animal cells, the infectivity drops sharply shortly after infection, and it is difficult to detect the presence of infection units. This is known as the eclipse phase. When dealing with the inoculation of leaves with virus, Bawden (1964) has indicated that after an interval, the latent period, newly produced virus becomes detectable and the content of successive extracts then rapidly increases. Bawden (1964) has indicated that this latent period has close similarities to the eclipse phase evident in animal cell virus infection.

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M e must now consider the facts known about the fate of virus pinocytosed by isolated protoplasts; but before considering this further, it is useful to discuss what is known about the fate of virus pinocytosed by animal cells. As we have seen, uncoating of the virus involves the intracellular release of viral nucleic acid from its protective coat. Joklik (1965) has discussed the two main experimental methods for following the release of viral nucleic acid. First, in

FIG. 20. Freeze-etched outer region of a regenerating protoplast similar to that in Fig. 19. Note the layering of the newly formed wall (W) (lamellae pile). The plasmalemma (PL) is highly granular with some marked depressions (arrows) which are probably pinocytic. Ice crystals (IC) (Willison and Cocking, unpublished observations).

certain systems it is possible to follow this release of viral nucleic acid by measuring the appearance of naked infectious nucleic acid; and second, and more directly, it is possible to follow the fate of labeled virus adsorbed to cells. Studies with 32P-labeled poliovirus added to HeLa cells growing in suspension cells showed that a high proportion of adsorbed virus particles eluted from the cells. Most of the virions that do not elute are uncoated within the cell, The work of Joklik and Darnell (1961) showed that the kinetics of uncoating indicated that over 50% of the virus particles were uncoated within 20 minutes after adsorption and that the liberated viral RNA was largely hydrolyzed to acidsoluble material. Very little of the RNA released from input virus is recoverable

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I17

in a macromolecular RNase-sensitive form; and Joklik and Darnell (1961) have concluded that “this is most probably the uncoated viral genome which escapes enzyme attack and goes on to initiate infection.” This analysis of the fate of polio virus thus provided for the first time an explanation for the very low efficiency with which some viruses initiate productive infection. In plant cell

FIG. 21. Freeze-etched outer region of a fruit protoplast after 5 days of regcncr.itiiig a new wall. Note similarity of the multilayered wall to that in protophsts after i days of cell wall regeneration (Fig. 2 0 ) . A few fibrils, probably of cellulose are, ho\vever, present to the outside of the lamellae pile (LP). Cellulose fibrils (arrows). Nntc graw ular plasmalemma (PL) with pinocytic depressions (Willison a i d Cocking, unpublished observations).

infections, it is possible that a single infectious virus particle is sufficient to initiate an infective center, and a discussion of the data available on the relationship between the number of lesions and inoculum concentration led Siege1 and Zaitlin (1964) to conclude that the available evidence was in favor of thc hppothesis that a single virus particle was capable of initiating infection in most plant host-virus systenis but that the interaction of virus particles with infcctible sites is not a well-understood phenomenon. Evidence that a pinocytosis-like process is involved in infection of leaves has been discussed by Mundry (1963). In experiments on the dilution of infective virus by ultraviolet-inactivated virus, WU et al. (1962) showed that when the same concentration of infective virus

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was inoculated in the presence or absence of up to ~ O O - ~ O O times O the concentration of inactivated virus, no difference in the numbers of infections found was observed. One interpretation of these results is that infection involves the occlusion of a certain voltone of inoculum, as would be the case for a pinocytic vesicle, rather than the adsorption of virus particles which tends to be concentration dependent. It should be noted, however, that Kleczkowski (1950) has

a

b

C

FIG. 22. Scheme of the subsequent steps in cell wall formation of the green alga Chloyella sp. ( a ) A cortical region of the cell with the plasmalemma, covered with particles and a subjacent Golgi complex. ( b ) Accumulation of matrix material, carried to the cell surface by Golgi vacuoles. The plasmalemma particles become detached and move to the outer periphery of the matrix. ( c ) Primary wall formation in the region where the particles are concentrated (Miihlethaler, 1967).

suggested that infectible sites are of variable sensitivity and that Dijkstra (1964) has suggested that viral nucleic acid and intact TMV have different infectible sites on Nicotiunu glzltinosu which seems to argue against a simple theory of nonspecific uptake. Moreover, Jedlinski (1964) has reported that infectible sites for T M V and tobacco necrosis virus are distinct on leaves of Nicotiana sylvestris. The experiments of Shaw (1967), who used T M V reconstituted from 1.Klabeled T M V protein and unlabeled nucleic acid to determine the interval between inoculation and breakdown of the inoculated virus, are particularly pertinent to the question of the rate of uncoating of plant viruses during infection. He demonstrated that within 7-8 minutes 25% of an inoculuin of the 14Cprotein labeled TMV had been uncoated and that this value rose to 50% after several hours. This suggests that uncoating occurs regardless of the infectivity of the virus since, as we have seen, it seems likely that merely one virus particle, under suitable conditions, is capable of initiating infection. Shaw himself concluded that the critical phase of establishment of infection occurs later in the

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infection process. From the work of Cocking and Pojnar (1969), it seems likely at least in the case of isolated fruit protoplasts that uncoating also occurs regardless of the infectivity of the virus. Protoplasts were suspended in a high concentration of TMV (0.1%) for 6 hours to allow pinocytic uptake to occur. They were then washed free of excess T M V and incubated in the cell wall regeneration medium of Pojnar et al. (1967). Virus was detected in pinocytic vesicles in the cytoplasm of protoplasts incubated in virus for 6 hours. Serial sectioning of cells was employed in this fine-structural study and it was shown that at this time there was an average of 239 virus particles per protoplast. By 30 hours, with the onset of cell wall regeneration, all virus taken up by pinocytosis in the first 6 hours had disappeared from the pinocytic vesicles. Even at the 6-hour stage there was some slight suggestion from the appearance of the virus that the virus particles were being degraded in the pinocytic vesicles in a fashion somewhat reminiscent of the degradation of viruses in animal cells after pinocytic uptake. In this electron microscope study, because of the technical difficulties of serial thin-sectioning, only one concentration of virus was used (0.1Cjo) and saniples were taken at rather large time intervals. After an average of 120 hours’ culture, 262,000 T M V particles were present in the cytoplasm of the “inoculated” regenerated fruit protoplasts. It was clearly evident that protoplasts were capable of becoming infected by TMV. These electron microscope studies also strongly suggest that infection was being initiated by the formation of pinocytic vesicles. Moreover, it appeared that the level of infection seemed to be of the same order as that of systemically infected tomato fruit locule tissue (cf. Cocking and Pojnar, 1968b). This work needs to be extended using much lower levels of input virus, and the rate of multiplication followed in detail by local lesion assay. Under these conditions, the effects of adsorption of virus to the newly formed wall should not be a major complicating factor (Kassanis, personal communication) and fewer virus particles will be present in the pinocytic vesicles. The ability of this isolated protoplast system to support virus multiplication means that we can now begin to extend the experimental approach that has been so productive in work on animal virus multiplication in animal cell cultures to plant tissue cuItures. Considerable improvements in tissue and single-cell cultures of higher plants have been made in recent years (Hildebrandt, 1962) ; but although meristem culture has aided greatly the elimination of virus infections, and tissue cultures have helped in other respects as well (Raychaudhuri, 1966), as Kassanis (1967) has emphasized, the present usefulness of tissue cultures to plant virus research is limited. These virus-infected isolated single cells formed from regenerated protoplasts have distinct experimental advantages over infected callus cultures in which the concentration of virus in the cells is very frequently much less than that found in cells of systemically infected plants and

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in which often only 50% of the cells are infected (Hansen and Hildebrandt, 1966). Although work on this infected protoplast system is, as yet, only at an early stage of development, the studies of Takebe et ul. (1968) indicated that virus multiplication can readily be obtained in isolated single cells in culture. These workers developed a procedure using a fungal pectinase which

D ooo

Cutin

5%

Hemisubstances Pectin Wax

0 .mi pjqa

FIG. 23. Simplified scheme of the outer wall of an epidermal cell. D = Ectodesmata as nonplasmatic structures (modified from Franke, 1967).

rapidly released mesophyll cells from tobacco leaves. These isolated cells from TMV-inoculated leaves supported multiplication of the virus during subsequent incubation. It was shown by local lesion assay methods that the virus titer of the extract of cells isolated from TMV-inoculated leaves increased 7-to 1.3-fold during incubation of the cells for 2 hours. Moreover, as would be expected for TMV multiplication, 2-thiouracil markedly reduced the increase in TMV titer, whereas actinomycin D had practically no effect. Intact plant tissues, whether they be the organized tissues of the leaf or callus tissues, are far too complex

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for detailed investigations of the rate of virus multiplication or detailed studies of the early stages of virus infection. A brief glimpse at the complexities of the outer wall of a leaf epidermal cell (Fig. 23) (Franke, 1967) will help to indicate the advantages, at least in the first instance, of dealing with isolated protoplasts to obtain clues as to the early stages of virus infection. Changes in the titer of viral antigen in extracts of leaves (Fig. 2 4 ) (Shalla and Amici,

Days after inoculation Titer of viral antigen in extracts of tomato leaves measured at various intervals after inoculation with TMV (Shalla and Amici, 1968).

FIG. 24.

1967) provide only an average value for cells at various stages of infection and tell us little about the rate of multiplication of virus in the cells of the various tissues of the leaf. As Shigematsu et ul. (1966) have emphasized, cells infected with TMV are only a small part of the total cells of a leaf during the early stages following inoculation with TMV and, moreover, there are major technical difficulties in the leaf system in following the incorporation of a labeled precursor into metabolites of a leaf cell. By isolating protoplasts from plant tissues, a less complex system is obtained in which to study both the initiation of virus infection and subsequent virus multiplication in a uniform population of cells. As our knowledge of such protoplast systems improves, cell regeneration from a whole range of different protoplast types infected with various viruses, both plant and animal, may be possible, as well as the formation of hybrid cell aggregates. The recently observed cytopathic effect of viruses such as TMV in regenerated protoplasts may also come to be the basis for a plaque assay for plant viruses (Cocking and Pojnar, 1969). Ultimately, these studies on virus uptake, cell wall regeneration, and virus multiplication in regenerated protoplasts

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The Meiotic Behavior of the Drosophiln Oocyte’ ROBERTC. KING Depniftment of Biological Sciences, Northwestern University, Evanjton, I l h o i s

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. The Morphology of the Drosophjla Ovary . . . . . . . . . . . . . . 111. The Cytology of the Drosophila Oocyte Nucleus . . . . . . . . A. The Light Microscopy of the Prophase Stages of Meiosis B. The Ultrastructure of Meiotic Prophase . . . . . . . . . . . . C. The c ( 3 ) G Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The sbd105 Mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Mitotic Crossing-over without Synaptonemal Complexes . . V. The Formulation of a Hypothesis concerning the Origin and 1:unctioning of Synaptonemal Complexes . . . . . . . . . . . . . . . . A. The Synaptomere-Zygosome Hypothesis of Synapto-

125 127 129 129 129 131 133 3

14

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. . . . . . . . . . . . . . . . . . 136 n the Nuclear Mein-

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of Recombinases . . . D . Nonspecificity of Synaptomere-Zygosome Interactions . . E. Intersynaptomerjc Distances and Travel Times F. The Biochemistry of Meiotic Prophase . . . . . . . . . . . . . G. Factors Influencing Crossing-over . . . . . . . . . . . . . . . . . Nondisjunction in Meiotic Mutants Affecting Crossing-over Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

142 I45

brane

I45 147 148 160 1A 4 165

I. Introduction The chromosome number of a given diploid, sexually reproducing species would double with each generation had nature not arrived at a mechanism for halving the zygotic chromosome number at some other point during the life cycle. The production of sex cells containing the haploid chromosome number is brought about by a single chromosomal replication followed by two nuclear divisions. The entire process, meiosis, occurs in animals during gametogenesis and in higher plants during sporogenesis. The rules of gene transmission described in Mendel’s famous hereditary laws (see Stern and Sherwood, 1966) follow from the behavior of chromosomes during meiosis and fertilization. Mendel’s law of segregation refers (in modern terms) to the segregation into different gametes and then into different offspring of the members of a given pair of alleles residing on the homologous chromosomes of the diploid parental organism. A restatement of the law of independen/ 1 This essay is fondly dedicated to Professor Theodosius Dobzhansky on the occasion of his seventieth birthday, January 25, 1970.

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FIG: I. ( A ) Dorsal view of the internal reproductive system of an adult female D. melanogater. Two ovarioles have been pulled loose from the left ovary. Sperm are stored in the ventral seminal receptacle (which is drawn uncoiled) and in paired spermathecae. The uterus is drawn expanded as it would be when it contains a mature egg. ( B ) A diagram of a single ovariole and its investing membranes. The nurse celloocyte complexes are representative of the first six stages (Sl-6) of oogenesis. Within the vitellarium, sectioned egg chambers are drawn so as to show the morphology of the nuclei of the oocyte and 6 of the 1 5 nurse cells. The distribution of nucleolar material

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assortment is that the members of different pairs of alleles are assorted independently into gametes during gametogenesis (provided they reside on different chromosomes), and that the subsequent joining of male and female gametes occurs at random. In addition to providing a diploid organism with haploid gametes, meiosis also affords the mechanism whereby different pairs of alleles located upon the same chromosome can recombine by crossing-over. It is the purpose of this article to review the encyclopedic body of genetic information available concerning meiotic crossing-over and disjunction in the female of Drosophila melanagaster, as well as the pertinent data from cytological studies on oogenesis, and to erect a hypothesis as to the mechanism of crossing-over which is in harmony with most of the facts. Information froin other organisms will be used when available. 11. The Morphology of the Drosophila Ovary An ovary of an adult female consists of a parallel cluster of ovarioles, each of which is differentiated into an anterior germarium and a posterior vitellarium (see Fig. I A ) . The vitellarium is composed of a series of interconnected egg chambers which lie in single file. Each chamber is in a more advanced developmental stage than the one anterior to it, and each contains an oocyte and 15 nurse cells surrounded by a monolayer of follicle cells (Fig. 1B). The egg and its 15 nurse cells are fourth generation descendants of a single germarial cell called an ovarian cystoblast. The cells formed by the mitotic activity of a cystoblast have been named cystocytes, and it is within region 1 of the germariuni that such mitoses occur (Fig. IB). The cystocytes generated from a single germaria1 cystoblast form a branching chain of cells. The 16 cells are joined by 1 5 canals each surrounded by a ring which is attached in turn to the plasma is drawn in the starred nurse cell nucleus, whereas the other five nurse nuclei show the distribution of Feulgen-positive material. The distributions of both D N A and nucleolar RNA are shown for the oocyte and follicle cells. Fragments detach from the oocyte nucleolus during stages 4 through 6 . ( C ) A pro-oocyte nucleus (left) and a nucleus in an adjacent pronurse cell (right) from germarial region 3. Synaptonemal complexes are seen in nuclei of the two prooocytes. The nuclei of the 14 pronurse cells lack synaptonemal complexes and contain more nucleolar material (N). Clouds of particulate matter adhere to the surface of these nuclei. ( D ) The oocyte nucleus (left) and a nucleus from an adjacent nurse cell (right) from a stage-3 egg chamber in the vitellarium. The magnification is the same as in C. Nucleolar material is far more abundant in each of the 1 5 nurse nuclei than in the oocyte nucleus. The synaptonemal complexes have congregated into a central area which under the light microscope ( B ) appears as a Feulgen-positive mass adjacent to the nucleolus. From Koch et al. (1967).

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membranes of the interconnected cells (Koch st ul., 1967). The cystocytes can be characterized by the number of ring canals each contains. Two cystocytes (designated l e and 2e) are interconnected, and each possesses four ring canals; two cells (3e and 4e) contain three canals, four cells ( 5 through 8e) contain two ring canals, and eight cells ( 9 through 16e) have but one canal each. Cells l e and 2e undergo a different type of nuclear differentiation from cells 3 through 16e, possibly because cells l e and 2e possess a unique pattern of cortical structures (Koch and King, 1969). Since only these 2 cells enter meiotic prophase, they have been named pro-oocytes, while the remaining 14 are called pronzlrse cells. The production of fourth generation cystocytes and their differentiation into pro-oocytes and pronurse cells begins during the pupal stage and continues throughout adult life (King et al., 1768). In each 16-cell cluster, however, the anterior pro-oocyte eventually switches to the nurse cell developmental pathway (Brown and King, 1964; Koch et ul., 1967). It is within germarial regions 2 and 3 (see Fig. 1B) that the cluster becomes enveloped by follicle cells. Koch and King (1769) have suggested that competitive interactions between the plasmalemmas of a posterior cluster of follicle cells and cystocytes l e and 2e determine which becomes the oocyte and which a nurse cell. At the boundary between regions 2 and 3, an interleafing of follicle cells will result in the production of a stalk that will form the connection between the germarium and the vitellarium. This interleafing of follicle cells anterior to an egg chamber transfers it to the vitellarium (see Fig. 1B). The major growth of the oocyte and its accompanying cells occurs in the vitellarium. The development of the egg chamber has been subdivided into a series of consecutive stages ending with stage 14, the mature primary oocyte (King et al., 1956; King, 1964; Cummings and King, 1969). During the first six stages (shown in Fig. IB), all 16 cells grow at roughly identical rates. During stage 7 , the chamber elongates and the growth rate decreases. During stages 8 through 11 vitellogenesis occurs and the oocyte grows approximately 10 times faster than at its previous maximum rate. The endopolyploid nurse cells contribute most of their cytoplasm to the oocyte and then degenerate. Concurrently, the follicle cells secrete about the oocyte first the vitelline membrane and then the chorion (King and Koch, 1963). During stages 1 2 through 14, while no further increase takes place in the volume of the oocyte, a variety of chemical changes occurs both in the yolk organelles and in the background cytoplasm (King, 1960; King et al., 1966; Cutnmings and King, 1967). During oogenesis, the amount of cytoplasm per oocyte increases by 90,000 times. Under optimal conditions, the developmental time intervals from cystoblast to S1 and from S1 to SI4 are estimated as 5 and 3 days, respectively (King, 1957; Koch and King, 1966). At the electron microscope level, pro-oocytes may be rea&ly differentiated

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f rorn pronurse cells by their nuclear morphology, since synaptonemal complexes are seen only in pro-oocyte nuclei (Koch et al., 1967; King et al., 1968). In the nucleus of the pro-oocyte destined to become a nurse cell, all synaptonemal complexes degenerate during stage 1. 111. The Cytology of the Drosophiln Oocyte Nucleus

A. THELIGHTMICROSCOPY OF THE PROPHASE STAGES OF MEIOSIS In D. melanogaster, the nuclei of pro-oocytes and young oocytes are unsuitable for detailed cytological study at the light microscope level because of their small size. In the wild-type oocyte, the chromosomal filaments begin to condense during stage 3 and are incorporated into a compact karyosome by stage 4 (Fig. 2 ) . The karyosome persists and becomes still more compact during stages 11 through 13. Late in stage 13, the nuclear envelope breaks down and the karyosome is liberated into the ooplasm. This DNA-containing structure which lacks a nuclear envelope is called a karyosphere (King et ul., 1956).

B. THEULTRASTRUCTURE OF MEIOTICPROPHASE At the electron microscope level, pro-oocytes and oocytes can be readily differentiated from pronurse cells and nurse cells by their nuclear morphology (Koch et ul., 1967). Ribbonlike synaptonemalz complexes appear in the nuclei of pro-oocytes shortly after their formation by the division of third generation cystocytes Id and 2d. Fortunately, there are species with larger chromosomes in which a correlation is possible between light and electron microscope observations of cells in meiotic prophase. It is known, for example, that in the primary spermatocytes of Plethodon cinereus (Moses, 1958), in the primary oocytes of Aedes aegypti (Roth, 1966), and in L i h m longiflorum microsporocytes (Roth and Ito, 1967; Moens, 1968) synaptonemal complexes are first seen during zygonema, reach their maximum lengths during pachynema, and degenerate during diplonema. Wettstein and Sotelo (1967) have shown from reconstructions of serial sections that in the pachytene spermatocytes of Gryllus argentinus an uninterrupted synaptonemal complex extends the length of each pair of homologs. Furthermore, the morphology of the synaptonemal complex is the same in each of the different bivalents. In D . melunoguster, leptonema must immediately follow the postmitotic DNA 2 Three spellings of this adjective are found in the literature (synnptinemal, synaptene n d , and synaptonenial). The first spelling was used by Moses, who coined the term synaptinemal complex (Moses, 1958). W e suggest, however, that the last spelling is the nlma, thread). Throughout etymologically correct one (G. .rynuptos, fastened together this article the terms leptonemu, zygonema, pachynema, and diplonema are used as nouns; whereas leptoterze, zygotene, pachytene, and diplotene are used as adjectives.

+

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replication in cystocytes l e and 2e, since synaptonemal complexes appear in the nuclei of pro-oocytes shortly after their formation by the division of third generation cystocytes I d and 2d. Synaptonemal coniplexes increase first in number and then in average length. The period during which the synaptonemal complexes appear and grow must be zygonema. This stage thus occurs during

+ and

c(3)G

Sin

s11

I4

FIG.2. Drawings of the light microscope cytology of the nuclei from germarial prooocytes (PO) and oocytes from egg chambers in consecutive stages (S,-s,) from wildtype and c ( 3 ) G females. The nucleolus, which is drawn as a solid sphere, bredks into fragments which disappear by stage 9 . From Smith and King (1 9 6 8 ) .

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the time the pro-oocytes move posteriorly through the germarium and during stages 1 and 2. Pachynema is completed in oocytes residing in the anterior portion of the vitellarium, since the combined length of all synaptonemal complexes reaches a maximum between stages 3 and 4. The complexes then degenerate and by stage 7 none are visible. It follows that the oocyte nucleus in stages 7 through 13 is in a modified diplotene stage of meiotic prophase.

C. THEc ( 3 ) G MUTATION The spontaneous mutation referred to as the recessive crossovey si/ppre.rsov in chromosome 3 of G o w e n and symbolized c ( 3 ) G was discovered in 1917 by Marie and John Gowen. Subsequently, J. W. Gowen (1933) reported that the mutant gene was located on the third chromosome at about locus 5 5 . Subsequent data (Lewis, 1948) placed the genetic locus at 58 and the cytological locus between 89A1 and S9B3. Gowen showed that in females homozygous for c(3) G, crossing-over in the entire chromosomal complement is reduced to a small fraction of normal. Externally, the mutation produces no visible effect in either the homozygous or the heterozygous condition. In 1964, G. F. Meyer reported (without giving any details) that he could find no synaptonemal complexes in oocyte nuclei from homozygous c( 3) G females. A thorough study of the structure and functioning of the ovaries of c(3)G females was published subsequently by Smith and King (1968), who found that females homozygous for c(3) G oviposited during the third through the eighth day of adult life an average of 1.5 eggs per ovariole daily. This figure is well within the range observed for various wild-type stocks. The distributions of oocytes within the various developmental stages was determined for c ( 3 ) G / c ( 3 ) G and c(3)G/+ females of four ages (0.5, 3.5, 7, and 10 days). Since no significant differences were observed, it was concluded that oocytes of either genotype spent about the same length of time passing from stages I through 14. O n the other hand, a significant difference did exist between the number of 16-cell cysts in the germaria of c ( 3 ) G and in wild-type germaria. Wild-type germaria contained twice as many clusters of fourth generation cystocytes on the average as did mutant germaria. It was concluded that the only difference in developmental dynamics between female germ cells in c ( 3 ) G and in wild type is that relatively less time is spent by c(3)G oocytes in a stage equivalent to zygonema. That is, 16-cell clusters containing pro-oocytes take a shorter time to pass through the germarium in the mutant than in the wildtype female. The results of a study of the light microscope cytology of the oocyte nuclei of c ( 3 ) G homozygotes is summarized in Fig. 2. In the wild-type oocyte nucleus, the chromosomal filaments begin to condense during stage 3 and are incorporated into the compact karyosome by stage 4. In c(3)G, this process is

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speeded up, since a well-defined karyosome is first seen in stage 3. Thus, the stage in c ( 3 ) G equivalent to pachynema appears to be shortened. In both and c ( 3 ) G the karyosome persists from stages 4 through 13. Subsequently,

+

I50

I00

f IJ, c

3 50

0

FIG. 3. The combined length of all synaptonemal complexes as a function of nuclear ,, and SGS8 represent data from serially sectioned nuvolume. The points shown as S-S clei from oocytes in egg chambers belonging to stages 1-4 and 6-8 from wild-type and c ( 3 ) G females. Each point represents one nucleus. The numbers above the points give the number of sections analyzed per nucleus, and the numbers in parentheses indicate the number of sections passing through the karyosome. Each point labeled P represents the average value calculated from data derived from sectioned pro-oocytes and the number above the P gives the number of nuclei studied. From Smith and King (1968).

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electron micrographs were taken of serial sections of nuclei of pro-oocytes and oocytes residing in c(3) G ovarioles. The data derived from these photographs are presented in Fig. 3. It is obvious that synaptonemal complexes do not occur in c ( 3 ) G oocytes belonging to the same developmental stages where in wild type the formation of the ribbon begins, reaches a maximum, and ceases.

D. THEsbdlo5 MUTATION E. B. Lewis reported the discovery of stubbloidfo5 in 1948. This deficiency resulted from the X-ray-induced loss of a small segment of chromosome 3. The deficiency is lethal when homozygous, but it is viable in the heterozygous condition. The deficiency includes the locus of c ( 3 ) G . Hinton (1966) has shown that crossing-over is abolished in females of genotype c ( 3 ) G / D f ( 3 ) ~ b das~ ~ ~ , well as in c ( 3 ) G / c ( 3 )G. Crossing-over is reduced to one-half to two-thirds of the control value in females of genotype Df(3)sbd10:/+ (Hinton, 1966, 1967). Smith and King (1968) demonstrated that in these females, while synaptonemal complexes are present in germarial pro-oocytes, they are not present in oocyte nuclei in chambers in the vitellarium which correspond to stages 2 and 3 . Thus, the development of synaptonemal complexes is precociously terminated in Df (3 )sbd205/+ females. Smith found subsequently that coniplexes are also missing from all but the most posterior clusters of fourth gencration cystocytes. The above findings demonstrate that the c ( 3 )G gene behaves more like an amorphic than a hypomorphic allele. In the homozygote, not even the earliest stages in the formation of synaptonenial complexes can be detected. Furthcrmore, whether alone or in double dose, the phenotype is nearly the same i n terms of crossing-over. O n the other hand, c ( 3 ) G + appears to produce different phenotypes in single and in double dose. Another way to describe the crossover results is that c ( 3 ) G+ can function normally when c ( 3 )G is present in the same nucleus but not when its homolog is deficient for the segment missing in D f ( 3 )shd1O5. Two alternative explanations suggest themselves. Tlic first is that c(3)G+ cannot function normally unless it can pair with another c ( 3 ) G gene, but that either c ( 3 ) G or c ( 3 ) G C is acceptable. The second esplanation is that the formation of synaptonemal complexes requires the combined action of two closely linked genes, S and t. Only one dose of S, but two doses of t, are required for proper function. In this model, wild type = St, c ( 3 ) G = sat, and Df(3)sbdIo5= soto (is deficient for both). Females of genotype + / c ( 3 ) G ( S t / s a t ) behave normally, since one S and two t genes occur per diploid nucleus. Females of genotype +/Df (3) sbdl05 (St/s"to) show decreased crossing-over because the requirement of two t genes is not met. Since the development of synaptonenial complexes is shortened in D f (3) sbdl05/+

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females, the hypothetical t gene might determine the interval during which the subunits of the synaptonemal complexes are synthesized, and the length of the interval could depend upon the dosage of t . Since the growth of synaptonemal complexes is precociously terminated in sbd1,df05/+ females at stage 1 and crossing-over occurs at a reduced rate, it is probable (1) that some crossing-over occurs prior to stage 2 in the nuclei of sbd105/+ oocytes (and presumably in wild type as well), and (2) that some crossing-over also takes place after stage 2 in the wild-type oocyte nucleus. The premeiotic replication of D N A occurs within the nuclei of oocytes before they enter the vitellarium (Grell and Chandley, 1965; Chandley, 1966). Thus, meiotic crossing-over is completed long after the massive DNA replication that gives the oocyte its 4C DNA content. Such an argument renders untenable the hypothesis advanced by Pritchard (1960) which attributes recombination in eucaryotes to copying errors at the time of DNA replication.

IV. Mitotic Crossing-over without Synaptonemal Complexes Meiotic crossing-over does not take place in the male of D. nzelanogdster (Morgan, 1912), and synaptonemal complexes do not occur in the prophase nuclei of primary spermatocytes (Meyer, 1961). According to LeClerc (1946), pairing of homologous chromosomes is seen in oogonial metaphases and larval salivary gland nuclei in females homozygous for c(3)G, and somatic crossing over occurs. Somatic pairing and crossing-over take place between the homologous autosomes of male D. melanogaster (Stern, 1936). Salivary gland chromosomes have been investigated at the electron microscope level and, although they are somatically paired, there are no reports of synaptonemal complexes associated with them. Newton and Darlington (1930) have shown that when three or more homologous chromosomes are present in a nucleus at pachyneina only two are synapsed at any particular point. Thus, there is a “saturation of pairing forces” once homologs join by twos. On the other hand, the forces in a nucleus such as that of a larval DroJophiJa salivary gland cell, which bring about the association of chromosomes into a polytene bundle, obviously are not saturated once homologs pair. From the above data, it can be concluded that somatic and meiotic chromosome pairing are basically different phenomena and that somatic pairing and somatic crossing-over can occur in Drosophila in the absence of synaptonemal complexes. Crossing-over between homologs in somatic cells may be widespread in its occurrence in plants and animals, but it takes place at a frequency hundreds or thousands of times lower than is the case for germinal crossing-over. Perhaps somatic crossing-over is common in Drosophita because it is facilitated by the occurrence of pairing of homologs in somatic cells in these and related insects.

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In most eucaryotes, however, such somatic pairing does not occur between homologous chromosomes, and germinal crossing-over generally occurs in both sexes (although often at different rates). Electron microscopists studying a wide range of eucaryotic species (including protozoans, slime molds, fungi, flowering plants, annelids, molluscs, crustaceans, arachnids, insects, fish, amphibians, birds, lower mammals, and man) have observed synaptonemal coniplexes inside the nuclei of germ cells undergoing synaptic stages of meitoic prophase (Moses, 1968). In those species in which gametogenesis in both sexes has been examined with the electron microscope, synaptonemal complexes have been observed in both spermatocytes and oocytes in cases in which crossing-over occurs in both sexes. These facts taken together with the data from c(3)G and .tbdl05 make it seem likely that the formation of synaptonemal complexes is essential for meitoic crossing-over in most eucaryotes. It cannot be argued that the enzymes required for exchanges to occur between the DNA of adjacent chromatids are absent or inhibited in somatic cells, because Taylor (1 9 5 8 ) has demonstrated, using radioautographic techniques, that exchanges do occur between the D N A molecules of sister chromatids in the dividing cells of root meristems. It follows that the synaptonemal complex may orient the nonsister chromatids of homologs in a manner that facilitates enzymatically induced exchanges between their DNA molecules.

V. The Formulation of a Hypothesis concerning the Origin and Functioning of Synaptonemal Complexes The combined length of the interphase, polytene, salivary gland chromosomes of D. melanogaster is about 1200 p, and the combined length of all somatically paired chromosomes seen in oogonial metaphases is roughly 8 p (Bridges, 1935). The combined length of all synapsing meiotic prophase chromosomes should be equal to the maximal combined length of all synaptonemal complexes (110 y, see Fig. 3 ) . Therefore, it is obvious that between interphase and meiotic prophase the chromosomes must undergo some sort of folding which reduces their length by at least a 10-fold factor. The synaptonenial complex forms, and crossing-over takes place while the chromosomes are in this folded state. Subsequently, the chromosomes undergo further coiling and folding (see DuPraw, 1968, Fig. 18-6C) to reach the dimensions characteristic of metaphase. The condensation of chromosomes to metaphase dimensions is known to involve a lysine-rich histone which cross-links DNA-containing fibrils (Mirsky et ul., 1968). Synaptonemal complexes must not play a role in the formation of the karyosome or karyosphere or in the condensation of the oocyte chromosomes to metaphase dimensions, since all these processes occur in femules homozygous for c ( 3 ) G .

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A. THE S Y N A ~ ~ T ~ M E R ~ - Z YHVPOTHESIS G O S O ~ ~ . BO F SYNAPTONEMAL COMPLEXFORMATION Each synaptonemal complex is in the form of a tripartite ribbon consisting of parallel dense lateral elements surrounding a medial complex (Fig. 4 ) . Cytochemical studies at the ultrastructural level (reviewed by Moses, 1968) have

FIG. 4. ( A ) Drawing of a segment of a bivalent as seen under the electron micro. scope. c, Chromatin; cs, central space; le, lateral element; sc, synaptonemal complex; tr, transverse rods of the medial complex. (B) Model illustrating the postulated composition of the synaptonemal complex. The aligned transverse rods of the medial complex are areas where paired zygosomes are attached to synaptomeres. The highly folded chromatids form the masses of chromatin fibers surrounding the synaptonemal complex.

shown that the lateral elements are rich in DNA and proteins (among them histones). The medial complex contains protein, but DNA is scarce or absent. The presence of RNA in the synaptonenial complex is questionable. Moses (1958) was the first to show by combined light and electron microscope studies of adjacent thick and thin sections that the lateral elements lie in the central axes of the paired homologous chromosomes of a meiotic bivalent. The niedial complex contains a system of transverse rods oriented perpendicularly to the lateral elements and separated from them by a clear area, the “central space” (Roth, 1966; Smith and King, 1968). It is reasonable to assume that the transverse rods form a strutwork which holds the lateral elements in their parallel

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configuration. If this is the case, then the clear areas to either side must contain fibrils that bridge the gap, and such fibrils can occasionally be resolved in electron micrographs. Much of what is known concerning the synaptonemal complex can be fitted into a logical framework on the assumption that each chromosome contains polynucleotide segments (hereafter referred to as synaptomeres) scattered along its length. Synaptomeres are involved in synapsis and play no role in transcription. Under the appropriate conditions, a series of coils (similar to those seen in a telephone cord) may be generated in each chromosome. Once in the coiled state consecutive synaptomeres lie in close proximity, and they pair in the manner shown in Fig. 5A. The resultant shortened and thickened chromosomes are seen in earIy zygonema, and presumably correspond to the unpaired “axiaI elements” of the electron microscopist. The hypothesis continues with the proposal that the pairing of homologs is brought about by an association, in the manner shown in Fig. 5C, of rod-shaped subunits hereafter called zygosomes. Zygosomes are assembled in the nucleoplasm, and each is visualized as a protein molecule having a folded “head” end by which it can attach to the central portion of a synaptomere. The “tail” end contains charged sites (represented by four dots in Fig. 5C). The charge distribution allows the zygosome to bind laterally with other zygosonies, but only if they are pointing in opposite directions. The bipolar properties of zygosome bridges offer a reasonable explanation of the previously mentioned “saturation of pairing forces” once homologs join by twos. It is important for the working of our model that a newly synthesized zygosome does not immediately attach to another one. Therefore, it is postulated that the zygosome is synthesized in a coiled state, and that the binding sites on the tail end are exposed by uncoiling only after attachment to the synaptomere takes place (Fig. 5B). Thus, our hypothesis assumes that a synaptomerczygosome system has properties similar to the repressor-operator system of bacteria. Jacob and Monod proposed in 1961 that there exists a class of genes called operators which control the functioning of adjacent cistrons. Such operators can be switched on or off depending upon whether they are free or have repressor molecules bound to them. The repressor molecule is now known to be a protein which binds directly to a region of specific nucleotide sequence in a DNA molecule (Bretscher, 1968). A protein zygosome is postulated to function in a similar manner with respect to a specific polynucleotide segment, a synaptomere. A zygosome, upon undergoing this initial chemical reaction, is postulated to make a configurational readjustment which changes its ability to undergo a future reaction with a third molecule (another zygosome). Repressors are known to behave in a similar fashion, since once they react with molecules of another class (effectors) their reactivity toward operators is modified

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(Jacob and Monod, 1963). A repressor is synthesized under the control of a gene called a regulator. A zygosome is presumably also coded by a specific gene and, according to this argument, the cistron involved in D . melanogasfer is the normal allele of c(3)G. Of course, the DNA code of the cistron must be transcribed to an RNA message, and RNA transcription does indeed occur in meiotic nuclei during synaptic stages (Hotta and Stern, 1963; Henderson, 1964). The synaptomere-zygosome hypothesis of a synaptonemal complex formation is in harmony with the previously cited data provided from electron microscope studies on the morphogenesis of the complexes in Drosophila and other insects. These data indicate that the axial complexes form first, that they are then brought into a parallel arrangement by the synthesis of the medial complex, and that once the synthesis is complete a synaptonemal complex extends the length of each bivalent. The suggestion that synapsis begins at the telomeres (Fig. 5B) is in harmony with Henderson's (1961) observations on spermatogenesis in various orthopterans. For the separation of homologs, zygosome bridges must detach from synaptomeres. Roth and Ito (1967) have demonstrated that this separation of homologs can be prevented in the lily by poisoning microsporocytes at zygonema with deoxyadenosine. Earlier, Roth (1966) observed (in diplotene mosquito oocyte nuclei) organelles which he called polycomplexes (see Fig. 6A). According to his interpretation, these structures were formed by the fusion of synaptonemal complexes that had detached from diplotene chromosomes. These findings suggest that during diplonema the folded end of each tygosome is .. . .

_.

..

~

~

~~~~~~

FIG. 5. Postulated steps in the synapsis of homologs to form a meiotic bivalent. ( A ) The left and right telomeres of a pair of homologous chromosomes (TL and T,:) attach in the manner shown to specific areas of the inner membrane of the nuclear envelope (ne) . The chromosomes shorten and thicken because of the folding that results from the pairing of synaptomeres that are distributed along the chromosomes. In A and B, synaptomeres ( s ) are represented by segments which are slightly wider than the intervening chromosomal regions. ( B ) By the beginning of zygonema the folding is complete. Zygosornes are synthesized in the nucleoplasm, and they attach to synaptomeres and simultaneously uncoil. As a consequence, when each synaptomere possesses a zygosome, a peg extends from the base of each chromosomal fold. Interdigitation of the pegs initially produces short stretches of synaptonemal complex. Eventually, the chromosomes pair throughout their length, and an uninterrupted synaptonemal complex extends from left to right telomeres (see Fig. 4 ) . ( C ) A small segment of the model synapsed bivalent drawn at higher magnification to elucidate the structure and functioning of synaptomeres and zygosomes. Each synaptomere is composed of three segments A, B, and C. The lateral elements A and C will pair with the A and C elements, respectively, of any other synaptomere. The B element is the site where the base of a zygosome can attach. Zygosomes projecting from each homolog interdigitate and bind in an overlapping tail-to-tail fashion. Each chromosome is shown to be divided into two chromatids.

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modified so it can no longer bind to a synaptomere. After this modification has occurred, however, the folded end can bind to the folded end of a zygosome pointing in the opposite direction. As a result, once the ribbons of zygosomes are freed from a bivalent, they can stack up as shown in Fig. 6B.

FIG, 6. ( A ) Drawing of a portion of a polycomplex formed by the fu\ion of synalitonema1 complexes which have detached from diplotene chromosomes. ( B ) Drawing of a magnified section of the hypothetical polycomplex illustrating a way in which the zygosomes might be stacked.

It is the sister chromatids that remain paired during the opening up of il tetrad that occurs during diplonema (Belling, 1929; Callan and Lloyd, 1956). Since the breakdown of the synaptonemal complexes is accompanied by the separation of nonsister chromatids, it seems clear that the complexes hold homologs together. €3.

CHROMOSOMAL ATTACHMENT SITESON

THE

NUCLEAR MEMBRANE

Woollam et al. (1966) have shown from a study of the spermatocytes of various rodents that the pachytene bivalents are attached at both ends to the

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niiclear envelope and that no other parts of the chromosomes are so anchored. Wettstein and Sotelo (1967) reached similar conclusions from a study of the syiiaptoiiemal coinplexes of cricket spernlatocytes. There was no tendency for all of the chromosomal termini to adhere to a restricted area of the nuclear cnvelope, which indicates that in these species the points of attachment are distributed throughout the inner surface of the nuclear envelope. These observations lead to the suggestion that, prior to the formation of synaptonemal coinplexes, specific chromosomal telomeres become attached to specific sites on the nuclear envelope. For example, the left telomeres of two homologous chromosomes may have adjacent points of attachment that differ from the attachment points of the right telomeres of the same chroniosonies and from those of the right and left telomeres of all other chromosomes. Therefore, the left end of a given chromosome will be anchored near the left end of its homolog, and the same applies to its right end. Such an arrangement would facilitate the zipping together of the homofogs by zygosome bridges (see Fig. 5B), and eventually this strutwork would be built from one end of the chromosome to the other (Fig. 4 A ) . If there is any truth in this hypothesis, it should be possible to demonstrate in D. melanagaster that starting at zygonema a three-dimensional pattern of telomeric connections to the nudear envelope is formed. Such a study would require reconstructions drawn from stacked positive transparencies of electron micrographs of serial sections from oocyte nuclei of various karyotypes. l o r example, a normal nucleus should have eight telomeric attachment sites, whereas the nucleus carrying a compound ring X chromosome should possess only six. If allelic telomeres are found always to adhere to neighboring sites on the nuclear membrane, this observation would suggest that the membrane is a 1110saic containing different areas, each with a surface specific for a given telomere. The development of this idea has been influenced by the findings of the i n crobial geneticists, who have demonstrated that genetically different bacterial viruses attach to different specific sites on the surfaces of their hosts. Thus, a male-specific bacteriophage such as R-17 attaches to a different part of an Escherichia coli than does T, (Brinton et al., 1964). W e know from the study by Berendes and Meyer (1968) on the polytenc chromosomes of the larval salivary gland cells of Drosophila hydei that telomeres contain tightly packed DNA fibrils coated with a protein matrix. Perhaps it is this protein that gives the telomere its adhesive properties. The teloinere may also play a role in the synthesis of portions of the nuclear membrane, since it is known that acentric chromosome fragments often become invested in a nuclear envelope (Das, 1962). From studies on autotetraploids, Sved (1966) has come to similar coiiclti-

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sions concerning the binding of chromosome ends to the nuclear envelope. If synapsis begins with the association of allelic telomeres into twos, and the chromosomes then zip together ending at the centromeres, one would predict that in a 4N nucleus two-thirds of the chromosomes would form quadrivalents and one-third bivalents during meiotic prophase. This expectation is realized. Interlocked bivalents and quadrivalents are observed very rarely, however. Sved postulates that this interlocking does not occur because the telomeres are attached to the inner surface of the nuclear envelope. Comings (1968) has sug gested that in mammalian cells there is only one specific attachment site on the nuclear envelope for the X chromosome. In female cells, there is competition for this site between the two X chromosomes, and whichever one randomly becomes bound to this specific site first will be the one that remains synthetically active. The other X chromosome eventually attaches to the nuclear envelope at a nonspecific site and becomes a Barr body. C. CROSSING-OVER THROUGH

THE

ACTJONOF RECOMBINASES

In attempting to explain the sequence of events occurring during crossingover, one has to construct a theory in harmony with the genetic as well as the cytological data. W e know from the results of a series of classic genetic studies dealing with D. melanoguster (T. H. Morgan, 1910; Sturtevant, 1913; Anderson, 1925; Bridges and Anderson, 1925; L. V. Morgan, 1933; Beadle and Emerson, 1935) that meiotic recombination generally involves only nonsister chromatids, that it occurs during a stage at which four chromatids are present, that each crossover event involves only two of the four chromatids and produces precisely reciprocal products, and that during double or multiple crossing-over all four, only three, or only two of the four chromatids may participate in exchange. More recent studies (Chovnick, 1966) demonstrate that fine structure mapping with a resolution approaching microbial systems is possible in Drosophilu and, therefore, one nust conclude that recombination can take place between almost any adjacent nucleotides of certain cistrons. The data of Smith and King (1968) derived from studies of c ( 3 ) G and Df(3)sbd1Q5indicate that crossing-over is occurring throughout the time synaptonemal complexes are being constructed in the oocyte nucleus and that crossing-over cannot occur in the absence of such complexes. Taken together, the data provided by Creighton and McClintock (1931) on Zed, by Stern (1931) and by Smith and King (1968) on Drosophilu, by Taylor (1965) on Romalea, by Rossen and Westergaard (1966) on Neotiella, by Hotta et dl. (1966) on Lilium, and by Chiang and Sueoka (1967) on Chlamydomonas demonstrate that crossing-over occurs between already synthesized chromosomes. It follows from this conclusion that chromatids are en-

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zymically broken and, once they have exchanged segments, enzymically rejoined. Crossing-over is known to produce precisely reciprocal products, and it follows from this fact that the nonsister Chromatids must be in such perfect alignment that enzyme-induced breakage occurs at precisely the same points in both. This requirement suggests that the chromatids should be uncoiled and intimately paired. Yet the chromatids of a synapsing tetrad are obviously in some sort of condensed form, since they are short and thick enough to be observed under the light microscope. Furthermore, the homologs are separated by the medial complex and central space of the synaptonemal complex. The following hypothesis offers a way out of this dilemma. Let us assume that the enzyme system involved in meiotic exchange is incorporated into a single macromolecular structure, a recornbindse, and consider some of its required properties. A recombinase must accept only two chromatids at a time, and the chromatids chosen must be nonsisters. Next, by the time that enzyme-induced breakage occurs the chromatids must be in intimate register, so that each break occurs in the same place in each chromatid. The broken ends must be interchanged and enzymically rejoined soon after. Nothing is known about the molecular structure of recombinases, but it is assumed that they bear many resemblances to DNA polymerases (see Erhan, 1968). One function of the synaptonemal complex could be to hold certain regions of the homologous chromosomes in parallel, but at a specific distance apart. Perhaps only under such conditions can the recombinase attach unerringly only to nonhomologous chromatids. One could, for example, visualize a recombinase as a molecule the length of a zygosome bridge with active sites consisting of grooves, one at each end, which accept chromatids. Assume that such a molecule is positioned (as drawn in Fig. 7) alongside and parallel to a zygosome bridge, with each end projecting toward the region of a chromatid adjacent to the synaptomere. Under these conditions, the recombinase could accept only nonsister chromatids. The recombinase would then slide along the chromatids, and as it did so it would contract, pulling the two chromatids toward one another (see Fig. 7). If the synaptomeres were in proper register, the chromatid loops would contain homologous sequences of nucleotides and would pair. The recombinase would proceed along the loops formed by the paired nonsister chromatids. The loops would extend at right angles from the plane of the synaptoneinal complex. The recombinase would open upon reaching the next zygosome bridge and either detach or continue its journey along the bivalent. In the event that the synaptomeres were not in proper register, the chromatid loops would be nonhomologous. Under these circumstances, it is postulated that pairing of the chromatids would be disturbed, and the recombinase would detach prematurely. The average tetrad undergoes a limited number of exchanges.

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For example, Weinstein (1936) has shown for the Drosophib X chromosome that over 90% of the chromatids recovered originate from single and double crossovers and, consequently, most X chromosome tetrads have only one or two chromatid exchanges. Therefore, one must postulate that a given recombinase

FIG. 7. A drawing illustrating consecutive steps ( 2 through 5 ) i n the niovement of a hypothetical recombinase along nonsister chromatids in the regions between adjacent zygosome bridges. The recombinase is shown in 1 before its attachment. The two daggerlike structures on the recombinase represent the sites on the molecule that are responsible for breakage and recombination between the nonsister strands. The movement of the recombinase occurs above the plane of the synaptoneinal complex (see Fig. 5 f o r orientation). To aid in visualizing this, the zygosome bridges have been inoved apart and the loops which project upward are drawn larger (because they would be nearer the eye of the observer). Only the portion of each synaptomere to which a zygosome attaches is shown.

molecule has a low probability of attaching to homologous loops on a bivalent (because synaptomeres may often be out of register) and of subsequently catalyzing an exchange during its journey along the bivalent. Double exchanges involving three or four chromatids are quite common, and these doubles could be used to support the idea that reconibinases attached to

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different portions of the same tetrad may be moving simuItaneously along different pairs of nonsister chromatids. Double exchanges involving the same two strands could be the work of the same recombinase, however. W e know that the minimium distance between two such exchanges is 10 crossover units (Muller, 1 9 1 6 ) . If one divides the combined length of the genetic maps for the four chromosomes ( 2 5 4 crossover units) into the estimated haploid D N A value (see Section V, E ) , one finds that 1 map unit is equivalent to 7 x l o 5 nucleotide pairs. The minimum distance between exchanges ( 7 x 106 nucleotides) could be a function of the minimum distance a recombinase travels before it is ready to catalyze a second recombinational event and the probability that the enzyme will remain attached to the chromatids in question during the time required for the journey. The fact that interference does not extend across the centromere suggests that recombinases do not travel across centromeres. (For further discussion, see Section V, G).

D. NONSPECIFICITY OF SYNAPTOMERE-ZYGOSOME INTERACTIONS There is considerable evidence from light microscope studies that pairing can occur between nonhomologous chromosomal segments during meiotic prophase. For example, as early as 1931 McClintock reported detecting nonhomologous pairing during the pachytene stage in Zea mays microspores. Such nonspecific pairing occurs over small nonhomologous regions when synapsis is interrupted by structural heterozygosity (see also Section V, G, 5 ) . Nonhomologous pairing also takes place in some monoploid individuals when homologous partner chromosomes are absent. At the electron microscope level, it has been reported that synaptonemal complexes form at sites of such nonhomologous associations. For example, a short complex can form in the region where a univalent folds back and synapses with itself (Moens, 1968). Typical synaptonemal complexes are also seen in the microsporocyte nuclei from hybrids between Lycopessicon ercrllentnm and Solanum lycopersicozdes (Menzel and Price, 1 9 6 6 ) . In such hybrids, synapsis between nonhomologous chromosomes is a common event. Thus, one must assume in the hypothesis that zygosomes attach to any synaptoniere and that zygosome bridges can form a strutwork binding together homologous or nonhomologous chromosomal segments.

E. INTERSYNAPTOMERIC DISTANCES AND TRAVEL TIMES The salivary gland chromosomes of D. melanogaster contain a total of about 5150 bands (C. B. Bridges, 1935; Bridges and Bridges, 1939; P. N. Bridges, 1941a,b, 1942; Slizynski, 1 9 4 4 ) . According to Rudkin (1965b), the average band contains 3 x lo4 nucleotide pairs per haploid chromosome strand. Therefore, the haploid D N A content is approximately 1.54 x 108 nucleotide pairs. This value must be an underestimate, since the D N A of heterochromatic and

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interband regions is omitted from consideration. It is assumed that the euchromatic bands contain about 75% of the total DNA and, therefore, that the C value is 2 108 nucleotide pairs. According to Roth (1966), each transverse rod in the medial complex is about 65 A thick and is separated from its neighbor by a space about 75 A wide. If each rod represents a zygosome aligned as shown in Fig. 4B, the maximum dimensions of the portion of a synaptomere that binds to a zygosome would be about 200 A. This length corresponds to roughly 60 nucleotide pairs, if one assumes that the distance between consecutive pairs is 3.4 A. The value of 100 nucleotide pairs will be used as the length of the entire synaptomere in future calculations, since the synaptomere contains a segment to either side of the one that binds to a zygosome according to the model drawn as Fig. 5C. The maximum length of all synaptonemal complexes in the Drosophilu oocyte is about 110 y (Fig. 3 ) . It is, therefore, estimated that a total of about 8 x 103 zygosomes [1.1 106 A/(75 A 65 A ) ] attach to all the bivalents in the pachytene nucleus. Therefore, there would be an identical number of synaptomeres, making up a total of 8 x 105 nucleotides or about 0.2% of the diploid complement of DNA. The average internemd (the DNA segment between two synaptomeres) would contain 5 x 1 0 4 nucleotides. This value, computed by dividing C by the haploid number of s y m p tomeres ( 4 I@), will be an overestimate if the synaptonemal complex is folded laterally in a plane perpendicular to its long axis (see Roth, 1966, Diagram 2 ) . For example, if zygosome bridges were stacked four abreast, the number of zygosomes would be quadrupled and the average internema would be 12,500 nucleotides long. Let us ignore this further complexity and assume that the average synaptomere is 100 nucleotides long and that the average internema is about 50,000 nucleotides long. Since the internema is about 500 times longer than the synaptomere, synaptomeric pairing such as diagrammed in Fig. 5A and B will reduce the length of a chromosome by a 500-fold factor. Keeping these dimensions in mind, it becomes obvious that the DNA loops projecting from the lateral elements will be greatly convoluted and intertwined rather than neatly laid out as they are drawn in Fig. 4B. If the combined length of all pachytene chromosomes is 110 y, then each of the five arms of the major chromosomes measures 22 y. Therefore, the X chromosome would be about four times as long as the diameter of the nucleus in which it resided (Fig. 2), and chromosomes 2 and 3 would each be eight times as long as the diameter of the nucleus. The minimum distance between exchanges in a Drosophilu chromatid was calculated as 7 1 0 6 nucleotides in Section V, C. This value would amount to a distance of 2400 y on an extended DNA molecule, but to only 5 p if the folding described above took place. Thus, if it were possible to see chiasmata one would predict that no two would

x

+

x

x

x

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be closer together than 5 p. In Schistocerca gregaria, this “interference distance” has been determined by Henderson (1963) to be 7.3 p in diplonema. In a female producing two eggs per ovariole per day, an oocyte spends about 3 days in the germarium and 1 day in the vitellarium in stages during which synaptonemal complexes are growing. Crossing-over presumably takes place during this 4-day interval. Let us assume that it takes the entire 4 days for a recombinase to travel from telomere to centromere and consider only the major chromosomes. Dividing the haploid DNA content by 5 , we obtain 4 x lo7 nucleotides per chromosome arm. To make the trip in 4 days, it is calculated that a recombinase would have to travel at the rate of 100 nucleotides per second, or about 2 p per minute. A trip along one internema would take about 8 minutes. A rate of travel of 100 nucleotides per second is not unreasonable, since Levinthal et al. (1963) have calculated that under optimal conditions an E. coli RNA polymerase can insert 1000 nucleotides per second into an mRNA corresponding to a protein weighing 30,000 daltons. Presumably, the enzyme must travel along 1000 nucleotides of DNA per second in order to transcribe at that rate. F. THERICKHEMISTRYOF MEIOTIC PROPHASE

Herbert Stern and his colleagues (Hotta et al., 1966; Ito et al., 1967; Stern and Hotta, 1967) have conducted studies of the DNA metabolism of in vitrocultured microsporocytes of L. longiflorum passing synchronously through the various meiotic stages. Stern’s group demonstrated that approximately 99.7% of the DNA in these meiotic cells was replicated before they entered prophase. Mitra (1958) had previously shown that a lily chromatid becomes breakable by X-rays and capable of rejoining independently of its sister chromatid at about this time. Late in leptonema, however, according to Stern, there is synthesis of DNA that makes up only about 0.3% of the total. If synthesis of this DNA is inhibited by poisoning the microsporocytes with adenosine deoxyriboside, the cells are arrested in zygonema and synaptonemal complexes fail to form. The newly synthesized DNA was isolated and characterized by denaturation and hybridization experiments. It was shown to be double-stranded, and DNA of equivalent nucleotide sequence was found to be present in somatic cells. These molecules were shown not to be nucleolar DNA. Stern suggested that this DNA is distributed throughout the genome in the form of Iinkers which are not replicated during the S period prior to meiotic prophase, whereas the rest of the chromosomal DNA is. The chromosome thus comes to contain two chromatids joined together by the unreplicated linkers. It is the replication of these linkers that converts the zygotene bivalent into a pachytene tetrad. The synaptomere-zygosomehypothesis can be expanded to include this infor-

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mation by assuming that each unreplicated linker is a synaptomere, and that DNA synthesis occurring late in leptonema replicates the synaptoineres to which the zygosomes will later attach. If this synthesis is prevented, the zygosomes are unable to attach to the axial cores and synaptonemal complexes never form. In Fig. 5C, the chromosomes are shown as containing independent chromatids in the intersynaptomeric regions, corresponding to Mitra’s data. The separation of chromatids to a degree visible under the light microscope does not occur until pachynema. Thus, the D N A segments between linkers must have a mechanism of replication independent of the linkers. If these segments have an average length of 50,000 nucleotides, each could contain several genes. Among them would be included a gene involved in the replication of the segment (see Taylor, 1967, his replicon model), structural genes devoted to the synthesis of different messenger RNA molecules, and a controlling gene similar to the operator referred to previously. Indeed, we know from radioautographic studies, which demonstrate asynchronous D N A synthesis along Drosophila polytene chromosomes, that each chromosome is subdivided into hundreds of independently replicating units whose synthetic activities are coordinated in time (Plaut et ul., 1966; Howard and Plaut, 1968; Mulder et ul., 1968). The most recent paper to come from Stern’s laboratory (Hotta ef d., 1968) demonstrates that certain proteins synthesized at the synaptic stages of meiosis are physically associated with the DNA distinctively synthesized at the same time. If this protein synthesis is inhibited with cycloheximide, a failure of chiasma formation results. Perhaps some of the proteins synthesized specifically during zygonema and pachynenia are components of zygosomes. Bogdanov and his colleagues (1968) have recently reported the results of a quantitative cytophotometric study of D N A and histone synthesis during cricket spermatogenesis. They found that the D N A synthesis that converts the 2C to the 4C amount occurs prior to leptonema. Histone synthesis is delayed, however, so that during zygonema only three times the haploid amount of nucleohistone is detected. Between pachynema and diplonema, the histone concentration rises to 4C and remains at this value throughout the remainder of meiotic prophase. These results may be interpreted as an indication that during the early synaptic stages certain portions of the chromosomes are stripped of a histone that is present at all other times. Perhaps pairing of synaptomeres (Fig. 5A) can occur only in the absence of this histone.

G. FACTORS INFLUENCING CROSSING-OVER Any hyl2othesis bearing on the mechanism of crossing-over must take into account a voluminous literature which documents variations in crossing-over governed by numerous extrinsic and intrinsic factors. Many of these factors have been manipulated experimentally by Drosophilu geneticists.

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I . Cenfromeres

Although the various genes are found to lie in the same order on both the salivary chromosome map and the genetic map of D . nielaizogaJter, great discrepancies often exist in the relative distances between the two genes on the two maps. In particular, crossing-over seems to be lower per unit of chromosome length in the vicinity of the centromeres. This is particularly striking in the case of chromosomes 2 and 3. It is difficult to interpret these results, since we have no cytological maps of the chromosomes actually undergoing crossing-over (i.e. those of the primary oocyte nucleus). Therefore, one could argue that crossing-over is constant per unit length of D N A in the oocyte chromosomes, but that in the chromosomes of the salivary gland nuclei the D N A near the centromeres is relatively less compact than elsewhere in the chromosomes. If, on the other hand, one can obtain an accurate estimate of the relative physical distance between genes in the oocyte chromosomes from the relative distance between the same genes in the salivary chromosomes, then one is forced to assume that exchanges occur less often per unit length of D N A near the centromeres of chromosomes 1, 2, and 3. According to the synai~tomere-zygosoiiiehypothesis, this reduced exchange could be explained by assuming that the average internema is shorter in regions adjacent to the centromeres or that thc probability of a recombinase detaching as it travels along two lionsister chromatids increases as it approaches the centromere. 2. Heterochromatin

Since heterochromatin lies to either side of the centromeres, the reduced crossing-over observed near centromeres may really be a function of paracentric heterochromatin. Roberts (1965a) has pointed out that the recombinational map length (0.04%) for the paracentric heterochroniatin of the X chromosome of D . melanogaster is far less than would be anticipated from its mitotic bulk, which is almost half of the chromosonie at metaphase. The genetic map length is also considerably less than would be estimated from its length in the interphase, polytene chromosomes of the cells of the larval salivary glands (17 bands out of 1024). Crossing-over is also low in the paracentric chromatin of Drosophila v i r i h . If, as the result of a translocation, this heterochromatin is moued to a position distant from the ceiitromere, the heterochromatin remains refractory to crossing-over (Baker, 1958). The coiling and replication cycles of the euchromatic and heterochromatic portions of the chromosomes are often out of phase. For example, Barigozzi et al. (1966) have shown that during the D N A replication of somatic cells of D . rrzelanogater cultured in vitro the heterochromatic regions replicate after the euchromatic regions. Rudkin (1965a) reports that the ratio of the amount of

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heterochromatin to euchromatin for the X chromosome is about 3:7 as measured for metaphase chromosomes in ganglion cells of female larvae. The ratio for the major autosomes is about 2:s. In the polytene salivary chromosomes, the ratio is about 1:30 for the X chromosome, however. Rudkin concluded that the heterochromatic D N A undergoes fewer cycles of replication than the euchromatic D N A in these chromosomes. In larval salivary gland cells, all the paracentric heterochromatin adheres to form a chromocenter. Some mechanism must be present that insures the release of each recombinase molecule that has completed its journey along a given chromosome arm. The recombinase would thus be available for attachment elsewhere. A hypothetical method for accomplishing this would be to have the paracentric heterochromatin code for an RNA which causes the release of recombinases from neighboring DNA. The production of recombinase-release RNA (rcrRNA) by paracentric heterochromatin would explain why recombination is reduced in the euchromatin close to the centromeres and why recombination rarely occurs in the paracentric heterochromatin itself. Beadle (1932) and Graubard (1932, 1934) have studied the effects of translocations and inversions that move blocks of genes from positions normally distant from paracentric heterochromatin to positions close to it. Under such circumstances, crossing-over between the genes within the transposed blocks is reduced below the normal values. Since the reduction in crossing-over extends quite far from the breakage point, one can postulate that rcrRNA can diffuse away from its site of synthesis in the heterochromatin. Suzuki (1965) has found that the injection of actinomycin D into Drosophih females increases crossing-over in the euchromatic regions near the paracentric heterochromatin. These findings can be explained by assuming that actinomycin D abolishes the transcription of rcrRNA and, therefore, recombinases are allowed to continue their journeys toward the centromere.

3. Temperature In the mouse, in which elevated temperatures are known to reduce the frequency of chiasmata in spermatocytes, Nebel and Hackett (1961) found that transferring males from 22' to 35OC for 76 hours caused a reduction in the number of synaptonemal complexes in zygotene and pachytene nuclei. In treated nuclei, single axial elements persisted into pachynema (where they are never seen normally). Henderson (1966) has shown that in the desert locust Schistocerca gregariu a constant temperature of 40°C reduces the chiasma frequency and can lead to complete failure of bivalent formation. He determined the time of premeiotic DNA synthesis through radioautographic studies and concluded that the heat-sensitive events that led to a reduction of the chiasma frequency in treated material took place at least 2-3 days (at 40OC) after premeiotic DNA

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synthesis had terminated. In Neurosporo crassa, however, according to McNellyIngle et al. (1966), and in Z. mays, according to Maguire (1968), heat treatments administered at late premeiotic interphase as well as at meiotic prophase influence crossing-over. Plough (1917) was the first to show that crossing-over in D. melanogaster females was influenced by temperature. He found that departures from a certain normal range of temperatures (18-29Oc) in either direction increased recombination in chromosome 2. The euchromatic chromosomal regions proximal to the centromere seemed to respond the most to the treatments. Temperature shocks that increase crossing-over, decrease interference (Hayman and Parsons, 1960). Grell (1966) has made a study of this phenomenon in the X chromosome. The shocks she used generally involved a transfer of the insect from 2 5 O to 35°C for a period of 8-12 hours. The increase in crossing-over observed in regions close to the centromere was demonstrated to be the result of meiotic, not gonial, crossing-over. The cells responding to the treatment were oocytes undergoing their early development in the germarium, and some of them may have been in premeiotic interphase (Grell and Chandley, 1965). It is not certain from any of the above studies that temperature shocks act directly upon the recombinational events. A compound reacting during recombination could be synthesized prior to the time it is to function. A heat shock could prevent its synthesis and, thus, produce an effect at a subsequent period. The findings reported for the mouse and locust are in harmony with the hypothesis that meiotic synapsis and crossing-over depend upon the synthesis of synaptonemal complexes, and that the D N A replication of each chromosome of the bivalent precedes such synapsis. Temperature shock could inhibit the formation of zygosomes or cause their premature breakdown. Since axial complexes are observed to persist in heat-treated mouse spermatocytes, the postulated pairing of synaptomeres is not affected. In Drosophilu, one has to explain the differential response of paracentric euchromatin to both heat and cold shocks. Perhaps the enzyme transcribing rcrRNA (Section V,G,2) is stable only in the 18O-29OC temperature range and functions late in premeiotic interphase and early in meiotic propbase. At temperatures outside the specified range, the enzyme is inactivated and, as a consequence, recombinases later encounter lower concentrations of rcrRNA. Therefore, recombination is elevated in the paracentric euchromatin.

4. Age Bridges (1915) was the first to show that the amount of crossing-over bctween certain genes in D. melanoguster declines during the first week of female life. In the cases of the three major chromosomes, the euchromatic segments that showed most clearly this age effect lie close to the centromeres (Bridges,

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1927; Plough, 1917, Stern, 1926). In R. F. Grell’s more recent study (1966), daily measurements were made of the amounts of crossing-over that had occurred in the proximal halves of the X chromosomes in eggs laid over a 2-week interval. The crossing-over frequency was about 20% in the first sample. The daily values then fell until they reached lo%, a frequency which was maintained for the sixth and subsequent samples. The eggs from the first sample were laid about 2 days after eclosion, and they must have undergone crossingover during the pupal period. The eggs from samples 6 through 14 were laid 8-1 6 days after eclosion, and they underwent crossing-over during the female’s adult life. The above data demonstrate that during metamorphosis crossing-over in the chromosomal regions proximal to the centromeres occurs at a rate double that in the X chromosomes of the oocytes of adults. This conclusion raises the question as to whether or not there are differences in the milieu of pupal and adult oocytes that parallel the dissimilarities observed in crossing-over. One difference of possible significance would be in tissue ecdysone levels. The prothoracic gland degenerates late in the pupal period, but before it does, it secretes large quantities of ecdysone (see the discussion in Aggarwal and King, 1969). Therefore, the ecdysone titer should be much higher in the pupal than in the adult milieu. It is well established that this hormone when injected into the larvae of various dipteran species results in localized puffing of the polytene chromosomes in the cells of the salivary glands and other organs (see reviews by Kroeger and Lezzi, 1966; Clever, 1968). RNA transcription is a necessary requirement for such puff formation, and radioautographic studies demonstrate that this synthesis occurs in the puffs themselves. It follows from these data that ecdysone does influence RNA transcription by Drosophila chromosomes, that this hormone is present at the developmental stage when crossing-over in regions proximal to the centromeres is high, and that it is absent or present in a far lower concentration at a stage when crossing-over in the regions specified is low. Obviously, studies should be carried out on the effects of injected ecdysone upon crossing-over in adult females. Data provided by Redfield (1966) complicate the relation between maternal age and crossing-over still further. She studied crossing-over in the region spanning the centromere of chromosome 3 and found that the crossover values fell from 18% in the first eggs laid to 11% in those laid between the fifth and sixth days. Thereafter, the values rose again and by day 1 2 were back at 18%. The significance of this rise and the reason for its occurrence in chromosome 2 but not in the X chromosome are not understood. 5 . Chromosome Aberrations

The first studies of chromosomal aberrations that influence crossing-over were those by Sturtevant and Beadle (1936) on inversions. Fewer progeny represent-

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ing crossover Chromatids are produced by inversion heterozygotes, both because crossing-over is itself inhibited and because certain types of crossover chromatids are eliminated. It can be shown in the case of paracentric inversion heterozygotes, for example, that each single exchange within the inversion loop generates an acentric and a dicentric chromatid. Such aberrant chromatids were first observed by McClintock (1931, 1933) in corn. In female Drosophilu and other dipterans, such chromatids are eliminated because of their selective distribution to the polar nuclei during oogenesis (Sturtevant and Beadle, 1936; Carlson, 1946). Similar exchanges within pericentric inversion heterozygotes produce monocentric, duplication-deficiency chromatids which are not so eliminated. Since eggs bearing chromosomes with duplicated and deficient regions would be expected to be incapable of completing embryonic development after they are fertilized, females heterozygous for pericentric inversions should be semisterile. Roberts (1967) has documented this expectation by finding pericentric inversions in D . melanogaster which drastically reduce both crossing-over and egg hatch in heterozygous females. Pericentric inversions are known (Alexander, 1952) that greatly reduce the production of crossover progeny zuithozrt drastically lowering the frequency of eggs hatching, however. Here it appears that crossing-over itself has been inhibited, and such an inhibition need not be the result of a disturbance in the synaptic pairing between the homologs of a bivalent. In the very first cytological study on chromosome pairing in an inversion heterozygote, McClintock (193 1) showed that reverse loop pairing did not characterize all inversions. She found that in maize plants heterozygous for a long paracentric inversion most pollen mother cells did show reverse loops, but in the case of shorter inversions there was a nonhomologous association which led to straight pairing. In heterozygotes for pericentric inversions, reverse loops were seen in only 1% of the sporocytes examined. More recently, Nur (1968) reported the results of a study of pachytene spermatocytes of the grasshopper Cumnula pellucidu. Males were chosen that were heterozygous for a paracentric inversion occupying about 10% of one of the longest chromosomes. In 90% of the cells observed, straight pairing occurred. Asynapsis was seen 8% of the time and in the remainder of the cells inversion loops or a similar chromosomal configuration occurred. Obviously, nonhomologous pairing takes place the majority of the time between the inverted segments. According to the synaptomere-zygosome hypothesis of meiotic synapsis, the homologs of each bivalent are zipped together by zygosome bridges which first form near each telomore and proceed proximally (Fig. SB). Since the honiologous telomeres are anchored to nearby regions of the nuclear envelope, the zygosome bridges form in a sequential fashion which brings those synaptomeres into register that link allelic internemal segments. Consider, however, the problems brought about by a short inverted segment lying proximal to the telomere of one homolog. The homologs will zip together starting at each end. When

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the inverted segment is reached, nonhomologous pairing will generally occur. This too presumably results from zygosome bridge formation. In this case, however, the internemal loops brought into register will not contain allelic genes and will not pair intimately. Under these circumstances, recombinases are postulated to detach (see Section V, C) and, thus, exchanges between the inverted segments of the heterozygote would not be catalyzed. Crossing-over is often known to be markedly decreased in adjacent uninverted regions to the left and/or to the right of the limits of an inversion (Sturtevant and Beadle, 1936). This finding can be explained by assuming that, for example, if a traveling recombinase reaches the left limit of an inversion and detaches, the probability of an exchange occurring in the uninverted chromosomal region to the right of the inversion is lower than it would be if no inversion occurred nearby. This is because once the recombinase detaches from the chromosome it may subsequently attach to some distant area on the same bivalent or to another bivalent which happens to be nearby, rather than to the segment in question. Crossing-over within the inverted sections is decreased in inverse proportion to the length of the inversion. Thus, more crossover chromatids are recovered from longer inversions. This finding suggests that if the inverted regions are long enough the chromosome segments can sometimes form reverse loops (as demonstrated by McClintock, 1931). Crossing-over should be abolished at the breakage points because a recombinase traveling toward an inversion loop would be forced to halt its journey upon reaching the base of the loop. Similarly, a recombinase that attaches at some site within the loop would be forced to stop upon reaching the end of the loop. Outside or inside the inversion loop, however, zygosome bridges bring internal loops into register that do contain allelic genes, and crossing-over can then take place. In such cases, the more common single crossover chromatids would be acentric or dicentric and would be eliminated. Double crossover chromatids are recoverable from the two- and threestrand double exchange tetrads. While inversions change the order of genes in a chromosome and their distances from the centromere, they do not alter the length of the chromosome. Aberrations such as deficiencies, duplications, and insertional translocations do cause such alterations, however. In D. melanogaster, aberrations of this sort sometimes reduce crossing-over markedly when heterozygous. For example, Roberts (1966) has shown that a specific tandem duplication when carried in the heterozygous condition reduced crossing-over in the left arm of chromosome 2 from 44.2% to 7.3%. Rhoades (1968) has more recently reported the results of a cytogenetic study of the effects of an insertional translocation upon pachytene pairing and genetic crossing-over in maize. The transposition involved the removal of an interstitial piece from the long arm of chromosome 3 and its in-

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sertion into the short arm of chromosome 9. For purposes of discussion, the normal chromosomes will be symbolized by N9 and N 3 and the aberrations by Df3 and Tp9. Female parents of karyotype TpgN9;N3N3 bearing appropriate marker genes were testcrossed. A pronounced reduction in crossing-over within chromosome 9 was observed throughout all the regions tested (these normally had a combined map distance of about 50 crossover units). Cytological observations were made on pachytene bivalents of Tp9N9 constitution. The portion of the Tp9 chromosome that had no homologous counterpart in the N 9 chromosome was expected to protrude as a buckle from one side of the bivalent. Such buckles were observed, but they occurred at various positions along the short arm of chromosome 9, and the lengths of the buckles were not constant (Fig. SA). Every position of the buckle other than those where homologous regions were synapsed on either side obviously involved pairing of nonhomologous segments. Rhoades’ findings fit rather neatly into the synaptomere-zygosome theory of synapsis. Assume that a synaptonemal complex grew from the telonieres of the short arm of the TpN9 bivalent toward the centromere. The complex zipped together homologous chromosomal regions until it reached the translocated piece of chromosome 3. Subsequently, nonallelic chromosomal segments became attached. Once the transposition was passed, the synaptonemal complex continued to zip nonhomologous regions together; as a consequence, all allelic internemal loops would have been out of register until the synaptonemal complex was met by the synaptonemal complex growing toward it from the opposite telomere. When this meeting occurred a buckle was forced to protrude from the longer chromosome. Here, the zygosome bridges could form within the buckle itself. The position of the buckle would be expected to vary if the relative speed with which the synaptonemal complexes grew toward each other also varied in this chromosome arm. Since such a large amount of illegitimate pairing occurred, one would expect crossing-over to be greatly reduced. The same hypothesis can be used to explain Roberts’ observations on the effects of a tandem duplication upon crossing-over. In the case of Df3N3 bivalents, Rhoades found that the observed buckles were far more uniform in size and position (Fig. SB), and crossover studies showed that recombination was reduced only in regions that contained the deleted segment. These observations suggest that the linking together of nonhomologous segments by zygosome bridges rarely occurred in the Df3N3 bivalent. Since the buckle usually protruded from the middle of the long arm of chromosome 3, one must assume that synaptonemal complexes grew in opposite directions along the long arm of chromosome 3 at such rates that they usually met in the middle. Dobzhansky (1931, 1934) studied the effects of a number of translocations

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when heterozygous upon recombination. He concluded that the greatest reduction in crossing-over is in the neighborhood of the breaks, and that this reduction is least pronounced in those chromosomal regions most remote from the points of breakage. Autosomal translocations having one or both breaks in the

FIG. 8 . ( A ) Camera lucida drawings of Tp9/N9 bivalents at pachynema showing the frequently observed asynapsis and nonhomologous pairing. ( B ) Camera lucida drawings of pachytene configurations of Df3/N3 bivalents showing the fairly uniform position of the buckle. From Rhoades (1968).

chromocentral regions exhibit normal or even increased recombination in these regions (Dobthansky and Sturtevant, 1931). Roberts (1965b, 1968) has found that the most commonly recovered crossover suppressors from X-irradiated Drosophild sperm are not inversions, but reciprocal translocations. When heterozygous, some of these reduce crossing-over more than any known inversion.

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Roberts (1965a) has made a study of translocations in which one break was always in the tiny fourth chromosome. He found that when the other chromosome involved in the translocation had a break close to the centromere, recornhination within the arm was little affected. Distally placed breaks reduced crossing-over drastically, however.

FIG. 9. Diagrammatic representation of the postulated pairing behavior of the second and fourth chromosomes of D. melanogaster under normal conditions ( A ) , in a 2L:4 translocation in which the break in 2L is distal to the centromere (B), and in a 2L:4 translocation in which the break in 2L is proximal to the centromere ( C ) . Centronieres are represented by open circles, telomeres by smaller solid circles. Legitimate pairing is represented by I ] 1 I, illegitimate pairing by X X X . In reality, the length of the second chromosome would be eight times the diameter of the nucleus.

These findings support the idea that the telomeres are attached to the nuclear envelope during the synaptic stages of meiosis. Translocations that have their breakage points near these chromosomal attachment points would lead to difficulties in the synapsis in heterotygotes. For example, if the left telomeres of the two second chromosomes were attached to the nuclear envelope at a point distant from the telomores of the fourth chromosome, synapsis would proceed by the formation of zygosome bridges to the point where the translocation occurred. Hereafter, for most of 2L, the pairing that does occur will be illegitimate (see Fig. 9B). If, on the other hand, the break occurs far enough away from the telomere of 2L, the synaptonemal complexes that form will hold the

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synaptomeres in proper register and hence recombination will show little reduction (see Fig. 9C). 6. Interchromosomal Effects

Sturtevant (1919) first observed that an inversion in the third chromosome of D. melanogaster when heterozygous increased crossing-over on chromosome 2. In subsequent years, many investigators noted that heterozygous inversions in any pair of major chromosomes in Drosophila increased crossing-over in the other pairs (see review by Lucchesi and Suzuki, 1968). According to Rendel (1957), increased crossing-over caused by heterologous inversion heterozygosity is accompanied by reduced interference. White and Morley (1955) reported that in the grasshopper Trimerotropis suffusa pericentric inversions suppressed chiasmata formation within the regions heterozygous for the aberrations. The lowered chiasmata frequency in these chromosomes was compensated for by an increased frequency in other bivalents. Thus, the phenomenon appears to be widespread. An explanation of the interchromosomal effects of inversions can be found in the postulated behavior of recombinases. According to the speculations advanced in Section V, C, recombinases remain attached only to those nonsister chromatids that pair properly, and intimate pairing of the type required only occurs if the nucleotide sequences are nearly identical (i.e., if the genes brought into register are alleles). “Liberated recombinase” molecules, which have detached from sister chromatids that fail to meet these specifications because of an inverted segment, are available to attach to other bivalents. Thus, the probabilities of crossing-over occurring in those normal bivalents of the genome would be greater than those normally observed. Roberts (196513) has demonstrated that an autosomal inversion which when heterozygous almost triples the amount of crossing-over in euchromatin proximal to the centromere of the X chromosome has no effect on proximal heterochromatin. Since it is postulated that this chromatin synthesizes rcrRNA, crossing-over would not be expected to increase here. Translocations that act as crossover suppressors should also promote crossingover in other chromosomal regions for the same reasons that inversions do. In Drosophila, numerous instances of translocations enhancing crossing-over in heterologous chromosomes have been recorded (Hinton, 1965), and Hewitt (1967) has reported that a translocation exists in the grasshopper Cibolucris parviceps that increases the frequency of chiasmata in all chromosomes, including the interchange itself. Chromosome rearrangements that attach homologs or arms of homologs to the same centromere are known in Drosophila. An example of such an aberration is the reversed acrocentric, compound X chromosome studied by Hart and

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Sandler (1961). They found that recombination in chromosome 3 was increased in the presence of this compound X chromosome. One would expect that the intensity of the enhancing effect of any aberration upon crossing-over within the chromosomal elements not involved in the aberration would be proportional to the degree to which crossing-over is suppressed within the chromosomal elements involved in the aberration. This would be because the larger the suppression of crossing-over, the larger the number of liberated recombinases available to attach elsewhere in the genome and catalyze crossing-over. Liberated recombinases would be more concentrated in one region of the nucleus than another, however, and each bivalent may be in a characteristic area. Hence, some may be accessible to recombinases liberated from a given aberration and others inaccessible, and a simple relation between crossover suppression in one bivalent and crossover enhancement in another may not always be found. The euchromatic regions of chromosomes that seem to be particularly sensitive to the enhancing effects of heterologous aberrations upon crossing-over lie near the telomeres and centromeres (Lucchesi and Suzuki, 1968). This enhancement pattern can be accounted for on the basis of the hypothesis presented earlier. Synaptonemal complexes would form first in the telomeric regions of chromosomes and, therefore, the total period of time during which these regions would be available for the attachment of liberated recombinases would be the longest. Recombinases reaching the euchromatin proximal to centric heterochromatin would encounter rcrRNA molecules which would bind to them and cause them to detach from the Chromosome. With the concentration of recombinase molecules raised by the addition of liberated recombinases, the centric heterochromatin may be unable to produce enough rcrRNA to bind to all incoming recombinases. As a consequence, the journey of the average recombinase in the proximal euchromatin would be lengthened, and crossing-over enhanced in this region. 7. Recently Discovered M i i t d o n s Influencing Recombination

Lindsley et al. (1968) and Sandler et al. (1968) have recently described three mutations in D . melanogaster, each of which influences crossing-over in a unique way. Since the wild-type alleles of these mutations presumably control meiotic processes, they have all been given the symbol mei. In the case of meiS51, recombination is reduced uniformly to about half the control value. The mei-S282 mutant, on the other hand, reduces crossing-over in a polarized fashion, the reduction being most pronounced distally. The mei-S332b mutant increases recombination in comparison with controls. Cytological investigations of the salivary gland chromosomes demonstrate that none of the mutations are associated with chromosomal aberrations. As yet, no studies have been made of the chromosomes in mutant oocytes utilizing the electron microscope.

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KING

According to the hypotheses developed in this chapter, in order for meiotic crossing-over to occur normally, synaptonemal complexes must form in a fashion that prevents illegitimate pairing. The formation of zygosonie bridges between synaptomeres that are in proper register is postulated to require the attachment of homologous telomeres to nearby sites on the nuclear envelope. In the case of females homozygous for c ( 3 ) G, crossing-over is abolished because synaptonemal complexes fail to form. In females heterozygous for .ibdlQz, crossing-over is reduced because the formation of the synaptonemal complex starts late and is precociously terminated. It remains to be seen whether or not mei-SSl behaves in a similar fashion. In the case of mei-S282, crossing-over is reduced to the greatest degree in the regions distal to the centromere. This finding suggests that in this mutant synapsis proceeds distally from the centroineres. Perhaps the adhesion of telomeres to the nuclear envelope is inhibited in mei-S282 oocytes. It must also be assumed that synapsis proceeds distally from the centromeres in the case of ring X chromosomes, since crossing-over occurs normally in such telomere-deficient chromosomes (Sandler, 1 9 5 7 ) . One would also expect to recover meiotic mutants that influence the behavior of the recombinase molecules themselves. The oocytes of females honiozygous for vzei-SS 1 may be shown to possess morphologically normal synaptonernal complexes at the appropriate stages. It could then be suggested that naei-S51 codes for a mutant reconibinase that is less efficient in catalyzing crossover events. For example, it might tend to detach prematurely from bivalents or to take longer to catalyze an exchange. The recombinase produced by mei-S332b could be more efficient than wild-type recombinases. Another possibility would be that the recombinase has a longer time to operate. If this suggestion is correct, one might expect to find synaptonemal complexes persisting past stage 4 (see Fig. 3) in mei-S332b oocyte nuclei. The same sort of argument can be used to explain why the genetic maps are so much longer in D . virilis than in D. melanogaster. For example, the distance between y and bb in D. vivilis is 2.5 times longer than in D. melanogaster (Chino, 1937; Bridges, 1398), whereas the lengths of the sex chromosomes in larval salivary gland cells are very similar in the two species (Hughes, 1 9 3 9 ) .

VI. Nondisjunction in Meiotic Mutants Affecting Crossing-over In addition to showing that meiotic crossing-over was almost completely abolished in female D. melanogaster homozygous for c(3)G, Gowen ( 1 9 3 3 ) reported that among the offspring of such homozygotes were matroclinous females and patroclinous males, haplo- and triplo-IV males and females, metamales and metafemales, intersexes, and triploids. H e concluded that the c ( 3 ) G females produced eggs some of which were diploid for the X, for the fourth, for all

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the autosomes, or for all four chromosomes, and others that lacked the X or the fourth chromosome. Gowen suggested that nondisjunction during the first meiotic division was the phenomenon producing the exceptional offspring because disomic eggs formed by c(3)G females always contained both grandparental chromosomes. Although the rate of nondisjunction in primary oocytes was very high, however, it was not elevated to the point where the chromosomes were being distributed completely at random. Notidisjunction was not increased in c(3)G/+ females. One would expect zygotes containing one or both major autosomes in single or triple dose to be inviable, and such lethal embryos presumably account for the majority of the eggs that fail to hatch (700/, in the experiments of Smith and King, 1968). If it is concluded that the absence of synaptonemal complexes is the cause of the abolition of meiotic crossing-over, it seems reasonable to use the same argument to explain the elevated rate of nondisjunction. Hinton (1966) has shown that nondisjunction in D f ( 3 ) s b d l ” / c ( 3 ) G is increased above the control rate by almost 300 times, whereas the rate of nondisjunction in Df(3)rbd105/+ females is only about 40 times that of controls. It is known that the formation of synaptonemal complexes starts late and is prematurely terminated in Df(3)sbdl05/+ oocytes (Smith and King, 1968). It follows that even if synaptonemal complexes are present for an abbreviated time, the rate of nondisjunction is reduced below the value seen when none are formed, Nondisjunction at the first meiotic division also occurs at an elevated rate in females homozygous for mei-S282 (Lindsley et ul., 1968). The high frequency in man of serious disease associated with chromosomal abnormalities arising from nondisjunction has only been appreciated recently (Turpin and Lejeune, 1965; Court Brown, 1967). There are recent publications (Sarles et al., 1968; Atkins et al., 1968) citing family groups in which relatives show nondisjunction involving different chromosomes. Such reports suggest that genes similar to c ( 3 ) G , ~ b d l 0 5 ,and mei-S282 may exist in human populations. How do synaptonemal complexes function to promote the normal disjunction of bivalents ? One obvious explanation would be that synaptonemal complexes, by promoting crossing-over, insure that each bivalent contains at least one chiasma, since, as Brown and Zohary (1955) have shown, each crossover event generates a chiasma. Darlington (1929) was the first to suggest that homologs must be held together by chiasniata at meiotic metaphase I in order to pass to opposite poles at anaphase I. H e assumed that homologs that were not so attached behaved independently, and since each could go to either pole at anaphase, half of the secondary gametocytes produced would be disomic or nullisomic for a given chromosome. Thus, the presence of one or more chiasniata in each metaphase bivalent was necessary to prevent homologs from assorting

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independently. While Darlington’s hypothesis is in harmony with most of the available data, it fails to explain in D. melanoguster females (1) why normal fourth chromosomes disjoin properly, although crossover chromatids are rarely detected (Grell, 1964) and ( 2 ) why under certain experimental conditions nonhomologous chromosomes shown nonrandom assortment (Grell, 1957, 1967). Perhaps crossing-over does not occur within the euchromatin of the fourth chromosome because high concentrations of rcrRNA produced by the nearby centric heterochromatin prevent the attachment of recombinases. Presumably, the fourth chromosomes still undergo synapsis through the formation of a synaptonemal complex. However, synaptonemal complexes disappear from the nuclei of the oocytes of D. melanogaster prior to stage 6, i.e., at least 40 hours before metaphase I is reached (Koch and King, 1966; Cummings and King, 1969, Table 1). Therefore one must seek further for the reason why chromosomes like the fourth should remain associated. Grell (1962, 1967) has put forth a hypothesis on the nature and sequence of meiotic events in the D. melanogaster female. She postulates that exchange pairing precedes exchange and is restricted to homologs and homologous regions of chromosomes. Regular segregation follows for those chromosome5 that undergo exchange. Chromosomes that fail to undergo exchange, either because they lack an independent homolog, because rearrangements interfere with exchange, or because they are too small to do so, are left behind. These chromosomes form the distributive pool, and they then undergo a second type of pairing called distributive pairing. Distributive pairing may involve homologs or nonhomologs, and chromosomes that are distributively paired always pass to opposite poles at first meiotic anaphase. According to Grell, exchange pairing occurs at an early diffuse stage and distributive pairing at a later condensed stage of meiotic prophase. The critical concepts leading to the recognition thnt distributive pairing differs from and follows exchange pairing in DrOJophZld arise as a result of Grell’s experimental demonstrations ( 1 ) that a given cliromosome undergoes distributive pairing with a nonhomolog only if it has not crossed-over with its own homolog and (2) that the frequency of exchange between a specified pair of homologs is not altered whether or not one of these homologs also participates often in distributive pairing with a nonhomologous chromosome. It is proposed that the exchange pairing of meiotic chromosomes referred to by Grell is the result of telomeric pairing at leptonema (or late premeiotic interphase), following during zygonema and pachynema by the construction of synaptonemal coniplexes. Recombinases attach to such bivalents and tntalyLc exchanges. When the synaptonemal complexes break down, the bivalents remain attached by chiasmata. Although synaptonemal complexes can form be-

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tween nonhomologous chromosomes (Section V,D) , it can be assumed that the probability of assembling a lengthy complex is rather small. Presumably, construction of complexes is hampered because the nonallelic telomeres are attached to sites on the nuclear envelope distant from one another. Of course when it does occur, pairing will be illegitimate. In the case of homologs that carry different gene sequences, illegitimate pairing through zygosome bridges is once again postulated to occur (Section V,G,4). Recombinases are thought to detach from bivalents when they reach regions of illegitimate pairing, and they are supposed to do so in Drosophilu in the case of the short fourth chromosome (see above). Thus, chiasmata will not be formed in any of these situations, and the probability that each chromosome will enter the distributive pool is greatly increased. Grell (1967) investigated the frequency of nondisjunction between chromosome 4 and X chromosome fragments of different lengths and found that disjunction was almost normal when the chromosomes were of equal or nearly equal length. It is assumed that the formation of terminal adhesions would be facilitated by having chromosomes of similar lengths. Perhaps distributive pairing can be equated with an adhesion between telomeres of homologs or nonhomologs occurring late in meiotic prophase. The adhesion would be nonspecific, but in most cases homologs would be involved because they would normally be nearby. This would be the case, if they were joined by chiasmata, and it would also be true for bivalents once held together by a synaptonemal complex, even if they did not participate in an exchange. The fact that in the case of oocytes lacking c ( 3 ) G' the meiotic distribution of homologs is not completely at random (the values observed are about 0.7 that predicted for random assortment) suggests that the end to end association of homologs is facilitated to a small degree even if synaptonemal complexes never formed. Here the postulated specific attachment sites for telomeres on the nuclear envelope (Section V, B) might be responsible for telomeric associations, a few of which might persist until metaphase I. Synaptonemal complexes that are maintained until anaphase I have been described in males of Bolbe nigra (Gassner, 1969). In such mantids long-lived synaptonemal complexes may function directly in distributive pairing. The Y chromosome does not influence recombination between X chromosomes in XXY females (Grell, 1967). Therefore, during meiotic prophase it seems likely that the Y does not pair with either X in such a way that prevents a normal synaptonemal complex from forming between the X chromosomes. If this interpretation is correct, it can be concluded that zygosomes do not bind to the Y chromosome. Both wild-type males and females possess two c(3)G+ genes per nucleus. Presumably, the c ( 3 ) G+ gene codes for zygosomes and participates in transcription during meiotic prophase in females but not in males. Since recombination in females is not influenced by the addition of Y chromosomes, the inactivity of c ( 3 ) G + in males is not attributable to the presence

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of the Y in spermatocytes and its absence in normal oocytes. More likely, the critical factors are the different ratios between X chromosomes and autosomes for males and females. It will be interesting to see if synaptonemal complexes reside in the young oocytes of triploid intersexes. Since nondisjunction is far less common in normal Drosophila males than in c ( 3 ) G females, it is clear that the mechanism of distributive pairing evolved by the male is far more specific than that in the female.

VII. Summary The events that are known or hypothesized to take place during meiosis can be summarized as follows. Prior to meiotic prophase, the chromosomes attach to specific sites on the inner surface of the nuclear envelope. Allelic telomeres have adjacent sites. The chromosomes replicate all their D N A except for the synaptomeres. During leptonema, histone-deficient synaptomeres pair, reducing the length of each chromosome by a factor of 500 times. Late in leptonema, the synaptomeres are replicated. Next, the gene ( c ( 3 ) G + ) coding for the mRNA of zygosomes is turned on. Zygosomes are assembled in the nucleoplasm. During zygonema and pachynema, zygosomes (z) attach to synaptomeres (s) and subsequently form zygosome bridges. The reaction is shown in the following diagram. b

+

/

F

\

\

z-sI-3 c:: ;-c::zz z-s d I

Y

Y -

z-s3 c-L 5-2

/

-

z-s

L

The reaction serves to zip homologous chromosomes together, starting at the telomeres and ending at the centromeres (Fig. 5 ) . Eventually, a synaptonemal complex extends from one end of the bivalent to the other. As soon as the synaptonemal complex starts to form, recombinases attach to nonsister chromatids and move along them toward the centromeres, catalyzing exchanges as they go (Fig. 7 ) . The paracentric heterochroinatin transcribes rcrRNA, which causes nearby recornbiriases to detach from the chromosomes. Recombinases detach prematurely from chromosomes if the nonsister chromatids contain nonallelic DNA segments. During diplonema, the zygosomes detach and the bivalents open up. Separation of the bivalents is prevented by chiasmata, however. Chromosomes lacking chiasmata undergo an end-to-end pairing with chromosomes of similar

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size. Such attachments insure that the bivalent moves as a unit to the spindle equator during metaphase I. At anaphase I, each homolog moves to an opposite pole and is incorporated into a secondary gametocyte. In each gametocyte, the centromeres of the chromatids making up each homolog become functional. The second meiotic division distributes each sister chromatid to a separate gametic nucleus. ACKNOWLEDGMENTS I am grateful to Drs. J. Cassidy, A. Chovnick, L. T. Douglas, R. F. Grell, E. A. Koch, P. A. Roberts, R. Rosen, L. Sandler, P. A. Smith, D. T. Suzuki, C. Stern, and J. H. Taylor for their constructive criticisms of early drafts of this article. The line drawings were prepared by Mr. E. John Pfiffner. The research was supported by the National Science Foundation (Grant G B 7457).

REFERENCES Aggarwal, S. K., and King, R. C. (1969). J . Morphol. 129, 171-200. Alexander, M. L. (1952). Texa.r, Univ. Publ. 5204, 219-226. Anderson, E. G. (1925). Genetics 10, 403-417. Atkins, L., Bartsocas, C. S., and Porter, P. J. (1968). J. Med. Getzet. 5, 314-318. Baker, W. K. (1958). A m . Naturalist 92, 59-60. Barigozzi, C., DoIfini, S., Fraccaro, M., Raimondi, G. R., and Tiepolo, L. (1966). Exptl. Cell Res. 43, 231-234. Beadle, G. W. (1932). Proc. Natl. Acad. Sci. U S . 18, 160-165. Beadle, G. W., and Emerson S. (1935). Genetics 20, 192-206. Belling, J. (1929). Univ. Calif. (Berkeley) Publ. Botany 14, 379-385. Berendes, H. D., and Meyer, G. F. (1968). Chromosomu 25, 184-197. Bogdanov, Y . F., Liapunova, N. A,, Sherudilo, A. I., and Antropova, E. N . (1968). Exptl. Cell Rer. 52, 59-70. Bretscher, M. S. (1968). Nature 127, 509-511. Bridges, C. B. (1915). J . Exptl. Zool. 19, 1-21. Bridges, C. B. (1927). f. Gen. Phyriol. 8, 698-700. Bridges, C. B. (1935). J . Heredity 26, 60-64. Bridges, C. B. (1938). J . Heredity 29, 11-13. Bridges, C. B., and Anderson, E. G. (1925). Genetjcs 10, 415-441. Bridges, C. B., and Bridges, P. N. (1939). J. Heredity 30, 475-476. Bridges, P. N. (1941a). J. Heredity 32, 64-65. Bridges, P. N. (1941b). J . Heredity 32, 299-300. Bridges, P. N . (1942). J. Heredity 33, 403-408. Brinton, C. C., Gemski, P., and Carnahan, J. (1964). Pruc. Nutl. Acud. Sci. U.S. 52, 776-783. Brown, E. H., and King, R. C. (1964). Growth 28, 41-81. Brown, S. W., and Zohary, D . (1955). Genetics 40, 850-873. Callan, H. G., and Lloyd, L. (1956). Nature 178, 355-357. Carlson, H. L. ( 1 9 4 6 ) . Genetics 31, 95-113. Chandley, A. C. (1766). Exptl. Cell Res. 44, 201-215. Chiang, K. S., and Sueoka, N. (1967). J. Cell. Physiol. 70, Suppl. 1, 119-145. Chino, M. (1937). hTjppon Idengaku Zasshi 13, 100-120. Chovnick, A. (1766). Proc. Roy. Soc. (London) B64, 198-205.

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Clever, U. (1768). Ann. Rev. Genet. 2, 11-27. Comings, D. E. (1768). Am. J. Human Genet. 20, 440-460. Court Brown, W. M. (1767). “Population Cytogenetics.” North-Holland Publ., Amsterdam. Creighton, H. B., McClintock, B. (1731). Proc. Natl. Acad. Sci. US. 17, 492-477. Cummings, M. R., and King, R. C. (1969). J. Morphol. 128, 427-442. Darlington, C. D. (1929). J. Genet. 21, 17-56. Das, N. K. (1962). J. Cell Biol. 15, 121-130. Dobzhansky, T. (1731). A m . Naturalist 65, 214-232. Dobzhansky, T. ( 1934). Z. InduRtive Abstammungs-Vererbungslehre 68, 134-162. Dobzhansky, T., and Sturtevant, A. H. (1931). Carnegie lnst. Wash. Publ. 421, 29-59. DuPraw, E. J. (1968). “Cell and Molecular Biology.” Academic Press, New York. Erhan, S. (1768). Ndture 219, 160-162. Gassner, G. (1969). Chromosoma 26, 22-34. Gowen, J. W. (1933). J. Exptl. Zool. 65, 83-106. Graubard, M. A. (1932). Genetics 17, 81-105. Graubard, M. A. (1934). Genetics 19, 83-94. Grell, R. F. (1757). Genetics 42, 374. Grell, R. F. (1962). Proc. Nutl. Acad. Sci. US.48, 165-172. Grell, R. F. (1964). Proc. Natl. Acad. Sci. US.52, 226-232. Grell, R. F. (1966). Genetics 54, 411-421. Grell, R. F. (1967). J. Cell. Physiol. 70, Suppl. 1, 117-146. Grell, R. P., and Chandley, A. C. (1965). Proc. Natl. Acad. Sci. U.S. 53, 1340-1346. Hart, P., and Sandler, L. (1961). J. Heredity 52, 160-162. Hayman, D. L., and Parsons, P. A. (1760). Genetica 32, 74-88. Henderson, S. A. (1761). Chromosoma 12, 553-572. Henderson, S. A. (1963). Heredity 18, 173-190. Henderson, S. A. (1961). Chromosoma 15, 345-366. Henderson, S. A. (1966). Nutuie 211, 1043-1047. Hewitt, G. M. (1967). Chromosoma 21, 285-295. Hinton, C. W. (1965). Genetics 51, 971-982. Hinton, C. W. (1766). Genetics 53, 157-164. Hinton, C. W. (1967). Can. J. Genet. Cytol. 9, 711-716. Hotta, Y., and Stern, H. (1963). J. Cell Biol. 19, 45-58. Hotta, Y., Ito, M., and Stern, H. (1966). Pror. Natl. Acad. Sri. U S . 56, 118f-llyl. Hotta, Y., Parchman, L. G., and Stern, H. (1968). Proc. Nutl. Acad. Sri. U . S . 60, 575582. Howard, E. F., and Plaut, W. (1968). J. Cell Biol. 39, 415-429. Hughes, R. D. (1739). Genetics 24, 811-834. Ito, M., Hotta, Y., and Stern, H. (1967). Develop. Biol. 6, 54-77. Jacob, F., and Monod, J. (1961). J . Mol. Biol. 3, 318-356. Jacob, F., and Monod, J. (1963). In “Cytodifferentiation and Macromolecular Synthesis” (M. Locke, ed.), pp. 30-64. Academic Press, New York. King, R. C. (1957). Growth 21, 75-102. King, R. C. (1760). Growth 24, 265-323. King, R. C. (1964). I n “Insect Reproduction” (K. C . Highnam, ed.), pp. 13-25, Roy. Entomol. SOC.,London. King, R. C., and Koch, E. A. (1963). Quart. J. Microscop. Sci. 104, 297-320. King, R. C., Rubinson, A. C., and Smith, R. F. (1956). Growth 20, 121-157.

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The Nucleus: Action of Chemical and Physical Agents RENB SIMARD Lahvratoire de Biologie MolPculaire, Facult; de Midecine UniversitP de Sherbrooke, Sherbrooke, Canada I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Normal Nuclear Fine Structure . . . . . ............... A. The Nucleolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Structural Support for RNA Synthesis in the Nucleus . . . . IV. Agents That Primarily Affect the Nucleolus . . . . . . . . . . . . A. Nucleolar Segregation and Actinotnycin D . . . . . . . B. Nucleolar Degranulation and Supranormal Temperature C. Nucleolar Hypertrophy and Thioacetamide . . . . . . . . . . D. Nucleolar Fragmentation and Ethioniiie . . . . . . . . . . . . V. Agents That Primarily Affect the Nucleus . . . . . . . . . . . . . . A. Margination of Chromatin and Proflavin . . . . . . . . . . . . A. Perichromatin Granules: Aflatoxin and Lasiocarpine . . C. Interchromatin Granules . . . . . . . . . . . . . . . . . . . . . . . . D. Nuclear Inclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary and Concluding Remarks . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

160 170

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188 193 19s 195 198 200 200 203 205

I. Introduction An increasing number of new antimetabolites and antibiotics have been studied in recent years by means of biochemical and biophysical methods. Their effects have permitted the production of specific lesions in the cell, thus clarifying our understanding of the behavior and metabolism of macromolecules. As a result thereof, the biochemist is now able to block or to modify the rate of a given reaction at will in order to create ideal experimental conditions pertaining to his research on the cell wall, nucleic acids, or protein synthesis. So far, however, the lack of parallel systematic investigations at the ultrastructural level has made it impossible to carry out structure-function studies that would help considerably to elucidate the complex structural arrangement of cellular organelles. In this chapter, an attempt will be made to demonstrate that chemical and physical agents can lead to selective cytological lesions provided that the treatment is suitably chosen in order to dissociate specific target effects from general cytotoxicity involving the whole cell. Special attention will be given to the nucleus and its ultrastructural modifications after treatment of cells with various antimetabolites. In some cases, the specificity of lesions induced by substances having similar molecular action will be used to explain the normal structural 169

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support by which nucleic acids and proteins are formed, stored, and transported from the nucleus to the cytoplasm.

11. Normal Nuclear Fine Structure A. THENUCLEOLUS Excellent reviews on nucleolar fine structure have been published in past and recent years (Swift, 1959a; Bernhard, 1966; Bernhard and Granboulan, 1968; Hay, 1968), and it is not our intention to duplicate them. There are four distinct nucleolar components in most mammalian cells: (1) 150- to 200-A dense granules randomly dispersed in the nucleolus, (2) a loose fibrillar reticulum composed of 50- to 80-A fibrils, (3) an aniorphous matrix of low electron opacity, and (4) nucleolus-associated chromatin with intranucleolar ramifications (Fig. la). Other terms used to describe nucleolar structures are nucleolonenza, which designates the loose anastomosing fibrillar network, and nucleolar body, which refers to the nucleolus without its associated chromatin. First described by Borysko and Bang (1951) and by Bernhard et al. (1952, 1955), the granular and fibrillar components are partially extracted with ribonuclease and disappear completely if digestion with the nuclease is followed by pepsin (Marinozzi, 1963, 1964, Marinozzi and Bernhard, 1963). They can be referred to as granular and fibrillar ribonucleoproteins (RNP) . The granular RNP are similar to the cytoplasmic ribosomes in their staining properties, but they are smaller, more irregular, and never arranged in subunits. Thin filaments about 20 A in width have been demonstrated in the granules; these filaments are RNase-sensitive (Smetana et al., 1968a). Transitional forms frequently occur between fibrillar and granular RNP (Marinozzi, 1963). The pars amorpha is completely extracted by pepsin digestion alone. The nucleolus-associated chromatin forms a ring around the nucleolus (Caspersson, 1950) with various amounts of intranucleolar ramifications according to the cell type (Swift, 1962b). It is extracted with desoxyribonuclease (Granboulan and Granboulan, 1964), but it can be best observed when ribonuclease is used to remove the RNP and enhance the contrast of the deoxyribonucleoproteins (Yotsuyanagi, 1960). The associated chromatin fibers, 70-100 A in width, are composed of coiled and uncoiled filaments 20-25 A in width (Smetana et al., l968b). The four components, including the associated chromatin, are integrally preserved during isolation procedures (Frayssinet et al., 1968) (Fig. l a and b).

B. THENUCLEUS The chromatin distributed along the nuclear membrane appears in a condensed form in cells fixed with aldehydes and osmium, thus delimiting an electron-

FIG. 1. Fine structure of isolated nucleoli from rat liver cells. (a) Nucleolar pellet fixed with glutaraldehyde followed by osmic tetroxide and embedded in epon. The granular component (g) is dispersed in a fibrillar network ( f ) with occasional amorphous material ( p ) . Arrows point to perinucleolar associated chromatin. x 40,000. ( b ) Nuclear pellet fixed in glutaraldehyde and embedded in GMA. Thin sections were digested with pepsin and RNase. Most of the nucleolar body ( N u ) has been extracted, while the contrast of the nucleolus-associated chromatin is enhanced (arrows). x 15,000.

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translucent interchromatin space in which numerous particles can be observed. This space contains a diffuse form of chromatin and probably a large amount of proteins. Within the condensed chromatin, Swift (1962b) described a special type of granules called “perichromatin granules” (Watson, 1962). These granules appear as isolated spherical bodies measuring 350-450 A in diameter and surrounded by a clear halo of 200 A that stands out in the condensed masses of chromatin. The perichromatin granules can also be found in the nucleolus-associated chromatin. Recent cytochemical studies have shown that these granules are highly contrasted by a preferential stain for RNP based on the use of chelating agents (Bernhard, 1968) (Fig. 2a and b ) . They resist DNase extraction in glycol methacrylate-embedded sections but are attacked by RNase and pronase digestions (Monneron et al., 1968). The cytochemical properties of perichromatin granules relate them to RNP particles. They are present in various amounts in the nuclei of most norinal or cancer tissues whatever the cell type but are less frequent in tissue culture cells. Similar granules have been described in differentiating embryonic cells (Hay, 1958; Hay and Revel, 1963) ; except for the fact that they are not always located in masses of condensed Chromatin, these granules are similar in size (350-450 A ) and cytochemical properties to perichromatin granules (Simard and Duprat, 1969). Amphibian oocytes (Gall, 1956; Lane, 1967), dipterian salivary glands (Swift, 1962b; Jacob and Sirlin, 1963), and Chironomus salivary glands (Stevens, 1964; Stevens and Swift, 1966) also have nuclear granules similar to perichromatin granules. Recent structural studies of the interchromatin space have shown that the organization in the nucleus is far more complicated than had previously been thought. Cytochemical and biochemical studies have demonstrated that a complex RNP network exists in most mammalian cells. Within this network, only one component has been characterized: the interchromatin granules (Swift, 1959b; Ris, 1962; Granboulan and Bernhard, 1961). These granules measure 200-250 A in diameter and have been shown to resist most enzymic digestions (DNase, RNase, and pronase) on thin sections embedded in glycol methacrylate (GMA) (Monneron, 1966). The presence of RNA in these granules, however, is suggested by the fact that they are strongly positive to the preferential stain for RNP (Bernhard, 1968) and react accordingly to various fixatives. These granules have been identified as ribosomes (Frenster et al., 1960; Sarnarina and Georgiev, 1960) although neither their structural nor their metabolic properties have so far proved to be similar to those of cytoplasmic ribosomes. Apart from interchromatin granules, a heterogenous population of particles, granular or fibrillar, are found in the interchromatin space, and a common denominator has yet to be proposed to classify them. Busch et al. (1963) and Smetana et al. (1963) have stated that the complex interchromatin network is composed of

FIG. 2. Nuclei of rat liver cell. (a) Typical aspect of nuclear structures after glutaratdehyde-osmium fixation and epon embedding. Arrows point to pericliromatin granules 10cated within the condensed chromatin. A large number of particles can be seen in the nucleoplasm (ncl). x 15,000. ( b ) Section contrasted by a preferential stain for RNP based on the use of EDTA. Interchromatin granules (ig) and perichromatin granules (arrows), as well as the nucleolus are strongly contrasted; chromatin masses (chr) have lost their usual electron density. Glutaraldehyde. Epon. (Courtesy of Dr. W. Bernhard.) X 30,000.

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RNA and proteins; fibrils which form a major part of the network are masked by the presence of nucleoproteins (Narayan et ul., 1966) and probably constitute the preferential site of synthesis of adenine uridine-rich RNA (Steele and Busch, 1966b) (Fig. 2a and b).

+

111. Structural Support for RNA Synthesis in the Nucleus Although numerous reviews have appeared in recent years on the biochemical steps involved in RNA synthesis in the nucleus and nucleolus (Georgiev, 1967; Perry, 1964, 1967; Penman et ul., 1966), few correlative studies have dealt with its ultrastructural support. The difficulty of this approach is obvious since most of the techniques for fractionation do not permit ultrastructural and cytochemical studies. In the nucleolus, the synthesis of RNA was first demonstrated by light radioautography (Harris, 1959; Amano and Leblond, 1960; Leblond and Amano, 1962). The associated metabolic events have been studied at the electron microscope level. Short pulses (10-minute) of uridine-3H result in heavy labeling of the fibrillar RNP components, and longer pulses (30-minute) of both the fibrillar and granular RNP components (Granboulan and Granboulan, 1965; Karasaki, 1965) (Fig. 3a and b ) . These experiments have now been repeated by different investigators in various laboratories, using a variety of material with similar results: cultured cells (Geuskens and Bernhard, 1966; Simard and Bernhard, 1967), ascites tumor cells (Unuma et al., l968), Chironomus (Von Gaudecker, 1967), and Smittia (Jacob, 1967). In ascites tumor cells, the same batches of cells labeled for 10 minutes with uridine-3H were processed through high-resolution radioautography and density gradient centrifugation analysis. Labeling of the fibrillar RNP component corresponded with that of the 45 S RNA in the nucleolus (Amalric et ul., 1969; Simard et al., 1969). There seems to be little doubt that the 45 S RNA is associated with the fibrillar RNP component in the nucleolus in the form of a nascent subribosomal particle sedimenting at 80 S (Tamaoki, 1966; Tamaoki and Mueller, 1965; Warner and Soeiro, 1967). Synthesized as a single polynucleotide chain, the 45 S is rapidly cleaved in 35 S (or 3 2 S) and 18 S RNA. The latter leaves the nucleus immediately, while the former undergoes a subsequent transformation resulting in a 28 S RNA fraction (Scherrer and Darnell, 1962; Scherrer et ul., 1963; Rake and Graham, 1964; Penman, 1966; Muramatsu et ul., 1966). Both the 35 S (or 32 S) and the 28 S RNA are found in the nucleolus of HeLa cells associated with particles sedimenting, respectively, at 5 5 S and 50 S (Warner and Soeiro, 1967). The association of these particles with either the granular or the fibrillar component of the nucleolus has not yet been determined. In the nucleus, the complexity arises from the apparent lack of fine-structural

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support for reactions that challenge biochemical analysis. Labeling of interchroinatin space begins after a 10-minute pulse of uridine-W, but the resolution of radioautography does not permit the association of radioactivity with any specific nuclear structure. A 30-minute labeling results in more pronounced radioactivity in the nucleus. After 1 hour of incorporation, cytoplasmic activity can be detected deriving from both the nucleolar and non-nucleolar sites of RNA synthesis (Fig. 3b). Several types of RNP particles have been isolated from the deoxyribonucleoprotein fraction and the nuclear sap fraction (Frenster et dl., 1960; Muramatsu and Busch, 1964; Samarina et al., 1965, 1966, 1967). From both fractions, these authors have isolated a polydisperse, rapidly labeled RNA which is DNA-like and possesses messenger properties. Mod6 and Chauveau (1968) have recently obtained a 40 S particle containing a rapidly labeled 3.4 S RNA. A complete cytochemical study at the electron microscope level revealed that this particle is comparable to interchromatin granules (Monneron and Moule, 1968).

1V. Agents That Primarily Affect the Nucleolus The search for cytological clues to correlate the function and structure of the nucleus encompasses the use of model systems and integrated ultrastructural and biochemical studies. The nucleolus has been a particularly rewarding field in this respect as it seems to react rather specifically to various chemical and physical agents. Four major ultrastructural modifications of nucleolar components appear to be directly related to a particular mode of action: ( 1 ) nucleolar segregution (actinomycin D) , ( 2 ) degrunuhtion (supranormal temperatures), ( 3 ) hypertrophy (thioacetaniide) , and (4) fragrnenfadtioiz (ethionine and 5-ffuOrOuracil). A. NUCLEOLAR SEGREGATION AND ACTINOMYCIN D 1.

T h e Effect of Actinomycin 1) on the Nucleolzis

The first cytological observations relating the action of actinomycin to the nucleolus were performed with time-lapse cinematography (Robineaux et ul., 1958) and phase contrast microscopy (Bierling, 1960). The development of actinomycin-resistant and -sensitive HeLa cells enabled Goldstein et ul. (1960) and Journey and Goldstein (1961) to describe nucleolar disruption and what is now known as nucleolar segregation affecting only the sensitive strain. Reynolds et al. (1963, 1964) observed the same phenomenon: they described it as “nucleolar cap” formation induced by both the carcinogen 4-nitroquinoline N-oxide and actinomycin D and suggested that the lesions could represent the morphological expression of a specific biochemical action. Cytochemical studies made by Schoefl (1964) demonstrated that actinomycin D causes coalescence of

FIG. 3 .

Ascites tumor cells labeled with Liridine-aH. Gevaert NUC 307 emulsion.

( a ) After 10 minutes of labeling, the silver grains are located in the fibrillar portion ( f ) of the nucleolus while the granular portion ( g ) is inactive. x 35,000. ( b ) After

30 minutes of labeling, the silver grains are located in both portions of the nucleolus. 14,000. Activity can also be noted i n the nucleus.

x

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three nucleolar components: (1) RNP granules embedded in a protein matrix, ( 2 ) the nucleolonema, and ( 3 ) an amorphous matrix. The effect of actinomycin D has since been shown to be more or less identical on various types of cells: rat liver (Stenram, 1965; Smuckler and Benditt, 1965; Oda and Shiga, 1965; Smetana et ul., 1966; Shankar Narayan et ul., 1966), rat pancreas (Jezgquel and Bernhard, 1964; Rodriguez, 1967) ; rat salivary gland (Takahama and Barka,

FIG. 4. Segregation of nucleolar components in rat embryonic cell induced by nogalainycin treatment (1 &in1 during 1 hour). The granular zone (g), fibrillar zone ( f ) , and amorphous zone ( p ) haye been redistributed. Osmium tetroxide. Epon. x 20,000.

1967), Chiyoizomus (Stevens, 1964), Smittirl (Jacob and Sirlin, 1964) ; amphibian tissue (Eakin, 1964; Jones and Elsdale, 1964; Siniard and Duprat, l969), and leukemic myoblasts (Heine et ul., 1966). It can be generalized, therefore, that the first cytological target effect of actinomycin D is separation and redistribution of nucleolar components; the term “nucleolar segregation” is now 1965) ( i i g . 4 ) . acceptable in describing this type of lesion (Bernhard et d., 2. Seyueiztial Descriptioiz o f the Lesioiz

In most instances, whether induced by actinomycin D or other substances, nucleolar segregation follows the same sequential steps if careful attention is given to both dosage and duration of treatment. (1) At very low doses, the nucleolus first takes the form of a compact sphere

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with condensation of the fibrillar portion and migration toward the periphery. Proportionally, the granular zone is of greater importance; ( 2 ) segregation of the nucleolar components then occurs, resulting in distinct granular, fibrillar, and amorphous portions; (3) longer exposure to the inhibitor causes dispersion

FIG. 5 . Morphological sequential events of the lesions of nucleolar segregation. The nucleolus first takes the form of a sphere (1); then, segregation occurs ( 2 ) . The granules leave the nucleolus ( 3 ) , and a fibrillar mass is the end phase ((I). RNP fibrils ( f ) ; RNP granules ( g ) ; amorphous portion ( p ) ; contrasted zone (sc) ; nucleolar chromatin (chr) .

of the granular zone which is seen to migrate from the nucleolus toward the nucleus; ( 4 ) subsequently, all that is left is a mass of closely packed fibrils with an occasional amorphous zone (Fig. 5 ) . This sequence has been observed repeatedly and constantly regardless of the agent used to induce segregation of nucleolar components in tissue culture. Enzymic digestion of each zone has been carried out on segregated nucleoli: both

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179

the granular and fibrillar zones are extracted by RNase while the amorphous zone is pepsin-sensitive (JCzCquel and Bernhard, 1964). Occasionally, a fourth zone is seen in the periphery of segregated nucleoli. This zone is electron-dense and granular and has been successively referred to as “blebs” or “satellites” (Schoefl, 1964), “dense plaques or caps” (Reynolds et al., 1964) , “new peripheral dense substance” (Stevens, 1964), “contrasted fourth zone of unknown etiology” (Bernhard et ul., 1965; Simard and Bernhard, 1966), and “microspherules” (Unuma and Busch, 1967). Agreement bas been reached as to the content of the zone, which has been shown to react strongly to pepsin and RNase (Schoefl, 1964; Stevens, 1964; Unuma and Busch, 1967). Monneron (1968), however, recently presented convincing evidence that these dense masses actually represent a clustering of perichromatin granules (see Section V, B) . 3. Specificity of NucEeoldr Segregution

In view of the variety of chemically unrelated substances capable of causing nudeolar segregation, a systematic study was undertaken in order to determine the level of the biochemical block responsible for morphological lesions. Cultured cells of rat embryos were treated with various antimetabolites, analogs, and antibiotics. The compounds were chosen because of their wide use, their known biochemical action, and their site of nucleic acid attack or protein synthesis. Substances were employed at the lowest concentration capable of inducing a characteristic cytological lesion. In most instances, higher doses resulted in nonspecific cytotoxic effects (Fig. 6). Two conclusions could be drawn from this study (Simard and Bernhard, 1966). First, nucleolar segregation is a specific lesion. Neither antimetabolites acting at the level of nucleotide precursors, nor those interfering with polynudeotide incorporation or protein synthesis were associated with the characteristic nudeolar lesion. One exception to this, although more apparent than real, was azaserine. This antibiotic is a known glutamic acid antagonist (Hartman et ul., 1955) but possesses an unsaturated diazo group capable of nucleophilic substitution. Furthermore, it behaves like an alkylating agent at appropriate doses, assuming mutagenic (Iyer and Szybalski, 1958), antimitotic (Maxwell and Nickel, 1954), and radiomimetic properties (Terawaki and Greenberg, 1965). All other substances causing nucleolar segregation were part of a group of compounds binding directly to the D N A molecule and interfering with its template activity. Second, nucleolar segregation corresponds to a blocking of nucleolar W A synthesis that is of nucleolar function. Among the 2 4 substances used, the compounds causing nucleolar segregation blocked the enzymic synthesis of RNA by

RENB

180

SIMARD

RNA polymerase by the formation of complexes with DNA in the manner of actinomycin D (Reich, 1964) or proflavin (Hurwith et al., 1962). These compounds are: echinomycin (Ward et al., 1965), nogalamycin (Bhuyan and Smith, 1965), chromomycin A (Ward et al., 1965), daunomycin (Di Marco et ul., 1963; Calendi et al., 1964), and proflavin and ethidium bromide (Lerman, 1961; Luzzati et al., 1961; Waring, 1966). I

TI dATP dGTP dCTP dTTP

Precursors ___c

4

Ribonucleotides

- - - - - - - - - - - _ -..-

I

DNA Polymerase

RNA

I

j

I

E

ll

I

Precursors

L

DNA

I

A

SN

II 5-Iododeoxyuridide 5-Bromodeoxyuride Cytosine arabinoside 5- Fluorourocil Thymidine (excess)

I

_____ -_____--- J

Amino acids

Aminopterin Azaserine (weak dose) 6-Mercaptopurine Alkaloids of vinca rosea

1

Polymerase

RNA

m

m Alkyloting agents Antibiotics Amino acridines Ethidiurn bromides Histones (excess) Po I y I ys I ne

=

Protein

Ip Azaguanine Chloramphenicol Puromycin Alkaloids of vinco rased

Proflovin Chromomycin A 3 Ec hinom ycin Nogalornycin Ethidium bromides Azaserine Dounomycin

Support for this hypothesis has recently been obtained. Using varying doses of actinoinycin D, Goldblatt et al. (1969a) observed that nucleolar segregation is a reflection or response of some alteration of DNA rather than the consequence of inhibition of RNA synthesis. Interference with RNA synthesis alone is unlikely to explain nucleolar damage (Goldblatt et al., 1969b). Other authors have reviewed the effects of selected hepatocarcinogens and stressed the coincidence of nucleolar segregation with the blocking of RNA synthesis in rat liver intoxicated by lasiocarpine, 3-methyl-4-dimethylaminoazobenzene, dimethylnitrosamine, and tannic acid (Reddy and Svoboda, 1968).

NUCLEAR A N D KUCLEOLAR LESIONS

181

4. Nzicleolur Segrexatioii Itzduced by Other Ageizts An increasing number of substances are known to affect the nucleolus in a specific manner. The potent carcinogen aflatoxin causes rapid segregation of nucleolar components in rat liver (Bernhard et al., 1965; Svoboda et ul., 1966). 112 vifro binding of the toxin to D N A has been suggested (Sporn et al., 1966; Clifford and Rees, 1767) in order to explain its action on RNA synthesis (Lafarge et ul., 1965; Clifford and Rees, 1967), particularly at the nucleolar level (Lafarge et al., 1966). Another carcinogen, 4-nitroquinoline-N-oxide (Endo, 1958), similarly affects the nucleolus (Reynolds et al., 1963), and recent works suggest that it reacts with nascent D N A (Malkin and Zahalsky, 1966) and blocks the action of RNA polymerase (Paul et ul., 1967). The alkylating antibiotic mitomycin C (Iyer and Szybalski, 1963) induces the same nucleolar lesions (Lapis and Bernhard, 1965) but only after long exposure, probably because its action on RNA synthesis is secondary (Kuboda and Furuyama, 1963). Ribonuclease (Robineaux et al., 1967), ultraviolet flying-spot irradiation (Montgomery et al., 1966), and a-amanitin, a toxin from “Aniulzitu phulloides” (Viume and Laschi, 1965), also belong to the same group of agents primarily affecting the nucleolus and RNA synthesis (Perry et ul., 1961; Fiume and Stirpe, 1966). The antibiotic mythramycin can also be added to this list (Kurne et ul., 1967). Hydroxyurea, a substance which reacts with D N A in vivo (Eisenberg et a1.: 1965), induces the same nucleolar lesions in amphibian embryos (Geuskens, 1968). Other stimuli for nucleolar segregation cannot be related to a direct action on RNA synthesis because of their as yet undefined mode of action; the antibiotic anthraiiiycin (Harris et al., 1968a), cyclohexinzide (Harris et al., 1968b), and puromycin aminonucleoside (Lewin and Moscarello, 1968) have recently been related to this phenomenon. Micrographs of typical nucleolar segregation have been obtained in cultured cells infected by herpesvirus (Sirtori and BosisioBestetti, 1967) and mycoplasma (Jkzkquel et ul., 1967). 5.

R N A Synthesis in the Presence

of Acfinomycin

D

Light microscope radioautography has revealed severely decreased incorporation of RNA precursors following actinomycin D treatment in tissue cultures (Schoefl, 1964) and rat liver (Stenram, 1965). High-resolution radioautography further demonstrated that when pulses of uridiiw3H precede actinomycin D treatment, accumulation of the radioactivity is found over the granular portion of the segregated nucleolus, although a persistent labeling of the fibrillar zone subsists (Geuskens and Bernhard, 1966). Microbeam experiments (Perry et al., 1961) and low doses of actinornycin D

182

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(Perry, 1962, 1963) provided biochemical evidence that the 45 S RNA synthesized in the nucleolus is a precursor of ribosomal RNA (Perry, 1963), a conclusion reached earlier by Scherrer and Darnell (1962). Although synthesis of the 45 S RNA is blocked by the antibiotic, transformation of the previously labeled 45 S RNA can still proceed, resulting in the accumulation of 28 S and 6 S RNA in the nucleolus (Muraniatsu et al., 1966) since the 18 S fraction is

FIG. 7. Hamster fibroblast treated with actinomycin D-3H for 60 minutes. Ilford L-4 emulsion. The silver grains are located mostly on the condensed portion of the nuclear chromatin and around the nucleolar body on the nucleolus-associated chromatin (arrows),

x

12,000.

not affected (Steele and Busch, 1966b). It seems that the action of actinomycin D is mediated through a preferential binding to the guanine residues of DNA (Reich, 1964). Nucleolar D N A has been shown to contain a high proportion of guanine and cytosine (McConkey and Hopkins, 1964). The expectation that actinoinycin D would bind preferentially to nucleolar DNA has been partly confirmed by highresolution radioautography. Incorporation of actinoinycin D-SH into cultured BHK cells resulted in accumulation of radioactivity in the condensed portion of the chromatin whether associated to the nucleolus or not, but with occasional ring formation around the nucleolar body (Fig. 7 ) . The amount of labeling was found to be time dependent, but concentration seemed to play only a minor role (Simard, 1967; Siinard and Cassingena, 1969). DEGRANULATION AND SUPRANORMAL TEMPERATURE B. NUCLEOLAR 1. Cytologicdl Effect of Supranorind Tempe?atuw Supranormal temperature has been used for a long time to synchronize cell culture (Juul and Kemp, 1933), following the work of Bucciante (1928) re-

NUCLEAR AND NUCLEOLAR LESIONS

183

lating the effect of temperature to the cell cycle, particularly to mitosis (Rao and Engleberg, 1965; Sisken et al., 1965). Fusion and condensation of nucleoli in Transdescmtiu after temperature exposure have been reported from phase contrast microscopy (Snoab, 1955). Systematic electron microscope and cytochemical studies were carried out on cultured hamster fibroblasts (BHK strain) after exposure to supranormal temperatures (Simard and Bernhard, 1967). At temperatures of 38", 39O, and 40"C, no noticeable lesion occurs even after an incubation of 120 minutes. The cells continue to grow normally when transferred to a new medium and cultured at 37°C for 24-48 hours. At 4 l o C , early changes appear in some nucleoli after 1 hour of incubation; there is a fading out of the nucleolus reticular aspect and a decrease in the granular RNP particles which present as fuzzy and cloudy spots. A critical point is reached at 42"C, with the appearance of striking nucleolar lesions. As early as 15 minutes after treatment, but of course more pronouncedly so after 1 hour, there is a complete loss of the granular RNP component and a disappearance of the nucleolar reticulum, associated with a complete retraction of the intranucleolar chromatin. The remaining material in the morphologically homogeneous nucleolus is a large amount of RNase-sensitive closely packed fibrillar RNP. The Iesions remain identical as the temperature is increased to 45°C. These alterations prove to be reversible when the cells are returned to 37"C, with the reappearance of an exaggerated amount of intranucleolar chromatin and granular RNP leading to nucleoli of considerable size. These nucleolar lesions occurred in otherwise well-preserved cells. Identical lesions were observed on normal diploid rat embryonic cells in exponential growth subjected to the same treatment. Ascites tumor cells were found to react similarly, but the critical temperature was 44.5"C (Pig. 8). DegranuIation of nucleoli was observed in enibryonic differentiating cells of the amphibian Plearodeles Waltlii incubated at 37°C for 5 hours (Duprat, 1969). 2.

Sequential Appearance of the Lesions

In ascites tumor cells, complete degranulation was obtained only after 30 minutes of treatment at 45 "C. Systematic cytochemical and morphological studies were carried out on the same batches of cells incubated at 44.5OC for periods of time varying from 5 to 30 minutes. After 10 minutes of heat shock, intranucleolar chromatin is compIetely absent from the nucleolus, while the granular RNP are still present; the granules disappear only after 30 minutes (Fig. 8). A similar sequence of events was found after 30 minutes of incubation at increasing temperatures of 39-40 "C. Retraction of intranucleolar chromatin was

184

RENB

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completed at 43"C, while the granular RNP disappeared as previously only at 44.5OC. It seems, therefore, that whenever cells are exposed to supranormal temperatures, retraction of intranucleolar chromatin precedes the degranulation of the nucleolus, an observation that has proved to be of significant importance for functional studies (Simard et al., 1969).

FIG. 8 . Nucleolar degranulation induced by supranormal temperature (44.5"C for 30 minutes) in ascites tumor cells. The nucleolus has rounded up and lost its reticular aspect as well as its granular component. It is now homogenous and consists of closely packed electron-dense fibrils. x 30,000.

3. Specificity of Nucleolar Degrdnulation

Whether or not supranorinal temperatures affect the iiucleolus in a specific manner cannot yet be ascertained. There exists for most biological reactions an optimum temperature responsible for a given equilibrium (Lwoff and Lwoff, 1961; Lwoff, 1962). The ultrastructural lesions induced by thermic shock are striking, however, and concern only the nucleolus; apart from a slight clumping of chromatin, the nucleus is not altered and no lesions have ever been observed in the cytoplasm. The lesions observed in the nuclei of degenerating and dead cells have been described by Trump et al. (1965). The reticular aspect of the nucleolus becomes

NUCLEAR A N D NUCLEOLAR LESIONS

185

blurred after 4 hours of autolysis, and the nucleolar RNP granules disappear after 8-1 2 hours. These changes, however, take place concomitantly with severe nuclear and cytoplasmic damage and are not reversible. Moreover, they involve the whole cell, not a specific organelle.

4. Nvcleolar Degranulation Indi*ced with Other Agents Depletion of the granular component has been reported to occur in all nucleoli that undergo segregation of their components (see Section IV, A ) . In most instances, an end phase is reached with entirely fibrillar but small nucleoli resembling those exposed to supranormal temperatures. Recently, Ganotte and Rosenthal ( 1968) have shown that rneth3.'lu~ox3.'rnethu~~o~, a hepatotoxin derived from cycusin, causes an aborted nucleolar segregation rapidly followed by degranulation of nucleoli which then assume a clumped reticulated pattern with wide open meshes. The picture closely resembles that of lesioiis obtained with supranornial temperatures. Entirely fibrillar nucleoli are also observed during amphibian embryogenesis. The formation of a nucleolus is observed during gastrulation, with the appearance of dense fibrous bodies within the chromatin material (Karasaki, 1964, 1965, 1968). These fibrous bodies resemble primary nucleoli of early gastrula in the anucleolate mutant of Xenopw, which lacks the nucleolar orgnizer (Jones, 1965; Hay and Gurdon, 1967). 5.

RNA Sy?zthesis ut Sapranormal Temperature

Incorporation of uridine-SH was studied by high-resolution radioautography in hamster fibroblasts. Following a thermal shock of 43OC for 1 hour, the uptake of uridine-SH by the altered nucleolus was almost absent after 5-minute or 30-minute pulses, while incorporation over the nuclear dispersed chromatin is much less affected. An approximate grain count revealed a 90% reduction in the nucleolar incorporation of the precursor of heat-treated cells as compared with control cells, whereas the extranucleolar nuclear uptake was lowered by only 20% (Fig. 9a and b). When the nucleoli were given a 30-minute pulse of uridine-sH just prior to heat treatment to label both the fibrillar and the granular RNP, subsequent chases for 1 hour with cold uridine at both 37" and 113°C resulted in a heavy nucleolar labeling in both experiments without any appreciable difference in grain count. Similar results were obtained in ascites tumor cells maintained at 44.5 "C for 30 minutes. No incorporation of uridine -3H was found after such treatment, and retention of radioactivity in the nucleolus was observed when incorporation of the precursor preceded the thernial shock (Simard and Bernhard, 1967). Density gradient analysis of nucleolar RNA's was performed systematically on ascites tumor cells. In correlation with the sequential events in nucleolar de-

186

RENB

SIMARD

187

NUCLEAR AND NUCLEOLAR LESIONS

granulation induced by thermal shock, the specific activity of nucleolar 45 S RNA was recorded after a thermal shock of 44.5OC for increasing periods of time and after a thermal shock of 30 minutes at increasing temperatures. In both cases, a rapid decrease was observed with a point of inflection corresponding to the disappearance of intranucleolar chromatin (Fig. 10).

\

5

Temp (min)

10

15 20 Temp (min)

25

30

FIG. 10. Specific activity of the nucleolar 45 S RNA following exposure to supranorma1 temperature of 44 5°C for increasing periods of time. The point of inflection of the curve is at 10 minutes, corresponding to the disappearance of intranucleolar chromatin. Blocking of RNA synthesis is complete after 10 minutes.

Qualitative analysis of nucleolar RNA’s performed after thermal shock failed to show any differences in the control cells even when the granular RNP coniponent had disappeared ultrastructurally. The same curves showed that while the synthesis of rapidly labeled 45 S RNA was severely affected by thermal shock, a labeling of the 8-10 S region persisted throughout the experiment. Ribosomal RNA was not labeled after a thermal shock except once again in the 8-10 S region. When cells were pulse-labeled 30 minutes before a heat shock of 30 minutes FIG. 9. Labeling of hamster fibroblasts with 5-minute pulses of uridine-3H. Gevaert NUC 307 emulsion. ( a ) Untreated cell growing normally at 37°C. The incorporation is located in the fibrillar portion of the nucleolus; extranucleolar incorporation is also evident. x 36,000. ( b ) Cell treated at 43°C for 1 hour. No incorporation is seen over the nucleolus which shows the structural degranulating effect of thermal shock. Extranucleolar incorporation is less affected. x 24,000.

RZNB

188

SJMARD

at 44.5"C, accumulation of radioactivity was found in the nucleolus and no transport was observed in the cytoplasmic ribosomes. When, however, the same pulse-labeling was followed by a heat shock of 30 minutes at 43°C (the granular RNP being still present, while nucleolar RNA syr,thesis was blocked to the extent of S o n / , ) , transport appeared unimpaired in the ribosomes (Amalric et al., 1969; Simard et al., 1969).

6. Conchsions Several conclusions can be drawn from this model of nucleolar degranulation at supranormal temperature. (1) The synthesis of nucleolar RNA is heat-sensitive, in a reversible manner provided that the system permits recovery harvesting of cells (in tissue culture for example). Other authors have observed the thermosensitivity of RNA synthesis in different biological systems (Moner, 1967; Gharpure, 1965; Stevens, 1966, 1967). The precise level at which temperature affects the nucleolar RNA synthesis has still to be determined and probably results from simultaneous alterations of several factors. In any case, the critical points of variation of the specific activity of 45 S nucleolar KNA (10 minutes at 44.5"C and 30 niiiiutes at 4 3 ° C ) are concomitant with the retraction of intranuclear chromatin, the presence and integrity of which appears to be necessary for the synthesis of 45 S nucleolar RNA. ( 2 ) The granular RNP are probably transitory configurational forms, unraueling after thermal shock. One of their main functions appears to be the transport of nucleolar RNA to the cytoplasm. This function is blocked at supranormal temperatures even if the nucleolus has been previously heavily labeled. (3) The fibrillar RNP, then, consist of a stable pool of KNA's in which all ribosomal and possibly nonribosomal precursors could be stored. N o modifications are indeed observed in qualitative analysis of nucleolar RNA after thermal shock when nucleoli are entirely fibrillar. Similar conclusions have been reached by other authors. Geuskens and Bernhard (1966) explained the persistent labeling of the fibrillar zone after actinoinycin treatment as being consistent with the hypothesis that fibrils contain a stable pool linked with ribosonial and nonribosomal nucleolar functions. A similar conclusion was reached by Jones (1965) and by Hay and Gurdon (1967) to explain the presence of a ilbrillar pseudonucleolus in the homozygote anucleolate mutants of Xeiaopirs, which do not synthesize ribosomal RNA owing to deletion of ribosomal cistrons.

c.

NUCLEOLAR HYPERTROPHY AND THI0ACBTAAIII)II

1. Cytological A c t i o n of Thloacetamide ( T A A)

The effect of TAA on rat liver is characterized by hepatic cell damage that progresses to centrolobular necrosis and cirrhosis with sufficient dosage (Klein-

NUCLEAR A N D NUCLEOLAR LESIONS

189

feld, 1957; Ruttner and Rondez, 1960; Gupta, 1956). The hepatocarcinogenic properties of TAA were first described by Fitzhugh and Nelson in 1948 and later by various authors (Gupta, 1955; Jackson and Dessau, 1961). The first detectable cytological action of TAA in hepatocytes is a remarkable nucleolar hypertrophy (Rather, 1951; Kleinfeld, 1957) that was related to an increase in nuclear RNA synthesis (Rather, 1951; Laird, 1953) although the RNA itself was not demonstrably chemically different from normal RNA (Kleinfeld and Von Haam, 1959). Electron microscope studies following TAA administration have emphasized that sublethally injured rat liver undergoes rapid nuclear enlargement with frequent cytoplasmic inclusions and striking hypertrophy of the nucleolus (Rouiller and Simon, 1962; Salonion 1962; Salomon et al., 1962; Thoenes and Bannasch, 1962) (Fig. 11). Cytoplasmic lesions include an abnormal increase of agranular endoplasmic reticulum, hypertrophy of the Golgi complex, and mitochondria1 lesions (Ashworth et al., 1965). The giant nucleoli were found to be particularly rich in RNP granules by planimetry (Shankar Narayan et al., 1966), but it was later demonstrated that the proportion between granular and fibrillar RNP remained approximately the same when segregation of nucleolar components was induced in giant nucleoli by actinoniycin D (Suter and Salonion, 1966). The mechanism by which TAA causes hypertrophy of the nucleolus and hepatocellular injury is not understood. Furthermore, it constitutes the sole example of such nucleolar enlargement. Therefore, the question of the drug specificity of TAA-induced lesions cannot be discussed here. Previous studies with isotopically labeled TAA-35S have shown that instead of binding with protein or alkylating nucleic acids, the compound is rapidly broken down since no increase in radioactivity is found in the livers of rats fed TAA-”S (Nygaard et al., 1954; Maloof and Soodak, 1961). Rees et ul. (1966) studied the metabolism of TAA labeled with 3H on the methyl group instead of with 35S and found that the carcinogen is metabolized within 24 hours and converted to acetate and hydrogen sulfate. Many carcinogens have been found to undergo metabolic changes to a form in which interaction with cellular components can take place. In the case of TAA, the problem still remains of identifying an active form of its metabolite capable of interaction in order to explain the hepatocellular lesions among which nucleolar hypertrophy is the most striking. 2. Niicleolar

Hypertrophy in Other Conditions

In addition to TAA, other conditions exist in which nucleolar hypertrophy can be observed. MacCarty (1928, 1936) was the first to recognize nucleolar hypertrophy as a pathognomonic sign of cancer cells. The ultrastructure of malignant cell nucleoli exhibits wide variations in the proportion of components (Bernhard and Granboulan, 1963; Busch et al., 1963), having inclusions of all

191

NUCLEAR A N D NUCLBOLAR LESIONS

sorts (Haguenau, 1960; Thoenes, 1964) and occasionally granular material of unknown origin (Shankar Narayan and Busch, 1965). None of these lesions, however, can be considered typical of cancer cells. Hypertrophy of the nucleolus is a common feature in rapidly growing tissues such as embryonic cells or regenerating liver cells following partial hepatectomy (Higgins and Anderson, 1931; Bucher, 1963; Stenger and Confer, 1966). Enlargement of nucleoli has also been described following chronic ethionine intoxication in liver cells (Miyai and Steiner, 1965; Svoboda et al., 1967), isoproterenol administration in salivary glands (Takahama and Barka, 1967), and protein deficiency (Svoboda et ul., 1966). Starved animals fed with a lowprotein diet (Stenram, 1958, 1963) or a threonine-devoid diet (Shinozuka et al., 1968b) show enlargement of liver nucleoli. In most of these cases, the changes appear to be related to an increase in RNA and protein synthesis (Barka, 1966; Stenram, 1958; Sidransky and Farber, 1958; Sidransky and Recheigl, 1962; Sidransky et ul., 1964; Kleinfeld, 1966). 3. R N A Synthesis irz Nucleolar Hypertrophy Induced with

TAA

The effect of TAA on RNA synthesis is characterized by a rapid increase in iiucleolar RNA synthesis, while at the same time a decrease in cytoplasmic ribosomal RNA is observed (Laird, 1953; Kleinfeld and Von Hamm, 1959; Koulish and Kleinfeld, 1964). The increased rate of RNA synthesis in the nucleolus corresponds to high-molecular weight ribosomal precursors (Steele et al., 1965), as the sedimentation profile of RNA of nucleolar fractions shows a six- to eightfold increase in the relative amounts of 45 s, 35 S, and 2 8 S RNA with the appearance of a new peak of 55 S RNA (Steele and Busch, 1966a). Most of this RNA, however, does not reach the cytoplasmic ribosomes (Kleinfeld, 1966). It is now known that TAA alters the nuclear enzymes considerably. The increased nucleolar RNA synthesis is associated with an increased activity of the nucleolar RNA polymerase system in vitro (Villalobos et ul., 1964a) and of latent ribonuclease (Villalobos et al., 1964b). A clear interpretation of the facts leading to giant nucleoli and increased nucleolar RNA content has yet to be proposed to explain TAA action. Kleinfeld (1966) considers the possibility that TAA does not alter the biosynthetic process per se but activates the initial phase of ribosomal RNA transcription simply by increasing the number of sites open for transcription. The subsequent events would then only be part of regulatory control mechanisms resulting in a piling of this RNA in nucleoli and its subsequent breakdown, as only a normal amount is transported to the cytoplasm. The use of this model would, in any case, be rewarding if it yielded more information regarding the active form or metabolite of TAA capable of interacting with nucleic acids.

192

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NUCLEAR AND ISUCLEOLAR LESIONS

193

D. NUCLEOLAR FRAGMENTATION A N D ETHIONINE 1. Cytological Lesions Indztced

by Ethionine

The ultrastructural changes induced by ethionine, a methionine analog, have been known for some time (Herman and Fitzgerald, 1962; Herman et al., 1962), but only recently have they been documented systematically in liver cells (Miyai and Steiner, 1965; Shinozuka et ul., 1968a). Within the first few hours after administration of ethionine, there is a progressive decrease in the nucleolar size, with the appearance of electron-opaque masses punctuating the nucleolonema. Later, 6-8 hours after ethionine injection, fragmentation and disorganization of the nucleolar architecture occurs with preservation of both the fibrillar and granular components. Nucleolar remnants can take the form of round or rug-shaped electron-opaque fibrillar masses with peripheral granular aggregates; in other cases, they sirnulate aborted segregation of nucleolar components. These changes are observed in most hepatocytes and end in complete dispersion of nucleolar components bearing little resemblance to what is accepted as a normal nucleolus (Fig. 12a). Other lesions in the nucleus include clumping of interchromatin granules and condensation of chromatin. Both methionine and adenine prevent fragmentation, and administration of adenine 8 hours after ethionine completely reverses the nucleolar lesions within 4 hours (Fig. 13) (Shinozuka e f ai.. 1968a). Protein synthesis does not appear to be essential for the restoration of nucleolar structure after fragmentation, but the presence of RNA synthesis proved to be an important factor (Shinozuka and Farber, 1969). 2. Nucleolar Fragmentation Induced by Other Substances

There are few instances of similar changes being induced by other substances. The term fragmentation has been used to describe light microscope observations of nucleolar changes induced by ribonuclease (ChPvremont et al., 1956) and 5 fluorodeoxyuridine (Love et ul., 1965). Treatment of monkey kidney culture cells with adenosine resulted in a dissociation of nucleolar structure somewhat similar to ethionine-induced nucleolar fragmentation (Stenram, 1966a). The action of 5-fluorouracil on nucleolar ultrastructure is somewhat similar to that of cthionine. The first lesions to appear are an increase in nucleolar size, with formation of dense granular aggregates (Stenram, 1966b; Lapis and Benedeczky, 1966). Longer exposure to the antimetabolite in tissue culture leads to FIG. 12. ( a ) Nucleolar fragmentation in rat liver cell 1 2 hours after ethionine injectiou. Nucleolar reinnants are indicated by arrows. x 20,000. (Courtesy of Shinozuka et al., 196%). ( b ) Nucleolar fragmentation in cultured cells treated with 5-fluorouracil during 2 4 hours at 100 pg/ml. The nucleolar remnants (arrows) are dispersed in the nucleus. Osmium tetroxide. Epon. x 12,000.

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194

SIMARD

d e g r a d a t i o n of the nucleolus and fragmentation of the fibrillar reticular network (Fig. 12b) (Simard, 1968). 3. T h e E f e c t of Ethionine in RNA Synthesis

The mode of action of ethionine is complex since it involves several levels in the synthesis of RNA and proteins. Being a substitute for methionine, it can be incorporated in its pIace or competitively inhibit metabolic reactions requiring methionine (Farber, 1963).

'

9

Ethionine

*

Adenine

8

'

4

7

5

6

FIG.13. Schematic illustration of the sequential changes of the nucleolar re-formation after adenine administration. 1, Normal nucleolus; 2, disorganization and fragmentation of nucleolus after the ethionine injection; 3-5, various structural forms encountered 2 hours after adenine administration; 9 , structure close to normal nucleoIus encountered 4 hours after adenine administration. (Courtesy of Shinozuka et al.,1968a). Villa-Trevino et al. (1963, 1966) have shown that ethionine severely inhibits RNA synthesis in rat liver and that this inhibition follows a decrease in adenosine triphosphate concentration but precedes the inhibition of protein synthesis. The administration of adenine and methionine prevented this inhibition or reversed it when administered after the injection of ethionine. According to several authors, it is through an excessive trapping of the adenosine moiety of ATP as 1-adenosylethionine that ethionine affects RNA polymerase requirements of ATP for the conduct of RNA synthesis (Farber, 1963; Smith and Salmon, 1965; Raina et a]., 1964). The parallelism between the biochemical and ultrastructural studies i s striking and led Shinozuka et al. (1968a) to suggest that the disorganization of nucleoli was either a reflection of the disturbance of cell metabolism because of

NUCLEAR A N D NUCLEOLAR LESIONS

195

ATP deficiency or a consequence of it resulting in a reduction of RNA synthesis. The mechanism by which 5-fluorouracil affects RNA synthesis is well known. This uracil analog is incorporated as a fraudulent base into RNA. It also affects incorporation of normal precursors into nucleic acids and inhibits thymidilate synthetase activity (Heidelberger, 1963). Severe inhibition of RNA synthesis is observed after 5-fluorouracil treatment (Heidelberger and Ansfield, 1963), but low doses give rise to high incorporation into heavy RNA molecules (Stenram, 1966b). The possibility exists that fraudulent RNA first accumulates in the nucleolus causing an enlargement of this organelle followed by a fragmentation of the reticulated fibrillar component because of subsequent blocking of nucieolar function. Although there are few similarities between the biochemical actions of ethionine and 5-fluorouracil, it should be stressed that the end results are similar, accompanied by fragmentation of nucleolar components.

V. Agents That Primarily Affect the Nucleus Compared with the nucleolus which is ultrastructurally highly organized, the interphase nucleus appears somewhat chaotic. Granules dispersed in chromatin fibrils in various stages of extension or coiling leave little room for cytological indications that could permit one to relate morphology to activity and chromosomal structures to duplication and transcription of genetic information. Recently, a number of drugs have been shown to induce characteristic lesions on the nuclear fine structure; some of these compounds affect the nucleolus as well, and have, therefore, already been mentioned in the preceding sections. Their action on the nuclear structures will be stressed in the following discussion. A. MARGINATION OF CHROMATIN

AND PROFLAVIN

1. Cytologicdl Lesions Induced by ProfEdvin

At low doses, proflavin completely modifies the ultrastructural aspect of the nucleus. When cultured cells are treated for 6 hours at 10 'pg/ml, the bulk of the chromatin mass forms electron-dense osmiophilic aggregates standing out in a nucleus that otherwise keeps its size and shape. There is an unusual unsticking and margination of the chromatin clunqx from the nuclear membrane. After 24 hours of proflavin treatment, the margination and clumping increase, as the nucieoplasm has lost an appreciable quantity of material and electron density. The perichromatin granules disappear during the treatment, while the interchromatin granules are grouped in clusters (Fig. 14a and b). At this moment, nucleolar segregation has taken place. As the treatment progresses in time, the sparseness of the chromatin markedly increases, leaving sticky-looking aggregates in a poorly defined fibrillar network. These striking nuclear lesions are observed in concomitance with nonspecific cy-

196

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NUCLEAR A N D NUCLEOLAR LESIONS

197

toplasmic alterations such as disorganization of endoplasmic reticulum and the presence of large cytoplasmic inclusions with myelin figures and osmiophilic material. N o mitosis has ever been observed in treated cells (Simard, 1966). Other authors reported nucleolar segregation induced by proflavin in cultured cells (Reynolds and Montgomery, 1967) and liver cells (Stenram and Willen, 1968) but did not emphasize the nuclear lesions. 2.

Margination of Chromatin Induced by Other Agents

Similar lesions were produced by the antibiotic daunomycin and the trypanocidal drug ethidium bromide in a different sequence. In cells treated with these two compounds, nucleolar segregation was the first lesion to appear, followed by margination and clumping of chromatin after a longer period of treatment (Simard, 1966). Ethionine has also been reported to cause margination of chromatin (Herman et al., 1962; Shinozuka et al., 1968a). Recently, we have studied new antibiotics that induce the same lesions in cultured cells after only 1 hour. In this case, the nuclear alterations appear so rapidly that no cytoplasmic modifications can be observed. The antibiotic U-12241 (Bhuyan, 1967), for instance, causes margination of chromatin and clumping of interchromatin granules after 1 hour at 10 vg/ml. The changes here are even more striking than with proflavin, as only interchromatin granules within a fibrillar network can be seen in the nucIeus (Simard, 1968).

3. Mode of Action of Propavin and Tentative Correlationj Proflavin is a mitotic inhibitor (Balis et a/., 1963) and potent mutagen (Freese, 1959; Lerman, 1964; Orgel and Brenner, 1961) that interferes with nucleic acids in viva (Bubel and Wolf, 1965; Franklin, 1958; Schaffer, 1962; Scholtissek and Rott, 1964) and in vitro (Hurwith et al., 1962). Proflavin binds to D N A by intercalation between adjacent base pairs (Lerman, 1961; Luzzati et al., 1961) and blocks, without specificity, the enzymic reaction leading to RNA and DNA synthesis (Hurwith et al., 1962). The antibiotic drug daunomycin possesses cytotoxic and antimitotic activity (Di Marco et al., 1963) and is also believed to bind to D N A by intercalation between base pairs (Calendi et ul., 1964) ; daunomycin inhibits RNA synthesis regardless of the base

FIG. 14. Margination of chromatin induced by proflavin treatment in cultured cells. (a) Proflavin, 10 pg/ml 6 hours. The whole appearance of the nucleus is changed. The chromatin forms electron-dense clumps with margination from the nuclear membrane (double arrows). The nucleoplasm has lost some electron density and clustering of interchromatin granules (ig) is seen. Osmium tetroxide. Epon. x 10,000. ( b ) Proflavin, 10 pg/ml during 2 4 hours. Clustering of interchromatin granules (ig) is striking. Glutaraldehyde. GMA. x 30,000.

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composition of the D N A template (Ward et al., 1965). Ethidium bromide is also known to form reversible complexes with both DNA and RNA and inhibits nucleic acid synthesis and nucleic acid polymerase activity (Elliott, 1963) ; this compound binds with DNA without preference for any base composition (Ward et al., 1965), probably by intercalation between base pairs (Waring, 1966). The mode of action of the antibiotic U-12241, a new antimicrobial agent isolated from the culture of Streptomyces bellus var. cirolerows, is postulated to result from the formation of stable complexes with DNA, with secondary inhibition of nucleic acid polymerase activity (Bhuyan, 1967). The relationship between the morphological effect of these compounds and their biological and biochemical action can only be speculative. It seems, however, that a certain class of DNA-binding agents alters the structural organization of the nucleus in a characteristic and reproducible manner. Alteration of the physicochemical properties of chromatin including its affinity for the binding sites on the nuclear membrane, stainability, and distribution have been proposed as a tentative explanation (Simard, 1966). Other possibilities, such as competitive displacement of histone-rich basic proteins from the chromosomal DNA by those agents that react strongly with nucleic acids, also appear as attractive hypotheses.

GRANULES : AFLATOXIN AND LASIOCARPINE B. PERICHROMATIN I . Cytological Actioiz of AFatoxin and Lasiocarpine

The ultrastructural lesions induced by aflatoxin in rat liver have been described by Bernhard et al. (1965) and others (Svoboda et al., 1966). When injected in low doses in hepatectomized animals during the regeneration phase, this potent carcinogen induced nucleolar segregation within 30 minutes to 1 hour. A “contrasted zone” of unknown nature was noted in association with the nucleolus and interpreted as a disruption of the nucleolar granular component (Svoboda et al., 1966). Another potent carcinogen, lasiocarpine, was found to produce the same lesions. This pyrrolizidine alkaloid caused segregation of nucleolar components within 30 minutes in rat liver (Svoboda and Soga, 1966) with, once again, the appearance of contrasted granular masses that were interpreted as disrupted nucleolar components (Reddy et al., 1968; Reddy and Svoboda, 1968). Both compounds are associated with the early appearance of helical polysomes in the cytoplasm of liver cells (Monneron, 1968, 1969). A recent ultrastructural and cytochemical study showed that both aflatoxiii and lasiocarpine induce an increase of perichromatin granules in rat liver, with early formation of dense masses measuring 0.2-1 p in diameter (Monneron el al., 1968). These masses are pepsin- and pronase-sensitive in their centers but their granular cortex is formed by RNP particles similar in structural and cytochemical properties to perichromatin granules. According to Monneron et dl.

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(1968), the formation of these dense masses is independent of the nucleolar lesions but closely related to the increase of perichroinatin granules (Fig. 15).

FIG. 15. Perichrornatin granules clustering in rat liver cells 3 hours after injection of lasiocarpine. Within the condensed chromatin (chr), perichromatin granules are grouped in granular masses (arrows). Segregated nucleolus in granular ( 9 ) and fibrillar ( f ) components. x 45,000. (Courtesy of Monneron et rtl., 1968.)

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2. iMode of Action of A/iatoxiu uiid Lasiocdvpiize

These two carcinogens almost completely inhibit nucleolar RNA synthesis (Clifford and Rees, 1967; Lafarge et al., 1966; Reddy et al., 1968; Mouli and Frayssinet, 1968) which, as seen previously, is accompanied by a segregation of nucleolar components. Little is known, however, of the action of these drugs on non-nucleolar nuclear RNA, the synthesis of which persists significantly after injection of the drugs (Lafarge et al., 1965, 1966). Monneron et al. (1968) proposed that the increase of perichromatin granules and formation of dense masses are linked to the persistent synthesis of certain types of RNP and the blocking of their transport to the cytoplasm. These authors conclude that, since no RNP particles are observed in the nuclear pores, the yerichromatin granules are either stocked iiz sitz or aggregate in dense masses as degradation proceeds, owing to a Iong sequestration in the nucleus. C. INTERCHROMATIN GRANULES Clumping of interchromatin granules has been reported in such a large variety of pathological conditions that it now appears to represent part of a nonspecific reaction related to cytotoxicity resulting from various injurious agents. In proAavin-treated cultured cells, there seems to be an increase of interchromatiii granules with cluster formation (Fig. 14a and b) ; this could eventually permit their isolation and characterization although in the present case the granules are somewhat larger than normal (Simard, 1966). In recent studies, increased granules have been observed in rat liver treated with aflatoxin, lasiocarpine, tannic acid, and TAA in acute stages of intoxication, and with dimethylnitrosainine and ethionine in chronic stages (Svoboda and Higginson, 1968; Miyai and Steiner, 1965; Shinozuka et dl., 1968b). Clustering was reported in several normal and cancer cells (Bernhard and Granboulan, 1963; Granboulan and Bernhard, 1961; Swift, 1959a), in degenerating and dead cells (Trump et al., 1965), and in cells exposed to irradiation (Andres, 1963) and supranormal temperature (Simard and Bernhard, 1966). It has been suggested that the interchromatin granules represent extrachromosoma1 RNA (Swift, 1963; Bernhard and Granboulan, 1963) or nucleolar RNA migrating to the cytoplasm (Smetana et al., 1963). The possibility that alterations of the interchromatin granules reflect activation or impairment of RNA synthesis at extranucleolar sites has been raised recently (Reddy and Svoboda, 1968). Since their biochemical nature, RNA and protein content, and function are not known, however, such conclusions are of limited significance at the moment. D. NUCLEAR INCLUSIONS The term nuclear inclusions is descriptive and used mostly to characterize the appearance of new structures in the interphase nucleus that are not known to

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result from a redistribution of normal nuclear structures or from a trapping of cytoplasmic material. The dense masses appearing after aflatoxin or lasiocarpine treatment and the clustering of nuclear granules after proflavin treatment are nuclear inclusions; but since they are clearly related to the peri- and interchroinatin granules, respectively, they have been discussed in preceding sections. Nuclear inclusions are not to be confused with nuclear bodies (Weber and Fromines, 1963; Weber et al., 1964) which were first described by de The et al. (1960). These bodies are found in normal and pathological conditions in different niorphological forms; they are probably normal cellular organelles related to cellular hyperactivity (Bouteille et al., 1967). On the basis of cytochemical and histochemical studies, the nuclear bodies do not contain D N A or RNA but may have proteins in their structure (Krishan et al., 1967). The presence of nuclear inclusions has been consistently reported in cultured cells treated with the carcinogen 4-nitroquinoline-N-oxide (Endo et al., 1959, 1961; Levy, 1963; Reynolds et al., 1963, 1964; Lazarus et a/.,1966). These inclusions appear as distinct spherical areas of low electron density after permanganate fixation for electron microscopy. Histochemical studies have stressed the presence of RNA in these inclusions (Endo et al., 1961), a conclusion also reached from studies with acridine orange fluorescence. Levy (1963) suggested that iiuclear inclusions do not represent RNA in transit to the cytoplasm but rather RNA trapped in the nucleoplasm by an inhibiting effect of these drugs on RNA transport. The inclusions would then originate from the redistribution of an already synthesized material (Lazarus et al., 1966). Nuclear inclusions were observed in embryonic cells of amphibians cultured in the presence of actinomycin D (Duprat et al., 1965). These inclusions are numerous (40 to 50 per nucleus after 8 hours of actinomycin treatment), but they seem to become confluent as treatment progresses in time; cytoplasmic differentiation is not affected by the presence of the antibiotic (Duprat ef al., 1966). A study at the ultrastructural level has shown that the inclusions are formed by agglomeration of coarse fibrillar elements 400-600 A long located in the interchromatin space (Fig. 16a and b) . Cytochemical studies suggest the presence of RNA and proteins in these inclusions, but no significant incorporation of uridine -3H could be obtained by high-resolution radioautography with various lxilsechase experiments. The RNP content is, therefore, not newly synthetized hut is probably the product of a stable “pool” or the result of accumulation of an RNA originating from excessive degradation. The appearance of the inclusions coincides with the development of nucleolar segregation and proceeds independently of it. They are believed to originate from a rearrangement in the RNP network of the nucleoplasm following actinomycin treatment (Siinard and DLIprat, 1969). Inclusions of another type were described by Jones and Elsdale (1964) and Jones (1967) in embryonic cells of lidna p ~ p ~ e nfollowing s actinomycin treat-

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ment. The antibiotic gives rise to the formation of bundles of relatively coarse threads measuring 200-250 A in thickness. The threads are arranged in crystalline arrays and several aggregates can be seen in a single section in the interchromatin region of the nuclei. The same lesions were induced by nogalamycin, chromomycin A3, and olivomycin, but not by ethidium bromide, daunomycin, and proflavin. Therefore, the thread formation appears to be connected with the binding site on the DNA molecule. It also seems to be related to the special nuclear structural organization of Rdm species since actinomycin did not produce the same lesions in other amphibious species. Intranuclear fibers resembling tonofibrils have been described in guinea pig epidermis following treatment with 4-hydroxyanisole, a compound that reacts preferentially with melanocytes and causes a rapid depigmentation effect (Riley and Seal, 1969). Heavy metals have been shown to produce inclusion bodies in rat kidney and liver ceIIs (Blackman, 1936; Wachstein, 1949). The inclusions contain fibrils embedded in a granular matrix and are formed by two distinct zones, periphery and core. They stand out in liver and kidney cells of rats treated with a series of intraperitoneal injections of lead. The inclusions have no relation to the nucleolus (Beaver, 1961; Galle and Morel-Maroger, 1965). A recent ultrastructural and cytochemical study has shown that the fibrils and much of the amorphous material of the inclusions are composed of protein other than histone and do not contain nucleic acid. The fibrils are believed to be derived from a protein originally associated with chromatin (Richter et d.,1968).

VI. Summary and Concluding Remarks

For the sake of clarity and comprehension, this review has correlated the effect of a drug with a particular nuclear structural component. But several chemical and physical agents can alter nuclear and nucleolar ultrastructure at different levels since their action on the DNA-RNA template system is mediated through a complex functional and structural machinery that depends on intensity, dose, and duration of treatment. Actinomycin affects the nucleolus and ribosomal RNA synthesis selectively at low doses, but can induce nucleoplasmic alterations as well. Proflavin, aflatoxin, and lasiocarpine are other examples of drugs that can induce fine nuclear and nucleolar lesions simultaneously. Indeed, such modiFIG. 16. Nuclear inclusions induced by actinomycin D (1 pg/ml during 12 hours) in embryonic cells of amphibians. The inclusions ( i ) are numerous and located in the interchromatin space. A nucleolar remnant ( N u ) is in terminal phase of segregation. The enlarged area ( b ) shows the differences between the inclusions ( i ) made of coarse fibers and the finely fibrillar nucleolar remnant ( N u ) . Osmium tetroxide. Epon. (a) x 4,000. (b) X 40,000.

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fications furnish unparalleled opportunities for correlative structure-function studies. The mc1eolz.r appears as a highly organized nuclear organelle composed of granular and fibrillar RNP, proteins, and associated chromatin. In the normal nucleolus, the synthesis of RNA is likely to follow the sequential ultrastructural steps outlined in the accompanying diagram. DNA h (0 [Fibrillar

12) R N P]e[Gr anul ar

(3)

1 O_

RNP

Cytoplasm

Reaction (1) coincides with the synthesis of 45 S nucleolar RNA and depends heavily on the integrity of intranucleolar chromatin which appears to be the structural framework of the nucleolus. Retraction of this chromatin, whether caused by actinomycin D, ethionine, or supranormal temperatures brings about a collapse of nucleolar architecture leading to segregation, fragmentation, or degranulation of the nucleolus as well as blocking of RNA synthesis. Actinomycin D permits reactions ( 2 ) and ( 4 ) to proceed until exhaustion of the nucleolus which is characterized at the terminal stage by a fibrous small nucleolar remnant. Supranormal temperatures favor reaction ( 3 ) , because when unraveling of the granular RNA occurs, transport of RNA from the nucleolus to the cytoplasm is blocked and accumulation of previously synthesized RNA in a fibrillar nucleolus takes place. In nucleolar hypertrophy caused by TAA, it is not certain whether the hypertrophy is the result of an augmented RNA synthesis [reaction (I)], a blocking of the transport of nucleolar products [reaction ( 4 ) 1, or a combination of the two. Further studies are required concerning the fragmentation of nucleolar components induced by ethionine in relation to its effect on RNA synthesis. Thus, we suggest that the granular and fibrillar R N P components have a close interdependent relationship in which the concentrations necessary for biological equilibrium is determined by the structural integrity of nucleolar chromatin, the rate of RNA synthesis in the nucleolus, and the requirements of cytoplasmic RNA synthesis. The fibrillar RNP probably contain a “pool” of nucleolar RNA’s, while the granular R N P are therrnosensitive configurational forms assuming the transport of 2 8 S RNA from the nucleolus to the cytoplasmic ribosomes. It is certain that this macromolecular equilibriuni involves the interaction of enzymic systems that are still to be determined. The situation is far more complex in the nucleus. The DNA-binding agents that belong to the proflavin group induce a series of uItrastructura1 alterations that can be summarized as follows: margination and clumping of chromatin, segregation of nucleolar components, and clustering of interchromatin granules. The carcinogens aflatoxin and lasiocarpine are associated with the early appearance of helical polysomes together with the increase and clustering of perichro-

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matin granules in rat liver cells; these lesions appear to be linked to the persistent synthesis of nonribosomal ribonucleoproteins and blocking of their trailsport to the cytoplasm. The clumping of interchromatin granules is observed in a wide variety of normal and pathological conditions; it probably consists of a nonspecific lesion related to cytotoxicity and cell death. A wide variety of nuclear inclusions have been described following treatment with 4-nitroquinoline-N-oxide and actiiioinycin D. The inclusions apparently contain RNA and a large number of proteins; they appear to be formed by a rearrangement of the interchromatin RNP network. Cellular intoxication with heavy metals such as lead can also produce nuclear inclusions of a different type. More research should be forthcoming on the structural arrangement of the nucleus which has been neglected by biologists because of its complex functional machinery and apparent lack of organized framework. The use of such models as drug-induced lesions and combined structure-function studies, however, should furnish more information in the near future.

ACKNOWLEDGMENTS I am greatly indebted to Dr. W. Bernhard of the Institut de Recherches s u r le Cancer, Villejuif, France, who provided me with stimulating advice and suggestions. This work was supported by a scholarship from the Medical Research Council of Canada.

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The Origin of Bone Cells MAUREEN OWEN Medical Reseurch Council External Scientific Staff, Bone Re.remch Luhma1oty, T h e Churchill Hospital, Oxfovd, Englund

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Osteoprogenitor Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Uptake of Tritiated Thymidine . . . . . . . . . . . . . . . . . . . B. Proliferative Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Kinetics of Differentiation . . . . . . . . . . . . . . . . . . . . . . . D. Histocheinistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Electron Microscope Studies . . . . . . . . . . . . . . . . . . . . . F. The Effect of Parathyroid Hormone . . . . . . . . . . . . . . . G. Cell Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. The Composition of Osteoprogenitor Cells . . . . . . . . . 111. Other Cells with Potential for Hone Formation . . . . . . . . . A. Experiments with Millipore Filters (Closed System) . B. Experiments with Direct Transplants (Open System) . C. Bone Fracture Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . 1V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

213 216 217 21 8 21 S 224 226 226 227 22s 229 230 211

235 231 236

I. Introduction The origin of all cells in the body is the fertilized ovum. Studies of the mechanisms that result in the development of different cell types, tissues, and organs from this single cell represent one of the most active fields of current research. For any particular tissue, whether in the embryo or in the postfetal organism, the problem in its siniplest form can be stated as follows. What is the nature of the cells and of the particular set of inductive stimuli that enable the two to react to form the tissue under consideration? In embryonic systems, this is known as the process of induction, although the term is also borrowed for similar situations in the postfetal organism. The process is not well understood and it is likely that there are many kinds of inductions taking place ,zt all stages of differentiation throughout the life of an organism. Cellular microenvironment and cell-to-cell and cell-to-substrate relationships are all probably iinportant in various inductive processes and in differentiation in general. This article is an attempt to review, for the case of bone, the progress that has been made on one small aspect of this problem in recent years, namely, which cells in the postfetal organism are capable of osteogenesis. This immediately raises the question of cell terminology. Cells were originally named accordiiig to their morphological appearance and in some cases this amounts to 110 inore than a difference in size, shape, or location. Cell terminology will, 213

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therefore, be kept as simple as possible and the emphasis will be on what the cells do, rather than their histological appearance. An attempt will be made, when relevant, to relate the results obtained by newer methods to the previous nomenclature. In classic histology we learn that connective tissue develops from the mesenchyme of the embryo. From the studies of Maximow many years ago, the con-

connective tissue stem cells

Increasing differentiation

I

t tissue stem cells

rl progenitors

(d)

Fully differentiated cells

FIG. 1. Diagram of differentiation of connective tissue.

cept arose that there is present in the body a small pool of undifferentiated mesenchymal cells which have the capacity to differentiate along any one of several lines. Although this concept has never been seriously disputed, there are still many unanswered questions, some of which are outlined in the following discussion with reference to the diagram in Fig. 1. Differentiation of any connective tissue in the body can be represented as occurring in four stages, Pig. 1. This is an obvious simplification since differentiation is more likely to consist of a gradation of stages. Using Fig. 1 as a model, we can ask the following questions concerning the nature of the pool of undifferentiated mesenchymal cells presumed to exist throughout connective tissues (Ham and Leeson, 1961). First, are the cells that compose it multipotential? In other words, are they connective tissue “stem cells”l (a in Fig. 1) and, as such, are they common 1 The definition of “stem cell” is after Caffrey-Tyler and Everett (1966). A stem cell is defined as a cell having the capacity for extensive proliferation resulting in renewal of its own kind as well as giving rise to fully differentiated cells. Strictly speaking this definition might also apply to some of the proliferating progenitors but in general the term is reserved for less differentiated cells.

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proliferative precursors of all connective tissues, i.e., hemopoietic tissue, bone, cartilage, fibrous tissue, and so on? Second, is the pool, on the other hand, made up of a mixture of distinct groups of stem cells for each of the different tissues (b in Fig. I ) , eg., hemopoietic stem cells, osteogenic stem cells, fibroblastic stem cells, and so on, each of which is capable of giving rise to all the different cell types in the tissue? The implication here is that each of the different stern cell types in b is already destined to differentiate in the direction of the tissue concerned. A third possibility is that the pool contains cells from both categories a and b. What is the distribution of these undifferentiated cells, whether they are equivalent to a, or b, or a mixture of both? Are they present in the circulating blood and, if so, what is the tissue of origin? The term “proliferating progenitors” is reserved for a further category of cells (c in Fig. I), which are the more immediate precursors of the fully differentiated cells (d) of a tissue. These cells have reached a more differentiated stage than those represented in a and b and are known to be already determined in the direction of differentiation of the tissue concerned. They still have proliferative capacity and, in addition, may exhibit some recognizable characteristics of the final differentiated tissue cell. A final question is, what is the relation between the ubiquitous pool of relatively undifferentiated mesenchymal cells in the body, represenled by a and b in Fig. 1, and the inore differentiated population of proliferating progenitor cells of bone (c in Fig. 1) ? One difficulty in studying the questions outlined above is the fact that the less differentiated cells in the scheme (a and b in Fig. 1 ) are not yet distinguishable with certainty under the microscope. The morphological appearance of the undifferentiated mesenchymal cell is unknown. Nevertheless, there are inany descriptions of it. In the literature on bone, it is often referred to as a cell with a pale, vesicular, oval, or fusiform nucleus and inconspicuous cytoplasm, but it may well take different forms depending on its surroundings. Neither can the different types of stem cells (b in Fig. I ) , if in fact they do exist as separate entities, be recognized under the microscope. In spite of their elusive morphology, there is nevertheless good evidence from other tests for the widespread existence of undifferentiated cells which can be induced to differentiate in the direction, for example, of either hemopoietic tissue or bone as the case may be. This can be inferred from various experiments which test the functional properties of these cells. In the case of hemopoietic tissue, for example, the existence of stem cells has mainly been demonstrated by their capacity to reseed hemopoietic centers in animals that have lost their own hemopoietic tissue by exposure to radiation (Micklem and Loutit, 1966; Loutit, 1967). In the case of bone, the main source of information on the capacity of undifferentiated cells for bone formation comes from experiments on heterotopic bone induction (i.e., induction of bone in sites outside the slteleton; Bridges, 1959; McLean and Urist, 1968). There is a very

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large literature on the subject of bone induction and no attempt will be made to cover it in detail. In particular, the nature of the environmental conditions that induce osteogenesis in sites outside the skeleton will not be considered. Only information relevant to the problem of which cell types have the capacity for osteogenesis will be reviewed. At the more differentiated end of the scheme (Fig. 1) are the immediate precursors of bone cells. They are the population of dividing cells situated near bone surfaces or sometimes in contact with them. Young (1962b) showed that they were the precursors of both the osteoblasts and osteoclasts, the two main differentiated cells of bone, and he named them osteoprogenitor cells. They belong to the category of proliferating progenitors of bone cells (c in Fig. I ) , but the two terms are not synonymous; proliferating progenitors is a wider term, although the composition of cells it covers is not yet fully defined. It includes osteoprogenitor cells and probably less differentiated precursors of these cells, which may exist particularly in marrow tissues. Osteoprogenitor cells will be used herein, as originally suggested (Young, 1962b), for those precursors of bone cells found near bone surfaces. These cells have been very actively studied in recent years, and an account of their characteristics will be given in Section 11. In Section 111, cells other than osteoprogenitor cells which are capable of bone formation, will be considered. Here, as already mentioned, the evidence is mainly from experiments on heterotopic bone induction. From this work, there is evidence to show that in addition to other cell types, relatively undifferentiated cells, which are mobile and have a widespread distribution in the body, can be induced to form bone. This immediately raises the question as to whether or not these ubiquitous undifferentiated mesenchymal cells are precursors of the proliferating progenitors of bone cells. One might envisage feed-in of cells from this compartment into the progenitor population as required. Whether this happens at all under normal physiological conditions in the postfetal organism is questioned. There is a possibility that the proliferating progenitors of bone are a self-perpetuating population, and that transitions from undifferentiated mesenchymal cells to progenitors of bone cells do not take place in the postfetal organism under normal conditions. It may be that in the mature organism the capacity of the undifferentiated cell for bone formation is reserved only for situations in which reparation or regeneration is needed. The evidence for these ideas will be presented and discussed.

11. Osteoprogenitor Cells These cells were first defined as the population of cells near bone surfaces that are labeled shortly after injection with thymidine-". The thickness of the

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layer of osteoprogenitor cells varies with the age of the animal and the part of the bone studied. The location of these cells, associated as they are with bone surfaces, is the main morphological criterion defining them. Their appearance is indistinguishable from cells previously described under the teriiis inesenchymal cells, spindle cells, fibroblasts, reticulum cells, endothelial cells lining blood vessels, and possibly some other components of bone marrow (Kembcr, 1960; Young, 1962a,b). Consequently, on morphological grounds it was thought these osteoprogenitor cells may be part of a pool of undifferentiated rnesenchymal cells with wider potential. Evidence from the kinetic studies of their differentiation (Young, 1962b), however, has shown that they are the immediate precursors of the differentiated cells of bone, and they have been called osteoprogenitor cells to give them a more meaningful name. Furthermore, recent work has indicated that they are largely made ~ i p of the progenitors of the two main differentiated cell lines in bone, osteoblasts and osteoclasts (Scott, 1967; Bingham et ul., 1969), each line of progenitors exhibiting some of the characteristics of its final differentiated form. Studies of the proliferative activity and the kinetics of differentiation of osteoprogenitor cells have been made using tritiated thymidine and radioautographic techniques. The results are described in the following discussion along with an account of the ultrastructure, histochecnistry, and some aspects of the behavior of these cells under the effect of parathyroid hormone.

A. UPTAKEOF TRITIATED THYMIDINE For a proliferating population, the cell cycle is divided into four phases (Howard and Pelc, 1953). G, is the resting period between the previous mitosis and the commencement of D N A synthesis. S is the period during which DNA is synthesized and the D N A content of the nucleus doubled in preparation for the next division. G2 is a short period before cell division, and &l is the period of mitosis. The total cell cycle time (GI S G2 M ) is represented by T,. Numerous studies (Quastler and Sherman, 1959; Cronkite et ul., 1959; Fry et ul., 1963) have confirmed the above scheme and have shown that in mammalian tissues S, G2, and M vary relatively little (Cattaneo ef dl., 1961; Owen, 1965) ; variations in T , are, therefore, mainly attributable to differences in GI. In proliferating cell populations, tritiated thymidine is taken up by cells during the S period. The fraction I; of the cell population that is labeled a short time after injection of thyniidine-:lH (i.e., before the labeled cells have had time to go through mitosis; about 1 hour is common) is an index of the proliferative activity of the tissue. Assuming a random distribution of the cells throughout the different phases of the cell cycle, it follows that f; is related to S and T , by the equation F = S/T,.

+ + +

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13. PROLIFERATIVE ACTIV~TY The fraction of osteoprogenitor cells that is labeled t hour after injection of thymidine-3H varies with the region of the bone being studied and the species and age of the animal (see Table I). Young (1962a) found that the proliferative activity F was different in different regions of the same bone, Results for the metaphysis of the rat (Keniber, 1960; Young, 1962a,b) showed that F fell dramatically with age, and this was also borne out by the results of Tonna (1961) for mice. Tonna also showed that the thickness of the osteogenic cell layer on the bone surface decreased in a similar way with age. Many of the studies, therefore, have been made in very young animals. As will be shown later, the proliferative activity of the osteoprogenitor cells is roughly correlated with the rate at which they differentiate into more mature forins. Values of S have been measured by several of the authors listed in Table I. Young found a value of about 8 hours for the cells in all three regions of the bones of 6-day-old rats, and a value of 6.2 hours was found by Owen and MacPherson (1963) for 2-week-old rabbits. These values are in very good agreement with previous measurements of S for other mammalian cells (Qnastler and Sherman, 1959; Cronkite et al., 1959; Lesher et al., 1961). Some values for the cell cycle time T , are also shown in Table I. It must be emphasized that only average values for T , can be determined. Individual members of the osteoprogenitor population are likely to be at slightly different stages of differentiation. Consequently, some cells may divide more frequently than others, but there is no information on how widespread the rnnge of values for T , might be for any particular osteoprogenitor population.

C. KINETICS OF DIFFERENTIATION

A study of the kinetics of differentiation of osteoprogenitor cells in 6-day-old rats was made by Young (1962a,b, 1963). His method was to determine the initial labeling ( 1 hour after thymidine-3H injection) of the osteoprogenitor cells and then to follow the rate of appearance of labeled nuclei in both osteoblasts and osteoclasts at later times after injection. Some of his studies in the metaphysis are described later. The metaphysis of young rats is a region of very active bone remodeling in which bone deposition and resorption take place side by side on the surfaces of a network of thin trabeculae. In the spaces between the trabeculae can be found the typical osteoprogenitor cells. A diagram taken from Young (1962b) of the cellular arrangement with regard to one nietaphyseal trabecula is shown in Fig. 2. Young’s results for the metaphysis of the tibia taken from Tables IV and VII of his paper (196213) are plotted in Fig. 3 . About 22% of the osteoprogenitor cells in the metaphysis were labeled after 1 hour, labeling of osteo-

VALUES OF F, S

F Species

Age

(%)a

Rat Rat Rat Rat Mouse Mouse Mouse Mouse Mouse Mouse Rabbit

6-8 weeks 6 days 6 days 6 days 1 week 5 weeks 8 weeks 26 weeks 52 weeks 4 weeks 2 weeks

7

a

b c

d

22 14 7

8.5

2.7 0.7 0.2 0.6 5

10

AND

TABLE I FOR OSTEOPROGENITOR CELLS

T,

S (hours) I

8 8 8

-

-

6.2

F = fraction labeled 1 hour after thymidine-3H administration. S = time required for DNA synthesis. T , = total cell cycle time. These results are for the “osteogenic layer” (osteoprogenitor cells

T, (hours) c

Region of bone

36 57 114 -

Metaphysis Metaphysis Endosteum Periosteum Periosteum

Kember (1960) Young (1962a,b) Young (1962a,b) Young (1962a,b) Tonna (1961)d

Periosteum Periosteum Periosteum Periosteum Metaphysis Periosteum

Tonna (1961) Tonna ( 1961) Tonna (1961) Tonna (1961) Simmons (1963) Owen (1963) ; Owen and MacPherson (1963)

-

-

62

+ osteoblasts)

Reference CI

Ei 8

5 2

B

m

n m P

K

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clast nuclei at this time was zero and that of osteoblasts very low. With increasing time after injection, the percentage of labeled osteoblasts and osteoclasts rose and leveled off at a value approximately equal to the initial labeling index of the osteoprogenitor cells. Since the rise in the percentage of labeled osteo-

RESORPTION O F TRABECULA

TRANSITION ZONE

APPOSITION OF BONE ON CALCIFIED CARTILAGE

RESORPTION OF CALCIFIED CARTILAGE

~

FIG. 2. Diagram of a metaphyseal trabecula. O.P., Osteoprogenitor cells (one of which is undergoing mitosis) ; O.B., osteoblasts; O.C., osteoclasts. Bone is depicted by cross-hatching, calcified cartilage is solid. The dashed arrows indicate the origin of osteoblasts and osteoclasts from osteoprogenitor cells (see Section 11, G ) . (From Young (1962b). Reproduced by permission.)

blasts and osteoclasts occurs at about the same time (Fig. 3 ) , Young concluded that these differentiated cells are both mainly derived from osteoprogenitor cells. In osteoclasts, it was found that one or more nuclei may be labeled, and this suggested that osteoclasts arise from the fusion of precursor cells, a conclusion that had also been reached by Keniber (1960). This disposed of, once and for all, the possibility that osteoclasts might arise either through cell division or through the fusion of osteoblasts, at any rate under normal circumstances. The results (Young, 1962b) showed in fact that there was a continual incorporatioil and shedding of nuclei by the osteoclasts, and it was concluded that the concept of the average lifetime of an osteoclast per se is meaningless-what is meaningful is the average lifetime of the nucleated components. As can be seeii from the width at half-height of the top curve in Fig. 3, the average lifetime of a nucleus in an osteoclast in the metaphysis in these young animals was about 150 hours. In the endosteum and periosteuni of the same animals, the turnover of osteoclast nuclei was slower. As is clear from Fig. 2, bone formation and resorption occur side by side in the metaphysis, and it is not possible to separate the osteoprogenitor cells associated with each proccss. In another system (Owen, 1963; Binghani et d., 1969), the midshaft of the femur of a 2-week-old rabbit, bone formation occurs only on the periosteal surface and bone resorption only on the endosteal surface, so that the two processes can thus be studied separately. This system is shown diagrammatically in Fig. 4. The fully differentiated cells of bone line

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the bone surfaces and behind them are situated the osteoprogenitor cells. Particularly on the periosteal surface, the cells are arranged in well-defined layers with the osteoblasts lining the bone surface; the osteoprogenitor cells (called preosteoblasts after Pritchard, 1952, Section 11, D) are in a layer behind them, Percent labeled osteoprogenitor

5

4 I

2 346810

20 140 60 I100 200 400 30 80 Hours

I

2

3 4 6 8 10

I

20 30 60 1100 200 400 40 80 Hours

FIG. 3. Percent of labeled osteoclast and osteoblast nuclei, ( 0 ) at different times after a single injection of thymidine-3H in the metaphysis of the tibia of a 6-day-old rat. Percent of labeled osteoprogenitor nuclei (U) at 1 hour after injection. (From Youllg (1962b). Reproduced by permission.)

and the whole is enclosed by the fibrous layer of the periosteuin. On the endosteal surface, the cells are not arranged in quite such well-defined layers. Osteoclasts cover about 40% of this surface (Owen and Shetlar, 1968). The osteoprogenitor cells are a layer of uninucleated cells within about 30 p from the endosteal surface. They have the undifferentiated appearance of typical mesencliymal cells. Although kinetic studies of the differentiation of these endosteal mesenchymal cells into osteoclasts have not been made, it is assumed

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that in this situation they are precursors of the osteoclasts. Under the light microscope, they are not distinguishable in appearance from the preosteoblasts on the opposite surface. As will be shown later, however, they are functionally distinguishable, and we have suggested the term preosteoclasts for the osteoprogenitor cells on this particular part of the endosteal surface.

-

Periosteum

FIG. 4. Diagrammatic representation of part of the cross-section of the bone wall from the midshaft of the femur of a 2-week-old rabbit, illustrating ( a ) the cells on the periosteal surface associated with bone growth and (b) the cells on the endosteal surface associated with bone resorption; endosteal mesenchymal cells are taken as equivalent to preosteoclasts.

In a previous paper (Owen, 1963), we have studied the kinetics of cell differentiation on the periosteal surface in the process of bone growth in young rabbits. Our method involved simultaneous injections of glycine-SH to label the position of the bone surface at the time of injection and thymidine-3H to label a proportion of the proliferating population, in this case the preosteoblasts. Information was obtained in two ways. First, glycine is rapidly incorporated into the collagen of bone matrix (Carneiro and Leblond, 1959; Young, 1962~;Owen, 1963) and is left behind as a narrow band of grains in the matrix as the bone grows (Fig. 5 ) . By counting the number of cells between this band and the first layer of fibroblasts of the periosteum at different times after injection, it was possible to determine the increase in the total number of cells and in the different categories of cells. Second, by an analysis of the

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distribution of thymidine-labeled cells with time after injection, it was possible to study the movement and differentiation of cells with respect to the bone surface. The system is advantageous in that bone growth is unidirectional during the period of the experiment (a few days), and it was possible to study

FIG. 5 . Radioautograph of part of the periosteal surface from a cross-section of the midshaft of the femur of a 2-week-old rabbit 2 days after a single injection of glycine-3H. The labeled material is a band of grains in the bone matrix on the right outlining the position of the periosteal surface at the time of injection, about 140 p from the periosteal surface on the left.

the fate and distribution of all cells that were originally on the bone surface at the time of injection. The results from this study were as follows. The main region of cell proliferation was the preosteoblast population on the periosteal surface (Fig. 4 ) . In this system, these cells are continually differentiating in an orderly direction

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to become osteoblasts on the periosteal surface. Subsequently, they become either osteocytes or cells on the surfaces of Haversian canals. The latter have a more flattened appearance than the more active osteoblasts on the periosteal surface. It was shown that cell division in the preosteoblasts on the bone surface could account both for the increase in size of this population attributable to growth and for the loss of cells from it in the process of differentiation. After 3 days, labeled osteocytes began to appear. The percentage of osteocytes incorporated that were labeled, approximately equaled the percentage of preosteoblasts and osteoblasts labeled at earlier times. Within the limits of the experimental results, it was concluded that all preosteoblasts differentiate; there was no evidence for cell death. The fibroblastic layer of the periosteum had a very low proliferative activity (F was about 1% compared with 10% in the case of the preosteoblasts). The increase in size of the fibroblast population by cell division was approximately balanced by the increase resulting from bone growth. It was concluded, therefore, that the fibroblasts contribute little to the preosteoblast population on the periosteal surface during normal bone growth. A similar conclusion was reached by Tonna (1961) and Tonna and Cronkite (1964, 1968) in their studies on mice. Osteocytes are never labeled at short times after thymidine injection. Labeled examples of this cell type are seen at later times, because of the cell having acquired its label when in the progenitor state. The time after thymidine injection at which labeled osteocytes are first seen within bone matrix has been recorded by several of the authors listed in Table I. In the work just described in 2-week-old rabbits (Owen, 1963), labeled osteocytes were first seen 3 days after injection. Young (1962b) reports 2 days in the metaphysis of 6-day-old rats, and Kember (1960) 5 days in the metaphysis of 6- to 8-week-old rats These figures suggest that there is a correlation between the proliferative activity I; of the progenitors of the osteoblasts (Table I ) and the rate at which the osteoblasts are incorporated as osteocytes, i.e., the rate of formation of bone matrix. This concurs with the rapid fall-off in I; with increasing age that has been observed in mice (Tonna, 1961), and the concomitant decrease in the thickness of the cell layers on the bone surface.

D. HISTOCHEMISTRY There are several excellent reviews of histochemical investigations of the pattern of enzyme activity and cell organelles in the different osteogenic cells (Pritchard, 1956; Cabrini, 1961). The results are disappointing from the point of view of tracing metabolic patterns related to differentiation or cell origin (de Voogd van der Straaten, 1966). The main differences are quantitative

THE ORIClN OF BONE CELLS

225

rather than qualitative. Depending on techniques and materials, practically all enzymes studied can be found in all cells. There are, however, several distinctive patterns which have been shown consistently. Alkaline phosphatase predominates in the cells of the bone-forming system, osteoblasts and preosteoblasts. Acid phosphatase and succinic dehydrogenase predominate in the cells of the bone-resorbing system, osteoclasts. Recent studies with the electron microscope (Doty et ul., 196s) have shown interesting variations in location of different phosphatases in the various bone cells, as well as quantitative differences. The association of alkaline phosphatase with the process of osteogenesis has been recognized for a long time (Robison, 1923; Bevelander and Johnson, 1950; Pritchard, 1956). In the embryo, the appearance of alkaline phosphatasepositive condensations of mesenchymal cells is taken as synonymous with the advent of preosteoblasts and subsequent bone formation. In the periosteum of growing bones, a histochemical study of the layer of cells behind the osteoblasts showed that these cells had many features similar to the osteoblasts including a positive alkaline phosphatase reaction. This led Pritchard (1952, 1956) to name these ceIIs preosteoblasts, since it was clear that they were in fact precirrsors of the osteoblasts. This has been confirmed in studies (Owen, 1963) of the kinetics of these cells on the periosteal surface as mentioned above. Apart from the studies of these progenitors of the osteoblasts, there has been little investigation of the histochemistry of osteoprogenitor cells until the recent work by BaIogh and Hajek (1965). In their paper, distribution of the oxidative enzymes of intermediary metabolism in healing fractures and associated bone was described. Several interesting new features emerged from this work. For the first time, certain variations in the enzyme content of different osteoprogenitor cells were observed. The osteoprogenitor cells of the periosteum (presumably preosteoblasts) differ in their content of glucose-6-phosphate dehydrogenase from the osteoprogenitor cells of fracture callus. O n the other hand, young developing chondrocytes of the fracture callus and preosteoblasts both contain a similar amount of this enzyme. It is tempting to conclude that in these cells the appearance of this enzyme may be connected with differentiation. Another striking result was the presence of mononuclear cells exhibiting a reaction for succinic dehydrogenase, thus distinguishing these cells from other osteoprogenitor cells. Succinic dehydrogenase is specific for osteoclasts among bone cells, and it was suggested that these cells may, therefore, be precursors of the osteoclasts. Walker (1961) has also described mononuclear and binuclear cells with strong succinic dehydrogenase activity in bones treated with parathyroid hormone. There is thus histochemical evidence for a “preosteoclast” stage among osteoprogenitor cells (Owen, 1968). This supports the more recent findings described in the next section,

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MAUREEN OWEN

E. ELECTRONMICROSCOPESTUDIES In a recent paper (Scott, 1967), a study has been made of the ultrastructure of the osteoprogenitor cells. The region studied was the proximal epiphysis of 18- to 21-day-old fetal rats, the mothers having been injected with tritiated thymidine 1 hour before sacrifice. Electron microscope radioautographs were made, and the observations were limited to labeled cells on the surfaces of the developing bone trabeculae and along the capillaries in the primary spongiosum. The results demonstrated that this population of proliferating progenitors is composed of two cell types distinguishable on the basis of their appearance under the electron microscope. Type A has extensive endoplasmic reticulum and other features generally associated with cells synthesizing proteins for export. Type B is characterized by an abundance of free ribosomes, mitochondria, and other features similar to those found in neutrophilic leukocytes or phagocytictype cells. It has been suggested that the term preosteoblast is applicable to cells of type A, and preosteoclast has been tentatively proposed for type-B cells. Both types of cell are present in a series of transitional forms, from the most primitive found in pericapillary sites to the most highly organized found nearer bone surfaces. It was also claimed that recognizable transitional forms between A and B subtypes were not observed. Fischman and Hay (1962), from their light microscope studies, also coilcluded that the osteoclast is derived from a cell with the characteristics of a “mononuclear leukocyte,” which is in agreement with the above results.

F. THEEFFECT OF PARATHYROID HORMONE In recent work (Owen and Bingham, 1968; Bingham et al., 1969), a study has been made of the effect of parathyroid extract (PTE) on the different cells of bone. The system used was the same as that illustrated in Fig. 4, i.e., the midshaft of the femur of young rabbits. It was possible to determine the effect of the hormone on RNA synthesis in the four different cell types, osteoblasts and preosteoblasts on the periosteal surface and osteoclasts and endosteal mesendiyma1 cells on the endosteal surface. The results showed that PTE has a similar effect on each differentiated bone cell and its corresponding progenitor cells, but opposing effects on cells of the bone-resorbing and bone-forming systems. There is a stimulation of RNA synthesis in the osteoclasts and endosteal mesenchymal cells and a depression of RNA synthesis in the osteoblasts and preosteoblasts. It was also interesting to note that the effect on the osteoclasts preceded the effect on their precursors, thus suggesting that the osteoclastic activity of the bone surface influences the activity of the immediate precursors. The effect of PTE on the uptake of thyniidine-3H has also been measured (Owen and Williamson, unpublished results) , Again, the results indicate a

THE ORIGIN OF BONE CELLS

227

stimulation of thymidine uptake in the mesenchymal cells on the endosteal surface and a depression in the preosteoblasts. These results show that the proliferating progenitors of the osteoblasts and osteoclasts respond differently to PTE and support the concept that there are two classes of progenitors of bone cells in functionally different states.

G. CELL TRANSFORMATION Young (1962b, 1964) has proposed the hypothesis that the cells of bone are different functional states of the same cell and that they can revert from one state to the other depending on the cellular microenvironment. The origin of osteoblasts and osteoclasts from osteoprogenitor cells, for example, is illustrated in his diagram in Fig. 2. There is one proviso, i.e., that transformations between osteoblasts and osteoclasts go through the osteoprogenitor stage. The arguments in support of the above hypothesis are very strong (Young, 1962b, 1963, 1964, 196S), although the evidence, particularly in vivu, is mainly circumstantial. For example, in the metaphysis of very young animals exceedingly dynamic remodeling of bone occurs. The situation has been diagrammatically represented in Fig. 2, and it has been reported that complete removal of a trabecula in a 6-day-old rat metaphysis, such as is represented here, occurs in 2 days (Young, 1962b). This must involve considerable shifts in cell populations. Since no evidence of cell death has been found in this type of material and since, as can be seen from Fig. 3, there is a rapid turnover of osteoblasts and of nuclei in osteoclasts, it has been concluded that transformations from one cell type to another can readily take place. In earlier papers (Heller et dl., 1950; Kroon, 1958), it was also concluded from morphological evidence that such transformations must occur in PTE-treated bone. The results from tritiated thymidine studies in the metaphysis of young rats treated with PTE (Young, 1964, 1968) have also been cited as evidence in support of the above hypothesis. During the first 24 hours following PTE treatment in young rats, there is a great increase in osteoclastic activity and a depression in osteoblastic activity. In the second 24-hour period, pronounced recovery of osteoblastic activity and a damping down of osteoclastic activity occurs. In Young’s first experiment (Young, 1964), thymidine-3H was given just before administration of PTE; 24 hours later, labeled nuclei were found predominantIy in osteoclasts. In a second experiment, thymidine was given 12-24 hours after PTE, i.e., toward the onset of the recovery period; 36-48 hours later, labeled nuclei were found predominantly in osteoblasts. From these experiments, Young concluded that osteoprogenitor cells are capable of specializing as either osteoblasts or osteoclasts depending on the changing microenvironment of the ceIIs. Although this is a possible explanation, the results are not unequivocal. In the

228

MAUREEN O W E N

previous section, it was shown that two types of osteoprogenitor cells exist and that PTE has an opposite effect on each, and any interpretation of the effects of PTE must take this into account. Further studies are necessary to elucidate these points. Studies of the healing of bone fractures (Tonna and Cronltite, 1961) have shown that osteoblasts can be stimulated to take up tritiated thymidine and presumably to divide. Consequently, in this situation osteoblasts are shown to be capable of reverting to the osteoprogenitor state. Other studies support the concept that the cellular niicroenvironment influences cell differentiation. For example, the availability of oxygen has been shown to play an important role in tissue development z n vitro (Shaw and Bassett, 1967). At low and medium oxygen tensions, chondrogenesis and osteogenesis occur, respectively, whereas at high oxygen tension, chondroclasia and osteoclasia predominate. There is also evidence (Holtrop, 1966; Crelin and Koch, 1967) that hypertrophic chondrocytes may transform into other bone cell types after dissolution of their matrix has taken place.

H. THECOMPOSITION OF OSTEOPROGENITOR CELLS The population of proliferating cells near bone surfaces contains two main cell types which have, to a varying degree, some of the ultrastructural and functional characteristics of their final differentiated forms, osteoblasts and osteoclasts. The extent to which this population of osteoprogenitor cells contains earlier undifferentiated forms, such as a common precursor of preosteoblasts and preosteoclasts or undifferentiated mesenchymal cells which are not yet determined in an osteogenic direction, is not known. If they are present, however, it is likely that these earlier undifferentiated coniponents represent only a small proportion of the osteoprogenitor population. The data available suggest that differentiation of osteoprogenitor cells to osteoblasts and osteoclasts at any one time involves the major proportion of the osteoprogenitor population (Young, 1962b). For example, the results in Fig. 3 show a rapid entry of labeled osteoprogenitor cells into the differentiated cell compartments and a rapid attainment by the differentiated cells of the initial percentage of labeling of the osteoprogenitor population. These two facts favor the conclusion that the proliferating cells near the bone surface behave, broadly speaking, as a single population from the point of view of differentiation, and also that they are comparatively well advanced along the pathway of differentiation. There is no information on the mechanisms that promote preosteoblasts as opposed to preosteoclasts; the key to this presumably must reside in the nature of the environment near forming and resorbing bone surfaces. The question now arises as to the origin of the osteoprogenitor cell in the body. Some authors attribute this role to the endothelial cells lining the small

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capillaries and blood vessels near bone surfaces (Trueta, 1963; Mankin, 1964). Even if this is the case, it only begs the question since the nature or potential of an endothelial cell in this situation is not known. Is it, for example, equivalent to an undifferentiated mesenchymal cell with several potential pathways of development, or i s it already a cell determined in an osteogenic direction? Another possibility is that precursors of osteoprogenitor cells are present in marrow tissues. They may, for example, be members of the “determined osteogenic precursor cells” that have been found in this tissue (Section 111, B) . At the present time, however, no experiments have been performed under normal physiological conditions which demonstrate that osteoprogenitor cells are derived from undifferentiated mesenchymal cells. On the other hand, under abnormal circumstances (e.g., the formation of bone in sites outside the skeleton), the fact that undifferentiated cells can be induced to form bone has been demonstrated many times. An intriguing question concerns the significance of this for normal physiological conditions. Some of the relevant results will be considered in the next section. 111. Other Cells with Potential for Bone Formation Osteogenesis in ectopic sites, i.e., extraneous to skeletal tissue, is an example of tissue induction in an adult organism and might be expected to provide information on the nature of the cells capable of bone formation. Induced osteogenesis always occurs in a connective tissue system. This system, usually a mixture of tissues with at least a proportion of fairly mobile cells can, in addition, be invaded and replenished by cells migrating from the blood. Osteogenic induction can be made to occur under a large variety of circumsta~ices-iinplantation of dead bone tissue, live bone grafts, trauma, and injections of alcohol being a few of the methods that have been successful in producing ectopic osteogenesis (Bridges, 1959; Urist, 1965). The question that is of relevance here, however, is which types of cells can be induced to differentiate in the direction of osteogenesis. In the past, some controversy has centered around whether the cells capable of osteogenesis are cells present locally in the host tissues near the site of induction, whether they are cells brought in by the blood stream, or, in the case of live grafts, whether they may be cells contributed by the donor tissue. Morphological techniques were mainly used, and from the evidence produced it was clear that, depending on the situation, cells of either host or donor tissue or both may take part in bone formation at sites of ectopic osteogenesis (Urist and McLean, 1952; Ray and Sabet, 1963; Burwell, 1964, 1966). Recently, several new techniques have been devised which have provided some more specific information pertinent to these problems. In particular, in

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MAUREEN OWEN

vivo implanted millipore filter chambers have provided a simpler system for the study of bone induction and they have been used to investigate the source of the cells taking part. Furthermore, experiments on direct transplants between animals whose cells can be distinguished by their chromosome karyotype (chromosome marker technique) are now beginning to be performed in connection with bone. Some of the results that have been obtained with these techniques will be described in this section.

A. EXPERIMENTS WITH MILLIPORE FILTERS(CLOSEDSYSTEM) Millipore filters, HA type, with a pore diameter of 0.45 p are impermeable to cells. They were first applied successfully by Goldhaber (1961) to the problem of bone induction. He cultured bone inside small chambers bounded by filters which were implanted in vivo and demonstrated the induction of bone on the outside of the chambers. This showed two things. First, that bone tissue itself was capable of bone induction and, second, that the substance responsible was transmissible across a millipore filter even one as thick as 150 1.1. This result has since been confirmed many times (Friedenstein, 1962, 1963-1964; Post et al., 1966; Buring and Urist, 1967; Heiple et ul., 1968; Friedman et al., 1968). A particularly interesting application of this technique has been the work of Friedenstein and his colleagues (Friedenstein et ul., 1967; Friedenstein, 1968) using the transitional epithelium lining the urinary tract, which is a well-known bone inducer (Huggins, 1930, 1968). Bone is induced in a connective tissue site under the influence of a direct transplant of transitional epithelium. Bone is also induced on the outside of a millipore filter chamber containing a living culture of transitional epithelium within. Bone is induced in certain cells within a millipore filter chamber in the presence of a living culture of transitional epithelium. Friedenstein’s experiments were designed to determine which types of cells were capable of induced osteogenesis. The system he used was as follows. Cells from different tissues in the body were cultured together with transitional epithelium in a millipore chamber implanted intraperitoneally. The tissues within such a chamber are able to derive nutrient from the host tissue but at the same time are isolated from the cells of the host. This constitutes what is called a closed system. It means that the processes of differentiation that develop in the population of cells present in the chamber at the time of its implantation into the recipient can be studied without being complicated by contributions from cells of the host. The different types of cells that have so far been investigated in this system are leukocytes from peripheral blood, peritoneal fluid cells, spleen cells, and cells of subcutaneous connective tissue (Friedenstein et al., 1967). All of these

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were introduced into the chamber as cell suspensions, consequently, cellular organization of the particular tissue concerned is not an important factor. A control experiment with only transitional epithelium in the chamber was also performed. In the case of leukocytes, peritoneal fluid cells, and spleen cells, bone was regularly formed within the chamber under the influence of transitional epithelium. From these results, it was concluded that tissues of hemopoietic origin contain cells that are responsible for bone induction. In the other two cases, subcutaneous connective tissue cells in the presence of transitional epithelium and transitional epithelium alone, no bone formation within the chamber was observed in this particular system (Friedenstein et ul., 1967). This does not necessarily imply that inducible cells are absent from these tissues; it may be that they are not present in large enough numbers. Other results from a different approach to this problem (Urist et d., 1969) demonstrate that the number of inducible cells in different tissues is an important factor in bone induction. In coniparison with cells from the hemopoietic tissues mentioned above, the behavior of marrow cells in this type of system shows several differences. Most important is the fact that the presence of an inducer, such as transitional epithelium, is not necessary. When a piece of marrow or a suspension of marrow cells was placed in a millipore chamber and implanted intraperitoneally, bone was formed inside the chamber even though there was no inducing agent present. On the other hand, the density of marrow cells in the chamber was an important factor; bone was not formed below a certain critical concentration of cells (Friedenstein et ul., 1966). In contrast to these results with marrow cell suspensions, osteogenesis could not be induced in peritoneal fluid cells solely by changing the concentration of cells in the chamber; the presence of an inducer, such as transitional epithelium, was always necessary (Friedenstein et al., 1967).

B.

EXPERIMENTS WITH DIRECT TRANSPLANTS (OPENSYSTEM)

When pieces of bone marrow are transplanted directly under the renal capsule in a host, bone formation with subsequent development of hemopoietic tissue at the grafting site occurs. This situation constitutes an open system since cells of the host are able to contribute. The experiments to be described were performed with a variety of inbred mouse strains and their F, hybrids (Friedenstein et al., 1968), and the questions asked concerned whether cells from the host or donor tissue served as precursors of both the hemopoietic and osteogenic tissue arising at the site of the bone marrow transplants. One of the interesting results is that the bone formed was found to be of donor type, i.e., derived from cells of the donor marrow, whereas the centers of hemopoiesis formed in association with this bone are composed of host-type cells. The experiment that demonstrates these phenomena is described below

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( Friedenstein et al., 1968) , although an earlier experiment by Mawdsley and Harrison ( 1 9 6 3 ) gave essentially the same result. A piece of donor marrow obtained from an inbred mouse strain, CBA-T6T6, was transplanted to a site under the renal capsule of an F, hybrid recipient (the I:, being a cross between the strains A and CBA-T6T6). It is known that, whereas tissues of an inbred animal can be successfully grafted to an F, hybrid, transplantation of I;, to either of the parent types, A or CBA-T6T6, induces an immunological response since the graft possesses antigens not possessed by the host. Following transplantation of the CBA-T6T6 type marrow, healthy bone and marrow were formed in the I;, recipient at the site of the graft. When these tissues were retransplanted back into the donor type CBA-T6T6 animal the hemopoietic tissue was immunologically rejected, whereas the bone tissue was not, thus implying that cells of the bone were of donor type and that cells of the associated hemopoietic tissues were formed from the repopulating elements of the recipient. Supporting evidence came from an examination of the chromosomes of the hemopoietic tissue formed in the F, recipient which showed that these cells were indeed of F, karyotype. Another interesting feature of bone formed from such marrow transplants is that it appears to have an almost unlimited capacity for self-maintenance. Healthy bone sites were still to be found 12-14 months after transplantation of marrow under the renal capsule. This is in contrast to the fate of bone formed under the influence of an inducing agent, such as transitional epithelium, once the inducing agent has been removed. For example, an allogenic transplant of transitional epithelium results in immunological resorption of the latter after a few weeks. This is followed by rapid disappearance of the bone induced under its influence even though there is no immunological action against this bone tissue which has been formed from the host’s own cells (Friedenstein, 1965, 1968). The main conclusion that Friedenstein has drawn from his rcsults, some of which are reported above, is that there are two types of cell in the body capable of bone formation. The evidence for them is briefly summarized below under the names that have been proposed for them (Friedenstein, 1968).

Inducible Osteogenic Precursos Cells (ZOPC) . These cells have been found to be present in some hemopoietic tissues. Osteogenesis occurs only in the presence of an inducer and for it to be maintained, repeated acts of the inducing agent are required. In the experiments so far performed, a self-perpetuating line of cells capable of protracted osteogenesis after the induciiih7 drent b has been removed has not been derived from IOPC cells. Wlicther under prolonged conditions of induction or any other conditions this is possiblc is ttot yet known.

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Determined Osteogenic Preczlrsor C e l b (DOPC). Marrow tissues contain cells that form bone without an inducing agent being present. The fact that cell suspensions are as effective as intact marrow is one factor that rules out the possibility of any bone fragments being present to act as inducing agents. The bone formed is relatively long-lived, and in an open system it was still of donor tissue even at 14 months. This indicates the presence in the original marrow graft of cells that are self-perpetuating and capable of prolonged osteogenesis. In terms of the scheme proposed in Fig. 1, IOPC might be equivalent to the undifferentiated cells represented by a or b or both, and DOPC to the proliferating progenitors of bone cells represented by c. The question to be asked is whether or not transitions between IOPC and DOPC occur under normal conditions in the postfetal organism in vivo? In this context, one may consider the situation that occurs in the case of bone repair, which might be expected to reflect to an exaggerated extent the processes normally occurring iiz oivo. This situation is considered in the next paragraph in the light of the above-mentioned results. C. BONE FRACTURF RFPAIR

The healing of bone fractures has been regarded as an example of bone induction (McLean and Urist, 196S), although this interpretation is not universally accepted. In the first stage, there is hemorrhage and increased vascularization at the fracture site with extensive infiltration of cells. Next, formation of fracture callus, usually of a fibrocartilaginous or bony construction, which bridges the gap between the broken ends of the bone, takes place. This is followed by increased activity, mainly of the osteoprogenitor cells of the periosteutn, with formation of new bone on the surfaces of the fracture callus. The final result is replacement of the callus by bone, ending with a bony union of the fracture pieces. The exact origin of the cells that contribute to the different stages of fracture repair is not known for certain. It is thought that formation of the callus occurs by induction of some of the cells brought in by the increasing blood flow to the fracture site. This might be effected either by trauma or by the broken ends of the bone (both being capable of induced osteogenesis in other circumstances). Formation of new bone and completion of union of the fracture is attributed mainly to the osteoprogenitor cells from adjacent bone surfaces. Subsequently, the callus is resorbed and replaced by this new bone. The temporary nature of the callus would be consistent with its formation from IOPC, i.e., cells capable of osteogenesis in the presence of an inductive stimulus. Formation of callus ceases and the tissue is resorbed when the inductive

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environment is no longer present. On the other hand, the more permanent nature of the bone that constitutes the fracture union would be consistent with its formation by cells which have the characteristics of DOPC, i.e., cells already determined in an osteogenic direction and capable of maintained osteogenesis. This interpretation of bone fracture repair in terms of these two cellular stages is highly speculative. Some aspects of it, however, are open to testing by experiment. Recently, by using the technique of chromosome markers, the origin of the fibroblasts that take part in the inflammation process occurring during skin healing (Barnes and Khrushchov, 1968) has been shown to be blood-borne components from marrow tissues. A similar experiment could be performed to determine the origin of the cells involved in callus formation during bone fracture healing. At the present time, however, there is no evidence to suggest that transitions between the undifferentiated cells and the more differentiated osteoprogenitor cells are part of the fracture-healing process.

IV. Concluding Remarks Certain hemopoietic tissues (peritoneal fluid cells and spleen, and blood leukocytes) contain cells capable of forming induced bone when exposed ro a suitable environment. The same tissues have also been shown to contain hemopoietic stem cells (Barnes and Loutit, 1967; Loutit, 1967). The question immediately arises: Are these hemopoietic stem cells the same cells as the inducible osteogenic precursor cells? In terms of the scheme shown in Fig. 1 are they both members of a pool of multipotential connective tissue stem cells (a)? Alternatively, are they two separate groups of cells each of which has already taken a step in the direction of differentiation of the respective tissxes concerned and in this case could be represented by b in Fig. 1 ? Whether a and b both exist as separate entities has not been resolved. What is certain is the widespread presence, in the circulating blood, hemopoietic tissues, and presumably throughout the vascular channels of connective tissue systems, of undifferentiated mesenchymal cells that can be induced to form either bone or blood. The very ubiquity of their distribution might favor the possibility that they are a pool of true multipotential cells. The bone formed from these undifferentiated mesenchynial cells is of a ternporary nature; repeated acts of inductive activity are required for osteogeiiesis to be maintained, and if the inducing agent is removed the boiie disaiipears. There is no evidence from any source that a self-maintained bone tissue system can be obtained from these cells. Marrow tissues also contain cells capable of bone induction. In this case, tlie bone formed is of a more permanent nature. A self-perpetuating population of

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bone cells capable of relatively unlimited osteogenesis can be derived from these cells, and the presence of an inducing agent is not necessary. It has been suggested, therefore, that these cells are already predetermined along the pathway toward osteogenesis (DOPC). No morphological or ultrastructural studies have been performed on them. The only information available about them is that they will form bone unassisted under favorable conditions and that since cell density is a crucial factor interactions between the cells may therefore be important. The extent to which they are determined in an osteogenic direction is not known. It is possible that they encompass a wide range of the stages of differentiation including both types of osteoprogenitor cell and less diff erentiated precursors of osteoprogenitor cells, and they have, therefore, been equated with the wider term proliferating progenitors c in Fig. 1. In summary, there are two types of cell in the body capable of induced osteogenesis. Our knowledge of them is very meager and can be summed up as follows. There are undifferentiated inesenchymal cells with widespread distribution and there are cells found in marrow tissues which are already predetermined in an osteogenic direction. The evidence for this has been illustrated from examples of the recent work of Friedenstein (Friedenstein et al., 1967; Friedenstein, 1968), although the results from earlier work give pointers in the same direction. Urist and McLean (1952), for example, distinguished between cells that may be “induced” to form bone and cells with inherent “osteogenetic” activity. The superiority of marrow tissues (which contain DOPC) for the purpose of bone grafting has also been realized for a long time (Burwell, 1964). What is still an open question, however, is the relation between these two cell types from the point of view of osteogenesis under normal physiological conditions iiz vivo. One can speculate on the kinetics of osteogenic cells in the body. The only certain feature of this is that replacement of the osteoblasts and osteoclasts, which takes place throughout life, occurs almost entirely by multiplication of the osteoprogenitor cells which are already quite well differentiated in the direction of osteogenesis. Supplementation of this population could take place from the less differentiated cells of the proliferating progenitors and these in turn might be replaced by cells from the ubiquitous compartment of undifferentiated mesenchymal cells. Another possibility is that the compartment of proliferating progenitor cells may be full-sized shortly after birth and the need for replenishment from the undifferentiated mesenchymal cells is zero. The purpose of these latter cells might then be to fill a temporary need for regeneration of osteogenic tissue when necessary, e.g., in the case of bone fracture healing. In the present state of knowledge, it is not possible to decide which of these two possibilities is the more likely. A difficulty is that our techniques for studying the function of undifferentiated incsenchymal cells in vivo are very limited.

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There is no evidence at present, however, for the transformation of iindifferentiated inesenchymal cells into precursors of bone cells and eventually into the mature cells of bone in the postfetal organism in v h o in normal circumstances. In the embryo, one must assume that these transformations do take place. If this no longer occurs in the postfetal organism, it represents an important new concept for osteogenesis in postnatal life.

ACKNOWLEDGMENT I am indebted to Dr. R. W. Young who has allowed me to use his data (Young, 1962b) in preparing the graphs in Fig. 3.

REFERENCES Balogh, K., Jr., and Hajek, J. V. (1965). A m . J . Amt. 116, 429. Barnes, D. W. H., and Khrushchov, N . G. (1965). Nutwe 218, 599. Barnes, D. W. H., and Loutit, J. F. (1967). Laizcet fi, 1138. Bevelander, G., and Johnson, P. L. (1950). h a t . R e c o ~ d108, 1. Bingham, P. J , , Brazell, I.A,, and Owen, M. E. (1963). /. Enil’ociiwol. 45, 357. Bridges, J. B. (1959). Intein. Rev. Cytol. 8, 253. Buring, K., and Urist, M. R. (1967). Clin. Ortholied. 54, 235. Burwcll, R. G. (1964). J . Bone Joint Surg. 46B, 110. Burwell, R. G. (1966). J . Bone Joint Sflrg. 48B, 532. Cabrini, R. (1961). Intern. Rev. Cytol. 11, 283. Caffrey-Tyler, R. W., and Everett, N. B. (1966). Blood 28. 873. Carneiro, J., and Leblond, C. P. (1959). Exj~il.Cell Re.r. 18, 291 Cattaneo, S. M., Quastler, H., and Sherman, F. G. (1961). Ncrruie 190. 923. Crelin, E. S., and Koch, W. E. (1967). h a t . Record 158, 473. Cronkite, E. P., Bond, V. P., Fliedner, T. M., and Rubini, J. R. (1c)59). l.‘i/i. Ini’c.it. 8. 263. cle Voogd van der Straaten, W. A. (1966). P,oc. .3;d E u . 011i.m Jywp L’~!c;/ied ‘ / i i r i i c \ , Dazjor, Switz., 1965, p. 10. Springer-Verlag, New York. Doty, S. B., Schofield, B. H., and Robinson, R. A. (196s). Liz “ParJthyroid Hormone and Thyrocalcitonin (Calcitonin)” ( R . V. Talinage and I-. F. Helanger. eds ) , 11, 160. Excerpta Medica International Congress Series No 159. Fischman, D. A,. and Hap, E. D. (1962). h a t . Reciiid 143, 129. Friedenstein, A. J. (1962). Nature 194, 698. Friedenstein, A. J. (1963-1964). Bull. Exp2’1. Biol. Med. ( U S S R ) ( l < q < b \ /’ /~‘ J , / I A / , ) 54, 1’555. Friedenstein, A. J. (1965). Bull. Exptl. Biol. M e d . ( U S S R ) (Etisyli.ih ‘ / ‘ ~ ~ I I / I / 59, .) lyi, Friedenstein, A. J. (1965). Clin. Orthoped. 59, 21. Friedenstein, A. J., Piatetzky-Shapiro, I.I.,and Petrakova, K. V. ( 1966). 1. L : : ~ h i~ d . Exptl. Moyphol. 16, 381. Friedenstein, A. J., Lalykina, K. S., and Tolmacheva, A. A. (1967). Acts d i i L i / . 68, >i? Friedenstein, A. J., Petrakova, K. V., Kurolesova, A. Y., and Frolwii, G. 1. ( 1 ~ ) . Tlztnrpluntaiion 6, 230. Friedman, B., Heiple, K. G., Vessely, J. C., and Hanaoka, H. (1965). Cliu. O i / h i i / ~ d . 59, 39.

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Fry, R. J. M., Lesher, S., Kisieleski, W. E., and Sacher, G. ( I 9 6 3 ) . 771 “Cell Proliferation” (L. F. Lamerton and R. J. M. Fry, eds.), p. 213. Blackwell, Oxford. Goldhaher, P. (1961 ) . Sciezce 133, 2065. Ham, A. W . , and Leeson, ’I+. S. (1961). “Histology,” 4th Ed., p. 2 4 2 . Lippincott, Phil:idelphia, Pennsylvania. Heiple, K. G., Herndon, C. H., Chase, S . W., and Wattleworth, A. (1968). J . H o u r Joint Suig. 50A, 31 1. Heller, M., iMcLean, F. C., and Bloom, W. (1950). A m . J . Anat. 87, 315. Holtrop, M. E. ( 1966). Proc. 3rd Eusopean Symp. Calcified Tissues, Davos, Szui~z., l96J, p. 32. Springer-Verlag, New York. Howard, A,, and Pelc, S. R. (1953). Heredity 6, 261 (Supplement on Chrotnosonie Breakage). Huggins, C. B. (1930). Proc. Soc. Exptl. B i d . Med. 27, 349. Hoggins, C . B. (1968). Cljn. Orthoped. 59, 7. Kcmber, N . F. (1960). J . Bone Joint Surg. 43B, 824. Kroon, D. €3. (1958). Acta Morphol. Need.-Scand. 2, 38. Lesher, S., Fry, R. J. M., and Kohn, H. I. (1961). Exptl. Cell Res. 24, 334. Idoutit, J. F. (1967). J . Clin. Pathol. 20, 535. McLean, F. C., and Urist, M. R. (1968). “Bone,” 3rd Ed., Chap. 13. IJniv. of Chicago Press, Chicago, Illinois. Mankin, H . J. ( 1 9 6 4 ) . J . Bone Joint Surg. 46A, 1253. Mawdsley, R., and Harrison, G. A. (1963). Nature 198, 495. Micklem, H. S., and Loutit, J. F. (1966). “Tissue Grafting and Radiation.” Academic Press, New York. Owen, M. (1963). J . Cell Biol. 19, 19. Owen, M. (1965). l’roc. 21211 EwuJieaiZ S y m p . Ca!rified l ’ i r r m s , Liege, Belgium, 1963, Congr. Colloq. Univ. de Liege, Vol. 31, p. 11. Owen, M. (1968). In “Biology of Hard Tissue,” Proc. 2nd Cotif. (A. M. Budy, ed.), 17. 147. Nail. Aeron. and Space Admin., Washington, D.C. Owen, M., and Bingham, P. J. (1968). In “Parathyroid Hormone and Thyrocalcitonin (Calcitonin)” (R. V. Talmage and L. F. Belanger, eds.), p. 216. Excerpta Medica International Congress Series No 159. Owen, M., and MacPherson, S. (1963). J . Cell Biol. 19, 33. Owen, M., and Shetlar, M. R. (1968). Nature 220, 1335. Post, R. H., Heiple, K. G., Chase, S. W., and Herndon, C. H. (1966). Clin. Oithoped. 44, 265. Pritchard, J. J. (1952). J . Anat. 86, 259. J’ritchard, J. J. (1956). In “The Biochemistry and Physiology of Bone” (G. H . Bourne, ed.), p. 179. Academic Press, New York. Quastler, H., and Sherman, F. G. (1759). Exptl. Cell Res. 17, 420. Ray, K. D., and Saber, T. Y . (1963). J . Bone Joint Surg. 45A, 337. I
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Trueta, J. (1963). J. Bone Joint Surg. 45B, 402. Urist, M. R. (1965). Science 150, 893. Urist, M. R., and McLean, F. C . (1952). J. Bone Joint Surg. 34A, 443. Urist, M.R., Hay, P. H., Dubuc, F., and Buring, K. (1969). Clin. Orthoped. 64, 194. Walker, D. G. (1961). Ball. Johns HopRins Hosp. 108, 80. Young, R. W. (1962a). Exptl. Cell ReJ. 26, 562. Young, R. W. (1962b). J. Cell Biol. 14, 357. Young, R. W. ( 1 9 6 2 ~ )Anat. . Record 143, 1. Young, R. W. (1963). Clin. Orthoped. 26, 147. Young, R. W. (1964). In “Bone Biodynamics” (H. M. Frost, ed.), p. 117. Little, Brown, Boston, Massachusetts. Young, R. W. (1968). In “Biology of Hard Tissue,” Proc. 2nd Conf. ( A . M. Budy, ed.), p. 167. Natl. Aeron. and Space Adrnin., Washington, D.C.

Regeneration and Differentiation of Sieve Tube Elements WILLIAM P.

JACOBS

Biology Department, Princeton University, Princeton, N e w Jersey

I. Introduction

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11. Regeneration of Sieve Tubes

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What Factors Control Sieve Tube Regeneration? .... Excision of Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Substitution of Chemicals for Effective Organs ...... D. Generality of Results ............................ E. Specificity of IAA for Control of Sieve Tube Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Are Sieve Tubes the Normal Path of IAA Movement? 111. Differentiation of Sieve Tubes in Organ or Tissue Culture IV. Normal Differentiation of Sieve Tubes . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. B.

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I. Introduction When one realizes that sieve tubes1 have been considered for 100 years to be the main pathways for the movement of organic food materials throughout the vascular plant, the relative paucity of critical developmental work should be questioned. The differentiation of sieve tubes has been studied much less than that of tracheary cells. The regeneration of sieve tubes has been studied even less than their differentiation. This is undoubtedly because sieve elements lack the easily distinguishable wall properties of tracheary cells and are typically tiny in diameter. Despite these difficulties, the absolute number of papers on the normal differentiation of sieve elements is sizeable, and even the regeneration of sieve tubes has been studied by several workers in recent years. The classic literature on the anatomy of normal differentiation of sieve tubes is summarized well in the reviews and books of Esau (1953, 1960, 1965), and there is no need to go over the material in detail. Figure 1 (Esau, 1953) shows stages in the development of sieve tube elements and associated companion cells. Nuclear disintegration and the development of slime and sieve plates are distinguishing features of sieve element development in the dicotyledons. Earlier work presents the pattern of sieve element differentiation in the angiosperm shoot apex as being very uniform, as well as strikingly different from that of tracheary differentiation. The first sieve tubes are reported to differentiate “continuously and acropetally” out into the young leaves, whereas the first tracheary 1 “Sieve tube” refers to a longitudinal chain of cells. The individual cells composing the sieve tube are called “sieve tube elements” (Esau, 1953, p. 2 6 8 ) .

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FIG. 1. Differentiation of sieve tube members in Cucurbita. ( A ) Transection of primary phloem with the different stages numbered as follows: (1) meristematic phloem cell just before division; ( 2 ) after division into sieve tube member and companion cell; (3) slime bodies have just begun to develop in the sieve element protoplast: ( 4 ) slime bodies are of maximal size and the thick (nacrk) wall is present in the sieve element; ( 5 ) slime bodies have dispersed; (6) sieve element is partly obliterated. Similar stages are depicted in the longitudinal sections in B-G. ( B ) Meristernatic phloem cells in division (above) and just after division (below) into a sieve tube member and precursor of companioii cells. (C) Young sieve element and precursor of companion cells. ( D ) Sieve tube member with slime bodies beginning to develop; precursor of companion cells has divided illto three companion cells. ( E ) Slime bodies of maximal size, nucleus highly vacuolated, thick walls in the sieve element. ( F ) Slime bodies partly fused into amorphous masses and nu. cleus absent. (G) Mature sieve element with thin parietal cytoplasm, large vacuole containing vacuolar sap and slime (mostly in the lower end of element). The protoplast is connected with the lower sieve plate but is partly withdrawn from the upper. Note the depressions (sieve areas) in the sieve element walls facing the companion cells in E-G. (From Esau, 1953, Fig. 12.5.)

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elements differentiate at an isolated locus near the base of the young leaf. The timing is different also; sieve tubes typically have been reported to differentiate in the young leaves before tracheary cells. The much sketchier information from studies of vascular regeneration in shoots suggests that the timing is different there, too, but in the opposite direction; sieve elements have been said to regenerate later than tracheary cells (von Kaan Albest, 1934; Simon, 1908). These differences in pattern and timing suggest that different chemicals control the differentiation and regeneration of these two vascular cell types. In this article, we shall emphasize recent studies of sieve tube regeneration and of the endogenous factors controlling sieve tube development.

11. Regeneration of Sieve Tubes Although tracheary regeneration had been investigated extensively as early as 1908 (Simon, 1908), it was not until 1934 that sieve tube regeneration was similarly investigated (von Kaan Albest, 1934). Eschrich’s elegant work in 1953 confirmed and extended von Kaan Albest’s. Both authors based most of their results on hand sections of regenerating vascular strands in elongating internodes of Impatiens. Impatiens was selected because Simon (1908) had used it for his studies of tracheary regeneration. Elongating internodes were used to avoid the problems of wound cambium formation that would be expected to occur if older, nonelongating internodes were used. von Kaan Albest reported that when the vascular strand was cut with a razor blade the first wound sieve tubes were not seen until 5 days later ( 1 day later than the first wound tracheary cells), when some were regenerated in direct connection with the phloem of the severed vascular strand. This regeneration was polar; it appeared first at the upper regeneration area (at the basal end of the severed vascular bundle). The wound sieve tubes differentiated in the unwounded cells of the procambial zone, running roughly parallel to the wound surface. After 5 days, the regenerating sieve tube strand bent around the edge of the wound and, still running more or less parallel to the wound, by 7-8 days had joined the apical end of the severed strand below the wound. (Except that the wound tracheids differentiated 2-3 days earlier than the wound sieve tubes, von Kaan Albest noted that the general course of regeneration was similar for the two types of vascular cells.) During the second week, more wound sieve tube cells regenerated, thickening the original regenerated strands. More cells differentiated above the wound than below it. A meristem finally developed in the callus 4-6 weeks after wounding and proceeded to cut off cells which differentiated into tracheids to the inside and sieve tubes to the outside. Seasonal effects were strong, the foregoing description applying to spring and summer. Eschrich (1953) confirmed this general pattern, adding that the rate of regen-

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eration varied with temperature and that Impatiens and Colezls regenerated sieve tubes faster than several other genera tested. The path of regeneration of the wound sieve tubes is shown in Fig. 2. They differentiate in the procambial zone, always interior to the starch sheath and

FIG. 2. Transverse section of cambial area of an Impatiens stem, showing cambial cells ( K ) , wound sieve tubes (a, b), recently laid down cell walls in early stage of sieve tube regeneration (c, d), and starch sheath ( S t ) . Several thick-walled wound tracheary cells are at the right of the cambial zone and two longitudinally running phloem strands have been cut through at A and B. (From von Kaan Albest, 1934, Fig. 12.)

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often just interior to, but not joining, the xylem-less phloem strands (Eschrich, 1953). There are only one to three undifferentiated cells of the procambial zone between the wound sieve tubes and the wound tracheids (Fig. 2 ) . The earliest reported sign of the differentiation of wound sieve tube elements is the occurrence of numerous nuclear divisions in the cells of the procambial zone (Thompson, 1967, Fig. 1). von Kaan Albest stated, without illustrations to confirm it, that whole cells may differentiate directly into wound sieve tube elements, particularly during the period immediately after wounding when cell divisions are still scanty. This has not been confirmed by later investigators.

FIG. 3. Diagrammatic reconstruction of a longitudinal radial section through cambial cell b of Fig. 2, showing the new curved walls. A transverse section cut through level I of this cell group would look like ‘c’ or ‘d in Fig. 2. A transverse section through level I1 would look like ‘a’ or ‘b’ in Fig. 2. (From von Kaan Albest, 1934, Fig. 12a.)

According to Eschrich, up to four nuclei may be present before new cell walls are formed (Eschrich, 1953, Fig. 5 ) . The first new cell wall is somewhat curved (Fig. 3), and the smaller subdivision of the original cell may then differentiate into a wound sieve tube element, or further cell divisions may occur within the smaller subdivision (Eschrich, 1953, Fig. 6 ) . Because the long axis of the wound sieve tube usually runs tangentially, its cross walls are typically part of the radial (longitudinal) wall of the original brick-shaped procambial cell. This portion of the radial wall that serves as the cross wall for the wound sieve tube usually includes several pits, and “it is probable” that the sieve plate differentiates from several of these neighboring pits (Eschrich, 1953, Fig. 10). Two wound sieve tube elements often differentiate from such subdivisions of a single brick-shaped cell of the procambial zone. von Kaan Albest thought it unlikely that the other daughter cells could be considered companion cells because they were too wide, were not particularly rich in cytoplasm, and did not have as many cross walls relative to the sieve tube elements as might be expected of normal companion cells (cf. Esau, 1953, pp. 281-284). Eschrich (1953), however, who used a variety of stains to investigate the cytoplasm and nuclei more closely, concluded that at least some of the other daughter cells were com-

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WILLIAM P. JACOBS

panion cells, judging by cell shape, as well as by the shape and staining properties of the nucleus. Before the pits on the radial wall of the procambial cells were changed into recognizable sieve plates for the new wound sieve tube element, they already showed a thin layer of callose, judging by resorcin blue stains (Eschrich, 1953). Even after the sieve plate was first differentiated, plasmolysis of wound sieve

FIG.4. Tangential section through the area in which wound sieve tubes are regenerating below a stem wound in Impatiens. (From Eschrich, 1953, Fig. 15.) tube elements with weakly developed sieve plates caused the protoplasm to pull away from the cell wall all around the cell, as well as from the thin sieve plate. The callose stayed on the wall. Only when the sieve plate had differentiated even thicker strands, judging by resorcin blue stain, did the cytoplasm fail to pull away from the sieve plates upon plasinolysis (Eschrich, 1953, Fig. 12). After sieve plate development there was a long pause before the nucleus of the wound sieve tube eleinent was resorbed. It did not begin until 10 days after wounding.

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Not all wound sieve tube elements are shaped like these narrow tubes. Some have three to four sieve plate areas instead of two (Eschrich, 1953, Figs. 7 and 8). Eschrich noted that nucleated wound sieve tubes that regenerated around the wound always connected to sieve tubes running longitudinally that still contained nuclei and, by that criterion, were probably differentiated after the

FIG. 5 . Diagrams of fluorescence observed in Inzputiens steins when fluorescein was added at different times before or after wounding. 1, Results obtained when fluorescein was added 2 hours before wounding; 2, fluorescein added at wounding time; 3, fluorescein added 2 days after wounding; 4, 4 days after wounding; 5 , 6 days after wounding; 6 , fluorescein added 8 days after wounding. (From Eschrich, 1953, Fig. 27.)

wounding (Fig. 4 ) . (When these longitudinally running sieve tube elements lacked nuclei, so did the wound sieve tubes connecting to them.) These nucleated “long sieve tubes” did not connect by sieve plates with the eiiucleate sieve tubes of the old vascular strand; they maintained an independent course in the strand, with the only histological sign of 3 relation being a slight development of callose on pits of the common longitudinal walls. Do the wound sieve tubes function in transport? von Kaan Albest (1934, pp. 34-35) presented some very indirect evidence that they do. She cut all the vascular strands of the five-sided Impatiens stem, by making cuts at five different levels. (Each large corner strand was thereby cut at two levels.) Seven days later all 10 plants looked fresh, and sections of two revealed that wound sieve tubes and wound tracheids had regenerated completely around the wounds, The other eight experimental plants continued their development, differing from the normal plants in no way noticeable to von Kaan Albest. Eschrich’s evidence was more direct and more convincing ( 1 9 5 3 ) . At various intervals after wounding, he added potassium fluorescein above the severed vascular strands (Fig. 5 ; note

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that these experiments were run in the winter, so regeneration was somewhat slower). His unwounded control plants gave results as expected from the work of Schumacher (1933) ; fluorescence was restricted to the sieve tubes and companion cells. With wounded plants, fluorescein added immediately ( 2 in Fig. 5 ) gave fluorescence in the sieve tubes down to about five to seven sieve tube elements above the wound edge (1.5 mm) . Four to six hours later, some fluorescence also appeared in phloem parenchyma cells adjacent to the most basal 1 mm or so of fluorescing sieve tube elements. Fluorescein added 2 days after wounding gave a pattern not much changed; as the callus formed over the wound, the fluorescence moved down into the basal 1.5 mm of sieve tubes and parenchyma ( 3 in Fig. 5 ) . If regeneration is allowed to proceed for 4 days before fluorescein is added, a striking and unexpected difference is found; the fluorescence is now strong in the cells of the starch sheath just above the wound, spreading from the cut middle strand out to the intact side strands but not entering their vascular elements. Transverse sections were said to show that the fluorescence was restricted to the cells of the one-layer-thick starch sheath, and was not visible in the procambial tissue (4 in Fig. 5 ) . Six days after wounding, the fluorescence in the starch sheath had spread down around the sides of the wound, and some wound sieve tubes were in their first stages of differentiation ( 5 in Fig. 5 ) . Sections of the new wound sieve tubes, comprised of two to five elements, showed fluorescence that was weak when the sieve plate was not fully differentiated and strong when it was. Because these fluorescing sections were not necessarily continuous with the cut middle strand, Eschrich presumed that their fluorescein came from the cells of the overlying starch sheath. Eight days after wounding, all the fluorescence was in the sieve tubes and companion cells, as in intact tissue (6 in Fig. 5 ) . Eschrich emphasized that the wound sieve tubes were apparently functioning to move fluorescein not only long before their nuclei were resorbed but to a noticeable extent even before their sieve plates were fully differentiated. This was a big change in viewpoint from that common in the earlier sieve tube literature in which theories (more than data) led investigators to expect that the only sieve tube elements functioning in transport were those in the enucleate, “late” stage.

A. WHATFACTORSCONTROLSIEVETUBEREGENERATION ? Although 44 pages of von Kaan Albest’s article are on physiological experiments, by modern standards this is an unsatisfactory section. The experiments were qualitative when they need to be quantitative. They were poorly designed; effects of developmental age were inextricably confused with effects of treatment; controls were not routinely used; and there was no sign of randomization. With these flaws it would be pointless to discuss the results in detail. Suffice it to say that von Kaan Albest followed up the conclusion of Kabus (1912) which

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stated that the presence of leaves or buds increased the chances of a graft being successful because of some “correlative influence” that came from them. von Kaan Albest excised leaves or buds above wounded internodes and found less regeneration of both sieve tubes and tracheary cells from the excisions. Tracheary regeneration was delayed by several excision treatments that had no detectable effect on the rate of regeneration of sieve tubes. A differential effect on the two types of vascular cells was also reported for the effect of leaves and buds below the wound; von Kaan Albest interpreted her experiments as showing sieve tubes to be unaffected by these proximal organs but tracheary regeneration to be inhibited by their presence (von Kaan Albest, 1934, p. 88). Decades later, von Kaan Albest’s pioneering study was extended by a group that had found it useful to apply a quantitative physiological approach to the study of tracheary regeneration. They had collected evidence that the normal limiting factor for tracheary regeneration was the auxin indole-+acetic acid (IAA) formed in young leaves (Jacobs, 1952, 1954, 1956). The amount of auxin coming from the leaves could be measured, this amount of synthetic IAA substituted for the leaves, and tracheary regeneration normal in amount and appearance obtained. To put the studies on a quantitative basis, existing techniques had been adapted so that whole mounts could be made of regenerating areas that had been made transparent after the tracheary cells were selectively stained. This allowed the entire tracheary regeneration to be seen at once and made possible the routine use of physiologically adequate sample sizes. Statistical techniques were used to obtain more information from the same amount of work, and genotypic variance was reduced by the use of a clonal stock. Because this approach had worked well for tracheary regeneration, it was natural to try to apply it to sieve tube regeneration also (LaMotte and Jacobs, 1962, 1963). At the time LaMotte started his work, there was no clue in the literature as to what the normal limiting factor for sieve tube differentiation might be. From the differences cited in the literature between tracheary cells and sieve tubes in timing and pattern, both in normal and regenerative development, one could only expect that sieve tubes would be controlled by a substance different from the IAA that controlled tracheary cells. Accordingly, his primary interest was in developing a technique capable of providing a quantitative and reproducible measure of the number of sieve tube elements regenerated. Coleus was used as experimental material because earlier workers had used it for studies of vascular regeneration and because its square stem facilitated the preparation of a flat “phloem strip” from one side of the stem. Regenerated sieve elements in Coleus were too smalI to be seen in whole segments of internode cleared by the same method employed in the studies on tracheary regeneration (Jacobs, 1952), so the anatomical technique of clearing

248

WILLIAM P. JACOBS

was combined with the physiological one of making a “phloem strip” (LaMotte and Jacobs, 1962). The phloem and the tissues external to it were separated from the xylem-pith cylinder on one side of the stem (Pig. 6). This strip, removed from an internode previously killed, fixed, and toughened, as well as

Phloem strip

Sieve t u b e s

Wound

-

FIG. 6. Diagram of the LaMotte technique for removing a phloem strip f r o m one side of the “square” Coleus stem.

cleared, in lactic acid, was then stained with aniline blue. The stain brought out the sieve elements strongly enough so that the strip could be accurately mxde thinner and more transparent by dissecting off the epidermis, cortex, and scar tissue. The thinned strip was then restained in aniline blue before being made into permanent mounts with the originally inner surface of the strip facing the cover-slip. [Aniline blue is the stain most frequently used for sic\ e elements (Johansen, 1940) .] Although fixing the internode before dissectiiig off the strip improved the regularity of the separation, the youngest internode to give easy separation was no. 5 (Fig. 7). This is also the youngest one to cease elongntioii, and from both criteria is presumably the youngest internode with well-developed cambium. For this purely technical reason, our first studies of thc regeneration of sieve elements were performed on internode no. 5. To improve the precision of counting the regenerated cells, we switched froin a V-shaped wound in the large corner strand (as used for our early xylem studies) to a slit wound in the strand running midway along one of the flat sides of the Coleus internode. This change to a “side wound” not only iiiakes the stripping easier, but causes all the sieve elements to regenerate in essentially one plane rather than in the three-dinaensional pattern of the “corner w o d . ”

249

SIEVE TUBE ELEMENTS

A microphotograph of such a preparation illustrates how easily the individual sieve elements can be seen through the cleared and stained strip (Fig. 8). The wound sieve tubes are seen through only one to three radially thin, undifferentiated cells of the cambial region. Apical bud Apical internode Internode no. I Internode no.

2

FIG. 7. Diagram of Colem plant to show the method of designating Ieaves and internodes. Internode no. 2, which is still elongating, was the internode used i n early studies on xylem regeneration and auxin transport. Internode no. 5 is the youngest internode with cambium. (The leaves, which are actually decussate, are here represented as being all in one plane.) (From Jacobs, 1954, Fig. 2 with permission of the editors of The Ameiicnti Naturalist and the University of Chicago Press.

The number of strands of regenerated sieve tubes was counted, using a counting convention designed to measure the number of actual sieve elements regenerated (LaMotte and Jacobs, 1962). A diagram of a typical strip, illustrating the counting convention, is shown in Fig. 9. Once the counting convention was established and demonstrated to give reproducible counts, it was determined that there was an average of 10 sieve elements per strand [9.9 i0.7 (mean standard error) (LaMotte and Jacobs, 1962, Table II)]. As usual, all slides were coded, to eliminate subconscious bias, before counting was done, and blind repeats were routinely run to check on the reproducibility of the counts. The plants were selected from the clonal stock for age and developmental stage, as in the earlier tracheary studies. To reduce the variation still further, the plants were grown in growth chambers with controlled light cycles, light intensities, and temperatures. Long-day conditions (16-8) gave more and faster

250

WILLIAM P. JACOBS

FIG. 8. Regenerated sieve tubes in Coleus stems, photographed in a phloem strip. A cross-strand with a branch strand joining it in the interfascicular region is shown. Note that many of the sieve tube members were formed diagonal to the long axis of preexisting cells. (From LaMotte and Jacobs, 1962, Fig. 3. @ 1962 The Williains & Wilkins Cn.)

251

SIEVE TUBE ELEMENTS

regeneration than short-day cycles (8-26), so long-day conditions were usually used (LaMotte and Jacobs, 1963, Table 1). B. EXCISIONOF ORGANS

In repeating von Kaan Albest’s experiments, but in this quantitative way, primary leaves and branches were cut off the plants in varying patterns both

apical

basal end 2

4 4.5

4.5

4

0

total 19.0 FIG. 9. Diagram of a phloem strip preparation (left) and its enlarged regeneration area (right) which show the original wound site and the various types of regenerated phtoem strands found in wounded CaZem internode no. 5. Assessment of the phloem strands in counting is shown by numerals. The letters a-g designate types of strands described in the original paper. The circles on the extreme left represent low power fields of view. (From Lahfotte and Jacobs, 1762, Fig. 1 . @ 1762 The Williams & Wilkins Co.) above and below the regeneration area, in the hope that lateral shoot organs of some age or position would be controlling the regeneration of sieve elements. When all leaves and buds were excised, a striking and statistically significant decrease occurred in the number of strands of sieve elements that regenerated (LaMotte and Jacobs, 1963, Fig. lo). The control group, with all leaves and buds intact, regenerated an average of 22.2 sieve tube strands in 5 days. The plants with only the root system and the main stem remaining regenerated only 4.7 strands. Thompson confirmed these findings (Thompson, 1966, Table I).

252

WILLIAM P. JACOBS

Leaves and buds above the regenerating internode were more important than those below; excision of proximal leaves and branches caused no change in the number of regenerated sieve tube strands, but excision of distal leaves and buds gave a statistically significant decrease (LaMotte and Jacobs, 1963, p. 88, foot-

C

CONTROL

ALL SHOOT

D

A L L BUDS OFF

ALL PRIMARY

ORGANS OFF

22.2 k 4 . 4

4.7?2

** I

LEAVES OFF

21 2

t 3.5

E

*

F l

Wound

ALL P R O X I M A L ORGANS OFF

I 5 6 _ f 30

ALL DISTAL ORGANS OFF

25.2 k 2 . 7

11.5 & 2.6

*

FIG. 10. The effects of excising various shoot organs on the number of sieve tube? regenerated in Coleus internode no. 5 (the mean and standard eiror for each treatment is shown). All plants were grown in long-day (16 hours) conditions; regeneration was for a period of 5 days; n = 4. Asterisks show significant differences from the control mean (A) (i.e., * = .05 and ** = .Ol). (From LaMotte and Jacobs, 1963, Fig. I.)

note 3). With this clue as to the role of the distal organs, one could expect signs of polarity of regeneration in time course experiments. When all leaves and buds were left on, more sieve tubes regenerated above the wound thaii below it during the first 3-5 days. By 7 days, this difference had disappeared (Fig. 11).

c.

SUBSTITUTION OF CHEMICALS FOR

EFFECTIVEORCANS

To our surprise, IAA replaced completely the striking effect of the distal shoot organs on sieve tube regeneration. The addition through the stem stumj;

253

SIEVE TUBE ELEMENTS

FIG. 11. The polarity of phloem regeneration above and below the wound rls shown by the time course. (Data from LaMotte and Jacobs, 1963, Table 2.)

of IAA at 2 ppm in aqueous solution, the concentration shown to replace exactly leaves 1 and 2 in their effect on tracheary regeneration in internode no. 2 (Jacobs, 1956), restored exactly the level of sieve tube regeneration to that found in intact control plants (Table I ) . When added mixed with lanolin, IAA at 0.1% and 1.0% evoked significantly more regeneration of sieve tubes than the attached organs themselves. To further isolate the reacting system, and specifically to make certain that the root system was not necessary for the restoration of the normal level of sieve tube regeneration by added IAA, internodes no. 5 were excised from the plant and treated at their apical ends with IAA in lanolin. A highly significant inEFFECTSOF IAA

TABLE I SUCROSE ON SIEVE TUBE REGENERATION IN Coleus PLANTS STANDING 5 DAYSWITHOUT SHOOTORGANS~

AND

Treatment Intact control plants All shoot organs excised Water Sucrose ( 2 0 gm/liter) IAA ( 2 mg/liter) TAA sucrose

+

5

I,

Number of regenerated strands, mean & S.E. 23.2 t 2.2 7.2 t 3.4b 7.6 2.5h 23.9 & 4.5 20.9 t 3.9

From Table 3 of LaMotte and Jacobs (1963); u = 4. Significantly different (at 5% level) from the values from the intact contmls.

254

WILLTAM P. JACOBS

crease in the number of sieve tubes was found after 7 days (LaMotte and Jacobs, 1963, Table 4 ) . The fact that isolated internode no. 5 did not give fewer regenerated sieve tubes than did the main stem with an intact root system remaining supports the view that the root system suppIied no significant amount of sieve tube-forming material.

I A A CONCENTRATION (%) FIG. 12. The effect of apically applied IAA at various “concentrations” in lanolin on xylem and sieve-tube regeneration around a wound in isolated no. 5 internodes of Coielts (Princeton clone). (From Thompson and Jacobs, 1966, Fig. 1.)

After this unexpected finding, it was obviously necessary to check, in the same internode, the effects of IAA on the regeneration of both sieve elements and tracheary cells. (Our earlier studies on tracheary regeneration had used elongating internode no. 2.) The LaMotte technique was used for sieve tubes, and a similar flat strip, stained for tracheary cell walls, was saved from the xylem side of the cambium. When excised internodes no. 5 were treated on their apical ends with different “concentrations” of IAA in lanolin, given the standard slit wound midway down the internode, then checked after 7 days for the number of regenerated tracheary cells and sieve element strands, the p o l e d results of all experiments were as shown in Fig. 1 2 (Thompson, 1965; Thompson and Jacobs,

SIEVE TUBE ELEMENTS

255

1966). When no IAA was added externally, no tracheary cells and only a few strands of sieve elements regenerated. As the concentration of IAA was increased to 0.01% IAA, there was a slow linear rise in the number of sieve elements; 0.1% IAA gave a sharp increase with still more sieve elements being regenerated when 1.0% IAA was added. When the number of sieve strands that had regenerated in an otherwise intact plant was determined and that value interpolated on the concentration curve for isolated internodes, the “intact” value was equivalent to an IAA concentration of 0.05% (asterisk in Fig. 1 2 ) . The number of tracheary cells that had regenerated in this same period, just across the cambial layer in the same internodes, is also shown in Fig. 1 2 . (The units on the two Y axes were selected so that the units represent the same iiumber of cells.) No xylem cells regenerated in untreated internodes, nor did they start to regenerate in noticeable amounts until 0.01 % IAA was added apically. There was the same striking increase in regeneration between 0.01 and 0.17; IAA that was noticed for the sieve elements. The values for tracheary regeneration in the intact plant fell at the same equivalent IAA concentration of 0.05% as did those for sieve strands. The regeneration of both sieve elements and tracheary cells were unaffected by IAA added at the original base of the isolated internode (Thompson and Jacobs, 1966, Table 11). Internode no. 5 , in other words, showed absohte polarity of IAA movement, as measured by vascular cell regeneration. A direct test of IAA-I4C movement in corresponding internode cylinders confirmed the strong basipetal polarity of IAA movement (Fig. 1 3 ; also see Jacobs and McCready, 1967). The fact that the curve for tracheary cells parallels so closely that for sieve elements in Fig. 12, but at a lower level, suggests that prospective sieve elements are either more sensitive than prospective tracheary cells to a given concentration of IAA, or that they have greater access to auxin in the stein-or perhaps both. If sieve tubes are the major path of normal transport for auxin, then the sieve tube side of the cambial layer would obviously have greater access to auxin after severance of the vascular continuity. The results with the untreated, excised internodes fit this hypothesis; the 60 or so sieve elements regenerated would be attributable to the small amounts of endogenous auxin available in the phloeni, while the lack of regenerated tracheary cells would reflect the lack of an auxin surplus to move radially and internally across the cambium. The time course of vascular regeneration was found to fit either hypothesis; regenerated sieve elements could be detected (with the stains used) a full day before tracheary cells in internode no. 5 (Thompson, 1967). The one fact that seemed to indicate that auxin first reaches the phloem side is that the first tracheary cells to regenerate are radially exactly opposite an already regenerated strand of sieve elements. The individual regenerated tracheary cell is so much larger than the individual cell of the regenerated sieve strand that it is startling

256

WILLIAM P, JACOBS

to see, in preparation after preparation, how exactly the path of differentiation of the tracheary strand follows the course already laid down on the other side of the cambial layer by the sieve tube (Fig. 1 4 ) .

I

I

FIG. 13. The polarity of movement through pith or vascular (“cornc‘r”) cylinders of Coleus internode no. 5 of 14C from IAA and 2,4-D, each supplied at an initial concentration of 5 pM. Radioactivity in receivers is shown for basipetal (solid lines) and for acropetal (dashed lines) receivers. The diagram in the upper left corner represents the transport set-up, with a 3-mm-long agar cylinder on each end of a horizontal, 5-mm-long cylinder of tissue. (From Jacobs and McCready, 1967, Fig. 1 with permission fi-om the editors of American Journal o! Botany.)

D. GENERALITY OF RESUJ-TS IAA stimulates the regeneration of both sieve tubes and tracheary cells in plants other than the Princeton clone of Coleus, as shown for the “Golden Bedder” variety of Coleus blumei and for the “Yellow Plum” variety of tomato (Lyropersicon esculentum Mill.) (Thompson and Jacobs, 1966, Table 111). In excised tomato internodes, 0.1% IAA in lanolin fully restored the numbers of vascular cells to those found in intact controls. Both tomato and “Golden Bedder” were like the Princeton clone in showing a strong basipetal polarity when vascular regeneration resulting from apical application of added IAA was compared to that from basal application.

258

WILLIAM P. JACOBS

E. SPECIFICITY OF IAA

FOR

CONTROL OF SIEVETUBE REGENERATION

Because the activity of IAA would be expected to be a function of its activity as an auxin, other synthetic auxins were tested for activity in isolated internode no. 5 (Fig. 15; data taken from Thompson, 1965). Each of the auxins was

0 0

10

I

I

NUMBER

OF SIEVE TUBE 30

I

I

STRANDS

70

50 I

I

I

Tryptophan Lanolin Control

0 tracheary cells

0

100

300 NUMBER OF TRACHEARY

500

3300

3400

CELLS

FIG. 15. Activity of various auxins in causing regeneration of sieve tubes and trachea0 ceiis in isolated Coletls internode no. 5. All compounds added as 0.1%) mixtures in lanolin (except for tryptophan, which was at 1.0%). Asterisks next to 2,4-D and 2,4,5-T indicate such proliferation of sieve tubes that counts were impossible to make accurately. (Data from Thompson, 1965, Table IX.)

active in causing the regeneration of both sieve tubes and tracheary cells. The two weed killers with auxin activity, 2,4-dichlorophenoxyaceticacid (2,4-D) and 2,4,5-trichlorophenoxyaceticacid (2,4,5-T), were at least as active as IAA in causing tracheary regeneration, and were so much more active in causing sieve tube regeneration that their counts for 7 days are minimum estimates only. We already knew that 2,4-D and IAA showed striking similarities in their polar movement through sections of whole internode or whole petiole (Jacobs, 1967). The unusually high vasculogenic activity of 2,4-D could be explained, however, if 2,4-D showed preferential movement through vascular tissue. By comparing movement through cylinders of tissue including only pith parenchyma with cyiinders including vascular tissue, we could show that 2,4-D did move preferentially through the latter-although basipetally polar movement occurred in both. On

SIEVE TUBE ELEMENTS

259

the contrary, more IAA moved through pith cylinders (Fig. 13; see Jacobs and McCready, 1967). This striking effect on sieve tube regeneration of 2,4-D added at hormonal levels seems to be a manifestation of the same action that causes “distortion of phloem” reported earlier as a typical result of using more nearly herbicidal levels of 2,4-D (Eames, 1950). Tryptophan, a presumed precursor of IAA in many plants including Coleus (Valdovinos and Perley, 1966) can also cause sieve tubes and tracheary cells to regenerate. Its lesser activity and more erratic effectiveness (Thompson, 1965) fit the idea that its activity depends on prior transformation to IAA. Several non-auxin compounds tested similarly on isolated internode no. 5 did not affect the number of sieve tubes or tracheary cells (Thompson, 1965). Thiamin and ascorbic acid, investigated because of an earlier report that they stimulated cambial activity in other genera (Kunning, 1950), were without effect at 1% in lanolin. So was nicotinic acid. Gibberellic acid (GA,), added to the top of internode no. 5 alone or in varying concentrations with 0.1% IAA, had no statistically significant effect on sieve tube or tracheary regeneration (Thompson, 1965). (When added with IAA, it decreased the average number of sieve tube strands regenerated in all four experiments, but never to the 5% level. In three of the four experiments, it increased the average number of tracheary cells regenerated, but again not to the 5% level by the “t” test.) Thinking that the stimulating effects of GA, on other actions of IAA-so often reported in the literature-might be the specific result of GA, affecting transported IAA (as had been shown in two legumes by Jacobs and Case, 1965; Pilet, 1965), Thompson added IAA-I4C alone or with GA, to internode no. 2 and checked the effect on vascular regeneration in internode no. 5 (Thompson, 1966). The GA, did not increase the amount of either vascular regeneration or of radioactivity in internode no. 5. (In all these experiments with GA,, IAA was added at a level low enough so that internode no. 5 would give more vascular cells if more IAA reached it.) When substituted for the distal organs, sucrose at 2 % had no effect on the number of sieve tubes regenerated in internode no. 5 , whether added alone or with IAA (LaMotte and Jacobs, 1963, Table 3). With such evidence from the older, nonelongating internode no. 5 that the regeneration of both sieve tubes and tracheary cells was controlled by the endogenous hormone IAA, some similar experiments were run on the younger, elongating internode no. 2. [This means that, in contrast to the earlier studies on tracheary regeneration (Jacobs, 1952, 1754, 1956), the internodes were wounded in one of their flat sides, rather than cutting a V-shaped hole from one of the large corner strands.] Excised wounded internodes no. 2 treated apically with various “concentrations” of IAA in lanolin during their week of regeneration gave the results shown in Fig. 16 (Thompson and Jacobs, 1966). The tracheary

2 60

WILLIAM P. JACOBS

cells responded much as they did in the older nonclongating internode, even to the interpolated values for intact plants falling at the same equivalent IAA concentration (vit., 0.05%). One of the few differences is that internode no. 2 showed no further increase as the IAA concentration was raised from 0.1 to l.OO/o.r This result fits expectations, however, from data on the transport of auxin added apically to sections cut froin the two internodes; internode no. 2 was found to show a plateau in the amount of basipetal transport as IAA coil-

'I5t

v

c

K

*

1 150

Intact plant

z 0 0

(Its sieve tubes= 175)

7

(0

X

100

2

/:I: I

0

m r

50

0 5 : 8 I 0001 IA A

001

CONCENTRATION

0

I

I

01

10

0

(OX)

FIG. 16. The effect of apically applied IAA at various concentrations on xylem and sieve tube regeneration around a wound in isolated no. 2 internodes of Coleus (Princeton clone). Each point is the average of two experiments. (Corrected data from Thompson and Jacobs, 1966, Fig. 5 . )

centration was increased, while internode no. 5 did not (Fig. 17; see Scott and Jacobs, 1963; Fig. 18; see Naqvi, 1963). Internode no. 2 showed a quite different response from internode no. 5 in its sieve tube regeneration. Although IAA added apically did cause an increase in the number of regenerated sieve tube elements, no IAA concentration tried fully replaced the rest of the plant in its effect on sieve tube regeneration. Presumably some other substance or substances in addition to IAA are needed to provide the normal number of sieve tube elements in this elongating internode. The hormonal auxin of Coleus is IAA and only IAA by a iiumber of criteria. In the work on tracheary regeneration cited earlier, Jacobs had shown that the young leaves of Coleus were the main endogenous source of auxin. This auxin was entirely IAA, judging by Rf in two solvent systems (Fig. 19), by activity in two bioassays, and by color tests and ultraviolet fluorescence (Scott and Jacobs, 1964). In addition, exact substitution of synthetic IAA for the endogenous 2 The original figure was in error in showing a larger decrease from 0.1 to 1.0% than does the present figure. (The error resulted from not using weightitig factors in averaging experiments of different sample size.)

-

,

25

I

I

v)

0,

-

e

pl

al 7J

W

3

a 3

0

P

2

k!

P

, ;51

50

/

f’.(

I

-.I

IAA added

Curvature

(ppm)

AV.+_S.E.

0.5 I

1.5k0.4

2 5 I0 20

0

-

(n) (2) (4)

-

2.0f0.8 (3) 17.02 1.6 (4) 19.62 1.8 21.4 5 1.6

-

(4) (5) I

PIG. 18. Basipetal transport through 7.O-mm sections cut from Coleus internodes nos. 2 and 5 (from Naqvi, 1963). Effect of different donor concentrations on the amount of IAA-14C transported through internodes nos. 2 and 5 in 8 hours. (From Jacobs, 1967, Fig. 8.)

2 62

WILLIAM P. JACOBS

auxin estimated to be IAA gave exact replacement of the effect of the young leaves on tracheary regeneration (Jacobs, 1956, Fig. 4 ) . Exact substitution by synthetic IAA also gave exact replacement of endogenous auxin in the abscission-speeding effect of the apical bud (Jacobs, 1955; Jacobs et al., 1957, Fig. 6). c

I

c

b w

lo-

'

z

-J

z

0

8-

b w

?-

b.

/

L

u) _I

a

i

9 - 5 and 5 0 p g

6-

lsopropanol -ammonia -water

Synthetic I A A R f = 0.38

I

./"

5-,

3

IAA (mg/liter)

FIG. 19. Evidence that diffusible auxin is I A A and only IAA. All the auxin activity of the agar diffusion extract runs to the R, typical of synthetic IAA (right side), Shading indicates significant difference from 2% sucrose control. Calibration curve of 0, 0.01, 1.0 mg/liter synthetic IAA is shown on left. Star indicates activity of elution controls of s and 50 pg synthetic IAA chromatographed like the unknown. Triangle indicates activ. ity of agar extract at Rf zone corresponding to synthetic IAA. (From Scott and Jacobs, 1964, Fig. 2.)

F. ARE SIEVE TUBES THE NORMAL PATHOF IAA MOVEMENT? As cited above, several pieces of evidence concerning timing and sensitivity of vascular regeneration in Coleas fit the view that sieve tubes are the major path of IAA movement in the intact plant. Although it is possible for IAA to move in polar fashion through isolated cylinders of tissue comprised solely of pith parenchyma from internode no. 5 (Fig. 13; see Jacobs and McCready, 1967), this is probably not the normal path. [We know that these pith cylinders act differently when isolated than in the intact plant; for example, they elongate considerably when isolated, a characteristic correlated with polar transport of auxin (McCready and Jacobs, 1963) .] Fischnich (1935) presented indirect evidence that IAA added to the intact Coleus leaf moved mostly in the vascular strand; root initiation on internodes below occurred only opposite the vascular strands. Camus' (1747) work on regeneration in cultured pieces of root (see Section 111) also supports the view that the cambial area is the preferred

SIEVE TUBE ELEMENTS

263

path of auxin movement (cf. Camus, 1949, Figs. 61 and 74). A particularly high content of endogenous auxin was found in scrapings from the general cambial zone of several trees (Soding, 1937). It was only recently, with the help of aphids, that the presence and movement of auxin in sieve tubes was demonstrated. Maxwell and Painter (1962) found auxins in extract of aphids that fed exclusively on sieve tube sap, and Eschrich (1968) found no labeled material in the aphids’ honey dew except the IAA-14C he had added to an intact leaf. At present, therefore, we know that IAA is present in large amounts in the cambial area (including young xylem, cambium, and young phloem), and that it can move in sieve tubes (although evidence is lacking so far as to the exclusiveness of this path). It takes 14 times as much IAA for a tracheary cell to differentiate in regeneration as in normal development (Jacobs and Morrow, 1957). To raise the endogenous level 14 times is apparently the reason why the vascular strand must be cut before regeneration occurs. Merely making a wound of about the same size will not suffice (von Kaan Albest, 1934). If sieve tubes are the normal path of IAA movement, then severing them should be the stimulus for vascular regeneration. von Kaan Albest cited 10 cases in which she had severed strands small enough to consist entirely of phloem. In three cases, no vascular cells regenerated; in the other seven, wound sieve tubes regenerated but no tracheary cells formed. [She interpreted these results as an indication that one must sever phloem strands to obtain phloem regeneration, and xylem strands to obtain tracheary regeneration. I think it more likely that the lack of regenerated tracheary cells was a result of the fact that much more auxin is required to regenerate tracheary cells (cf. Fig. 1 2 ) and that a small strand-such as would consist entirely of phloem cells-would not carry enough auxin to make anything but sieve tube elements.] Further evidence that it is the non-xylem part of the vascular strand that must be severed came from Roberts and Fosket (1962) ; pith punctures that damaged the xylem led to no xylem regeneration, but deep cortical punctures did. Also confirming the hypothesis that auxin movement is in the sieve tubes or cambium or both were the results of Thompson (1967). He physically separated the xylem from the phloem region of a severed vascular strand and inserted a very thin glass coverslip. Tracheary cells regenerated only on the phloeni side of the cover-slip and in strands that matched the path of the sieve tubes that had regenerated a few cells external to them. Thompson also noted a case among his treatments in which a strand containing no differentiated tracheary cells had been accidentally severed; a substantial number of tracheary cells regenerated, nonetheless (Thompson, 1965, p. 8 2 ) .

2 64

WILLIAM P. JACOBS

111. Differentiation of Sieve Tubes i n Organ or Tissue Culture There are many papers describing the growth and differentiation observed in sterile cultures of organs or tissues. In addition to their other points of interest, such cultures may be thought of by the developmental physiologist as representing a step beyond, for instance, isolated internodes in the direction of further isolating the reaction system that controls cell differentiation (Jacobs, 1959). Cultures do have a serious limitation, however. By cutting away most of the plant or animal from the cultured tissue, we are likely to have cut away sources of chemicals that are normally present in nonlimiting amounts. W e will have made some chemicals “artificially limiting.” For instance, it is reasonable to expect that in an intact organism of the many chemicals presumed to be necessary for the differentiation of a sieve tube, only one or two of these will actually be controlling sieve tube differentiation by their limiting availability. In the artificially deprived culture situation, however, the limiting factor may be one of the compounds always present in superfluity in the intact organism. To correct for this limitation of the culture technique it is necessary to relate quantitatively the results of cultures back to the limiting factor in the intact organism. Such a “correction for the intact organism” was not needed in one of the early papers on sterile culture, because the author grew an entire sterile seedliiig in a test-tube. Molliard (1907) found that adding sugars to the clearly noaoptimal medium increased the number of sieve tubes that differentiated. His Figures 45 and 46 of transverse sections of Ipoinuea show this in striking fashion. Most of the later workers who studied vascular differentiation in sterile cultures were, to the contrary, growing only small pieces of the organisin and, therefore, the relevance of their results to the limiting factors in the intact plant needs to be demonstrated. The elegant and extensive work of Camus (1949) showed that a bud grafted onto the “shoot-end” of a cultured piece of Cichorizm root induced a vascular strand that eventually joined with the cambial zone of the root tissue. Various synthetic auxins could substitute to a sizeable extent for the vasculogenic effect of the grafted bud, with IAA and indolebutyric acid giving results most like the bud itself. Soon after IAA had been shown to be able to control the regeneration of titcheary cells in young Colezls stems, it was shown to be able to speed the regeneration of tracheary cells in Syringd callus using the techniques of Camus (Wetmore and Sorokin, 1955), and in cultured Piszlm roots (Torrey, 1953). Both papers reported that there was no discernible effect of auxin on sieve tube differentiation. Further investigation of the cultured Syringu callus, however, colifirmed and extended Molliard’s results; Wetmore and Rier ( 1 9 6 3 ) reported that in the presence of auxin higher sucrose concentrations (4-5%) favored the de.

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velopment of sieve tube elements, whereas lower sucrose concentrations ( 1-276) led to fewer sieve tube elements and more tracheary elements. This has been confirmed in Phaeolzls callus cultures by Jeffs and Northcote (1966), who also stated that kinetin added along with IAA and sucrose increased cell divisionsits classic effect-and “phloem differentiation.” Wetmore and Rier said, “It is striking that the majority of vascular elements, whether xylem or phloem, are formed in nodular centers and not as isolated elements.” Jeffs and Northcote noted this also. What this indicates is that the auxin-sucrose treatments are essentially causing the differentiation of a piece of complete vascular strand that contains sieve tube dements toward the outside of the callus,3 tracheary cells toward the interior, and a cambial zone in between the two. We should remember that there are special difficulties in trying to study sieve tube differentiation in callus cultures; not only are the cells apt to be very small, but they do not form in such predictable locations as in regenerating internodes, nor do they usually differentiate as a strand of sieve tube elements. The difficulty of searching through a whole callus for the few cells that might show sieve plates or slime plugs undoubtedly explains why the work published so far has not included actual counts of sieve elements, nor shown a photograph of recognizable sieve tube elements induced by chemicals. [Both Galavati ( 1 9 6 4 ) and Wetmore and Rier (1963, Fig. 3) have published convincing photographs of sieve tube elements induced in cultured tissue by developing buds.] One other report on cultures should be mentioned because of the possibilities it raises. Gautheret (1961) reported that GA,, if added to Jerusalem artichoke cultures along with IAA, caused broad areas of xylem and phloem to form from the cambial area (Gautheret, 1961, Figs. 1-3). GA3 added without the auxin had little effect-the auxin requirement for GA, activity agreeing with many though not all earlier reports on other processes. Higher concentrations of IAA added with the GA, differentially inhibited cell formation on the phloem side. Because no evidence was presented that the new phloem cells included an increased number of sieve tube elements, this seems like the equivalent in tissue culture of the GA-stimulation of cambial activity in trees that had been reported earlier (Bradley and Crane, 1957; Wareing, 1 9 5 5 ) . Similar effects on cambial activity resulted from GA,IAA additions to a number of noncultured, regenerating woody stems (Wareing et al., 1964; Digby and Wareing, 1966), although the authors apparently did not know of Gautheret’s paper. These later authors interpreted their results as an indication that GA, caused the d i f w e c tiation of sieve tube elements, but their actual evidence was that the cells induced by GA,IAA on the phloem side of the cambium are not differentiated 3 The legend for Fig. 1 2 of Wetmore and Rier (1963) describes the reverse of this, but Prof. Wetmore (personal communication) stated that the legend was incorrect and that the contrary statement on their p. 423 was correct.

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into sieve tube elements nor into companion cells (Wareing et al., 1964). In their second paper, they were able to find some sieve plates in some cells on the phloem side of the cambium in one of the genera, but conspicuously refrained from claiming that the GA,-IAA treatment induced more of them. DeMaggio (1966) also reported more cells cut off on the phloem side of the cambium from GA, application to Pinus explants, but could not find the standard critical signs of sieve cell differentiation. (He could see more cells with walls that were birefringent in polarized light after GA, treatment, so some wall changes did occur.) To summarize the research on regeneration, at present it looks as if IAA is the hormone controlling the differentiation of sieve tubes in secondary tissues (i.e., those with a cambium, such as Coleas internode no. 5 ) , with GAS affecting the number of cells cut off by the cambium but not affecting the number that then differentiate into sieve tube elements. (Wareing et al., essentially counted cell number on the phloem side, whereas Thompson in Coleus counted sieve tube element differentiation.) In primary tissues (e.g., Coleus internode no. 2), IAA seems to be the sole and sufficient explanation for tracheary regeneration, but something in addition to IAA is required to fully replace the effects of the distal and proximal tissues on sieve tube regeneration. From the results with sterile culture, this additional factor may be sucrose. According to the work of Gautheret, Wareing et al., and DeMaggio, however, it might well be gibberellic acid. Although it was hypoth. esized many years ago that sugar reacted with auxin to determine the spatial and temporal pattern of normal vascular differentiation (Jacobs, 1752), sugar has not as yet been shown to actually limit vascular differentiation in intact healthy plants, Molliard’s sterile seedlings being semistarved according to his description. Favoring gibberellic acid as the second factor are the facts that the action of gibberellic acid is typically restricted to young tissue, and that GA, has been shown to be active in petioles no. 2-4 of this same clone of Coleus (Jacobs and Kirk, 1966). [A detailed and interesting argument for the importance of sugar in controlling vascular development is given in a review by Wetmore et ul. (1964).]

IV. Normal Differentiation of Sieve Tubes Our discussion of research on this topic will be brief and selective. Much of the recent work has been with the electron microscope, and I am not qualified to discuss that critically. Of the other papers on sieve tube differentiation published in the last decade, Esau has recently discussed most of them with her usual thoroughness and authority (1965). In a more recent paper, however, the

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quantitative methods used in the CoZeus regenerative studies were applied to the normal shoot tip. The results were illuminating and unifying. The rationale of the experiments was as follows. Our quantitative studies of sieve tube regeneration had shown that sieve tubes formed first, and that later and exactly opposite them tracheary strands regenerated. The earlier literature, qualitative as it was, had led us to expect quite a different spatiotemporal pattern. The literature had been similarly misleading as to the pattern of tracheary regeneration in relation to the polarity of auxin transport (Jacobs, 1952, 1954). Accordingly, it seemed reasonable to look with some scepticism on the accounts in the literature indicating that the pattern of differentiation of sieve tubes was “continuous and acropetal” and thus quite different from that of the first tracheary cells. W e investigated apical buds with quantitative methods and aroundthe-clock collecting. The latter was based on the fact that earlier anatomists had typically collected samples only during the daylight hours and on the belief that a sizeable percentage of the developmental activity would occur during the dark portion of the normal 24-hour cycle. Cell differentiation during the dark period would be particularly expected if auxin were the limiting factor, because auxin production had been reported to follow a diurnal cycle with a maximum at night (Yin, 1941). This expectation had already been confirmed for xylem differentiation (Jacobs and Morrow, 1 9 5 7 ) ; a new locus of tracheary differentiation was detectable only in collections made at night. The first applications of this method revealed that the first differentiation of sieve tubes in a leaf primordium was strictly correlated with the length of the leaf (Fig. 20; see Jacobs and Morrow, 1958), no leaf differentiating its first sieve tube element until it was 400-450 p long. All leaves longer than 470 p had a continuous strand of sieve elements extending up into them from the stem. It seemed appropriate that the leaves did not grow longer than about 0.5 inm before differentiating the cells specialized for the movement of organic food materials, because 0.5 mm is roughly the distance beyond which physical diffusion would inadequately provide for the growth of the primordium. In other respects, the first results were disappointing. There was no sign of a discontinuity in any one of the many “first sieve tube strands” differentiating in the young leaves (Fig. 2 0 ) ; the differentiation was apparently “continuous and acropetal” as the classic literature reported. For a leaf primordium too short to contain differentiated sieve tubes, the sieve tube strand destined for that leaf was presumably down in the stem differentiating acropetally toward the leaf base from the procambial cells of the leaf trace. This process of acropetal differentiation in the leaf trace occurred, at first glance, just as expected. As the young leaf grew progressively longer, the sieve tube (destined to connect it eventually with the mature part of the plant below) dif-

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ferentiated progressively closer to the leaf base (Fig. 2 1 ; see Jacobs and Morrow, 1967). Regression equations fitted to the data confirmed that there was a linear relation between the length of a young leaf and the distance from the base of the leaf to the top of the acropetally differentiating sieve tube in the leaf trace (Jacobs and Morrow, 1967, Table 1 ) . Scrutiny of the regression line

1

32r

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0 2

Immature sieve tube

4

6

8

10

LENGTH

12

14 16

18

20 22 24 26 28

OF LEAF (microns x 100)

Longitudinal distribution of first sieve tubes within the main vein of young Coleus leaves. Each solid vertical line represents the sieve tube of a different leaf. (From Jacobs and Morrow, 1958, Fig. 1 with permission from the editors of Scieiice.)

(as drawn through the data of Fig. 2 1 ) , however, revealed an unexpected problem; although the sieve tubes in the leaf traces were differentiating acropetally as the leaf grew, they were lagging progressively farther behind the leaf tip. At zero leaf length they averaged 442.9 p below the “tips” of the leaf; at 4SOp leaf length, they were 601 p below (450 f 101 p). This trend of iizcrediiiig distance of sieve tubes from the leaf tip somehow had to be reversed, because all leaves more than 470 p long had continuous sieve tubes out into the leaf (Fig. 20). To investigate how this increasing gap was closed we used our knowledge of the close relation between leaf length and sieve tube differentiation to select leaves just about to show sieve tubes out in the leaf. This technique uncovered a new phenomenon of sieve tube differentiation; in leaves 387-459 11 long a separate locus of sieve tube differentiation was found near the base of the young leaf (Fig. 2 2 ) . Within a very short span of leaf lengths, the sieve tube of the first locus connected basipetally with the acropetally differentiating sieve tubes in the stem-thus closing the gap. Why was this separate locus of sieve tube differentiation, found reproducibl) year after year in this clone of Coleus (Jacobs and Morrow, 1967), not discov-

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ered before among the angiosperms ? I A separate locus for sieve cells had been found in young leaves of Equisetzlm (Queva, 1907) and of Selaginella (Jacobs, 1947) but was thought to be correlated with the microphylls of those genera, in contrast to the megaphylls of the angiosperms.] Aside from the remote possi-

- 500‘

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I

too

I

200

I

I

300

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1 500

LENGTH OF LEAF (microns) FIG. 21. Data for leaf length plotted against the most distal position of sieve tubes in the corresponding leaf‘ traces, including linear regression line and its equation. (From Jacobs and Morrow, 1967, Fig. 3 with permission from the editors of Aineiicuii Jou17)d of Botany. )

bility that it occurs only in Coleus, there are three obvious reasons: (1) The separate loci were mostly found in nighttime collections; ( 2 ) even though our sample sizes were larger than those of most of our predecessors, we did not find the separate locus until the quantitative relations from the linear regression pointed out to us that the “differentiation gap” occurred and thereby directed us to the particular range of leaf lengths in which the locus was found; and ( 3 ) ColeuJ has a fairIy long leaf plastochron under our growth conditions-forming a new leaf pair once a week-and genera with plastochrons of 1 or 2 days, such as Xunthizlm, would presumably go through this developmental stage 3-7 times faster.

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V. Conclusions The quantitative studies of Coleus provide a unifying picture of vascular differentiation and regeneration. The pattern of first differentiation of sieve tubes in the young leaf is not different from that of tracheary cells in being “continuous and acropetal” as the literature would lead us to expect, but rather remark-

0 387

387

100 p

H FIG. 2 2 . Isolated loci of sieve tube differentiation shown in stereodiagrammatic chart of three different shoot tips (F, I, J ) , with leaves 387-459 p long (leaf pair 11). Stiypling shows expected level of sieve tubes as derived from the regrcssion line of Pig. 21. (For comparable xylem differentiation, see F, I, J, Jacobs and Morrow, 1957, p. 830.) Nodes, which are represented as flat plates labeled with Roman numbers, are shown as transparent where traces would end in this area. The scale shown is valid for vertical measurement but not for horizontal. (From Jacobs and Morrow, 1967, Fig. 5 with per. mission from the editors of American Jolrrnal of Botany.)

ably similar to that of tracheary cells. The first sieve tube element in the leaf forms at a separate locus on the outer side of the procambial strand near the base of the leaf. This is the region where the first tracheary cell will form on the inner side of the procambial strand several days later. Vascular regeneration in internode no. 5 shows the same pattern (again, contrary to expectations from the literature) ; sieve tubes regenerate first, then 1 day later and exactly opposite them on the inner side of the cambium the tracheary cells differentiate. [In elongating internode no. 2, regenerated strands of tracheary cells are also exactly opposite the regenerated sieve tubes (Thompson, 1967, Fig. 5 ) , but the timing has not yet been investigated in Colez~s.] The regeneration of both cell types in internode no. 5 is controlled by the amount of basipetally moving endogenous auxin, IAA, coming from the leaves.

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The IAA moves in the sieve tubes (and probably in the procambium-cambium also, particularly in locations where no sieve tubes have yet differentiated, such as in the youngest leaf primordia). When the sieve tube strands are severed, the local IAA concentration in the cells adjoining the cut end builds up enough to initiate the differentiation of wound sieve tube elements. Subsequently, tracheary cells regenerate opposite the wound sieve tubes. Recent radioautographic evidence confirms that IAA itself (or a derivative of it) moves across from the sieve tube-cambial zone to the differentiating tracheary cells (Sabnis et ul., 1969). When IAA labeled with tritium was added in normal amounts to Coleus internodes, the “acetone-insoluble” label was found to be specifically localized in the secondary walls of those tracheary cells that were in a stage of early differentiation. In such radioautographs, there was no localization of tritium in the sieve tubes, the basipetally moving IAA-3H presumably having been extracted with the acetone used to prepare the sections for ultramicrotoming. With regeneration in internode no. 5 so fully explained by IAA, it is reasonable to hypothesize that the very similar patterns of normal vascular differentiation in the shoot tip are also explainable by IAA, particularly since reasonable correlations have already been shown between normal IAA production and normal tracheary differentiation (e.g., Jacobs and Morrow, 1957, Fig. 16). Regeneration of sieve tubes in young elongating internode no. 2 is not fully understood. Whether GA, or sugars are interacting with IAA to limit sieve tube regeneration in internode no. 2 , we can not say at present. The lack of a cambium in that developmental stage may well be critical, however, because von Kaan Albest reported that several species with isolated procambial strands did not regenerate sieve tubes when their vascular strands were severed (although some did regenerate tracheary cells). There is a great need for quantitative and critical studies of cambial activity in relation to sieve tube differentiation. From our present knowledge, the relation between leaf growth and vascular differentiation seems reasonable; as a leaf grows more quickly, it produces more IAA, which in turn allows more vascular tissue to differentiate in the traces supplying the leaf with the wherewithal to grow. A declining growth rate is associated with declining IAA production (Jacobs, 1952, Fig. 5 ) and, therefore, with declining differentiation of vascular tissue in the leaf traces. ACKNOWLEDGMENTS The cost of preparing the manuscript and figures was aided by an NSF research grant. I am also grateful to Mrs. Mary Leksa for deciphering and typing the manuscript, and to research assistants Mrs. Paula Edwards and Mrs. Hannah Suthers for their help in assembling figures and legends. (Mrs. Suthers also constructed Figure 6.)

REFERENCES Bradley, M. V., and Crane, J. C. (1957). Science 126, 972. Camus, G. (1349). Rev. Cytol. Bid. Vegetales 11, 1.

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DeMaggio, A. E. ( 1 766). Science 152, 370. Digby, J., and Wareing, P. F. (1766). Ann. Botany (London) 30, 537. Eames, A. J. (1950). A m . J . Botany 37, 840. Esau, K. (1953). “Plant Anatomy.” Wiley, New York. Esau, K. (1760). “Anatomy of Seed Plants.” Wiley, New York. Esau, K. (1965). “Vascular Differentiation in Plants.” Holt, Rinehart & Winston, New York. Eschrich, W. (1753). Planta 43, 37. Eschrich, W . (1968). Planta 78, 144. Fischnich, 0. (1935). Planta 24, 552. Galavazi, G. (1964). Acta Botun. Need. 13, 420. Gautheret, R. (1961). Compt. Rend. 253, 1381. Jacobs, W . P. (1747). A m . J . Botany 34, 5 8 5 . Jacobs, W. P. (1952). A m . 7. Botany 39, 301. Jacobs, W . P. (1754). A m . Naturalist 88, 327. Jacobs, W . P. (1955). A m . J . Botany 42, 594. Jacobs, W . P. (1756). A m . Naturalist 90, 163. Jacobs, W. P. (1757). Develop. B i d . 1, 527. Jacobs, W. P. (1767). Ann. N . Y . Acad. Sci. 144, 102. Jacobs, W . P., and Case, D . B. (1765). Science 148, 1729. Jacobs, W . P., and Kirk, S. C. (1966). Plant Physiol. 41, 487. Jacobs, W. P., and McCready, C. C. (1967). Am. J . Botany 54, 1035. Jacobs, W . P., and Morrow, I. B. (1957). Am. J . Botany 44, 823. Jacobs, W . P., and Morrow, I. B. (1958). Science 128, 1084. Jacobs, W. P., and Morrow, I. B. (1967). A m . J . Botany 54, 425. Jacobs, W. P., Danielson, J., Hurst, V., and Adams, P. (1959). Develop. B i d . 1, 534. Jeffs, R. A., and Northcote, D. H. (1966). Biochem. J. 101, 146. Johansen, D. A. (1740). “Plant Microtechnique.” McGraw-Hill, New York. Kabus, B. (1912). Beitr. Biol. Pflanz. 11> 1. Kunning, H. (1950). Planta 38, 36. LaMotte, C. E., and Jacobs, W . P. (1962). Stuin Technol. 37, 63. LaMotte, C. E., and Jacobs, W. P. (1963). Deuelop. Biol. 8, 80. McCready, C. C., and Jacobs, W . P. (1963). N e w Phytologist 62, 360. Maxwell, F. G., and Painter, R. H. (1962). J . Econ. Entonzol. 55, 57. Molliard, M. (1907). Rev. Gen. Botan. 19, 241, 329, 357. Naqvi, S. M. (1963). Ph.D. Thesis, Princeton Univ., Princeton, New Jersey. Pilet, P. E. (1965). Nattlre 208, 1344. Queva, C. (1907). Soc. Hist. Nat. d’Auttin 20, 115. Roberts, L. W., and Fosket, D. E. (1962). Botan. Gaz. 123, 247. Sabnis, D . D., Hirshberg, G., and Jacobs, W. P. (1967). Plazt Phy.rjo1. 44. 27. Schumacher, W. (1733). Jahvb. IVa’ss. Botan. 77, 685. Scott, T . K., and Jacobs, W . P. (1961). Science 139, 589. Scott, T. K., and Jacobs, W. P. (1764). Proc. 5th Intern. Conf. Plan1 G u w l h Subitnwcel, Actes Collog. Intern. C.N.R.S., 1963 No. 123, pp. 457-474. C.N.R.S., Paris. Simon, S. (1908). Ber. Deut. Botan. Ges. 26, 364. Svding, H. (1737). Jahrb. Wi.rs. Botan. 84, 637. Thompson, N. P. (1965). P h D Thesis, Princeton Univ., Princeton, New Jersey. Thompson, N. P. (1766). Plant Physiol. 41, 1106. Thompson, N . P. (1767). A m . J. Botany 54, 588.

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Thompson, N. P., and Jacobs, W. P. ( 1 9 6 6 ) . Plant Physiol. 41, 673. Torrey, J. G. ( 1 9 5 3 ) . A m . J . Botany 40, 5 2 5 . Valdovinos, J. G., and Perley, J. E. ( 1 9 6 6 ) . Plant Physjol. 41, 1632. yon Kaan Albest, A. ( 1 9 3 4 ) . 2. Botan. 27, 1. Wareing, P. F. ( 1 9 5 8 ) . Nature 181, 1744. Wareing, P. F., Hanney, C . E. A,, and Digby, J. ( 1 9 6 4 ) . In “Formation of Wood in Forest Trees” (M. H. Zimmermann, ed.), pp. 323-344. Academic Press, New York. Wetmore, R. H., and Rier, J. P. ( 1 9 6 3 ) . A m . J . Botany 50, 418. Wetmore, R. H., and Sorokin, S. ( 1 9 5 5 ) . J . Arnold Arboretum (Haward Uvziv.) 36, 305. Wetmore, R. H., DeMaggio, A. E., and Rier, J. P. ( 1 9 6 4 ) . Phytomorphology 14, 203. Yin, H. C . ( 1 9 4 1 ) . A m . J . Botany 28, 250.

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Cells, Solutes, and Growth: Salt Accumulation in Plants Reexamined’ F. C. STEWARD AND R. L. MOTT Laboratory for Cell Physiology, Growth, and Development, Cornell University, Ithaca, N e w York Introduction-The Solutes in Cells: Their Composition and Accumulation ...................................... A. Contrasted Features of Plant and Animal Cells ...... B. Vacuolar Contents in an Autotrophic Nutritional System ..................... ............ C. The Genesis of Vacu e de nogo Creation of ...................... Solute Content . . . . . . D . Concepts of Active Transport: Their Origin and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. “Division of Labor” in a Highly Organized System . . 11. The System Involved: Its Membranes and Fine Structure . . A. Classic Concepts of Cytoplasm: Its Membranes and Their Role . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Cytoplasm: Its Fine Structure and Biochemistry . . C. Current Concepts of Membranes, Protoplasmic Surfaces, and Cytoplasmic Organelles-An Epitome . . . . 111. Physiological Studies on Salt Accumulation in Plant Cells: Past and Recent Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A, Period 1925-1945 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Period 1945-1960 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. More Recent Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Growth Requirements of Cells: Their Implications for Solute Uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Endogenous Capacities and Exogenous Requirements of Cut Discs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Endogenous Capacities and Exogenous Requirements of Cultured Tissue Explants . . . . . . . . . . . . . . . . . . . . . . . .

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1 This review departs somewhat from a typical format. Several topics are touched upon without exhaustive bibliographies. It is part review and part preview. In the latter context, it draws upon much work that has not yet been published in detail, so the essential evidence is here recorded diagrammatically. This part of the review gains coherence inasmuch as it draws upon much other work of this laboratory on different aspects of the behavior of cultured cells and tissues, for this provides the background against which the problem now needs to be viewed. It is this needed synthesis that explains why the treatment dwells so heavily upon the work of one laboratory, work which has been supported by grants ( GM 09609) from the Institute of General Medicine, Department of Health, Education, and Welfare, Bethesda, Maryland. Furthermore, during part of the period of the work, one of us (R.L.M.) also held a predoctoral research fellowship awarded by the National Institutes of Health.

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V. Absorption Studies with Cultured Cells and Tissue Explants A. The Responses of Different Carrot Clones to Different

317

.................. Stimuli . . . . . . . . . . . . . . . . . . The Time Course of Growth, Metabolism, and Absorption in Cultured Carrot Explants: The Typical Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Behavior of Carrot Explants in Simple Salt Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The Behavior of Potassium and Sodium Halides in Tissue Exposed to the Progressively Remnstituted Nutrient System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The Behavior of Potassium and Sodium Halides in Tissue First Stimulated to Grow, Then Deprived of Nutrients and Stimuli, and Subsequently Restored t o Growth . . . . . . . . . . . . . . . . . . . . . . . . F. Generalized Applications of the Co Accumulation in Relation to Growth ......... G. Salt and Solute Relations Viewed on a Cellular Basis: Effects Attributable to Endogenous and Exogenous Stimuli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Indicators of Salt Uptake in Cells and in the Plant Body I. Ion Uptake by Cultured Tissues: A Resume . . . . . . . . VI. Salient Features of Solute Accumulations in Cells: Perspectives and Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction-The

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Solutes in Cells : Their Composition and Accumulation

An essential of life is a degree of organization not encountered in the inorganic world. To perform the functions of the living state, cells and organisms create and preserve an internal composition very different from that of their immediate environment. This has prompted the view (Oparin, 1961) that barriers or membranes were an earlier evolutionary requirement than DNA, which was the later means by which more highly differentiated systems transmitted very specific information when they were self-duplicated (Oparin, I 96 I , p. 96 et seq.) . A. CONTRASTED FEATURES OF PLANTAND ANIMALCELLS It is a salient characteristic of mature living plant cells that their fluid contents are at a higher osmotic pressure than the ambient media in which they occur (i.e., higher than the fluids from which their solutes were obtained or against which they are retained). At one extreme, the internal aqueous fluids of some cells may consist almost entirely of inorganic salts, most prominently KC1 (e.g., as in the large coenocytic internodal cells of Nitelld or Chai,u or the

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coenocytic vesicles of Vdloizid) ; at the other extreme, plant vacuolar saps may be rich in sugars, in organic or amino acids, as well as in such nitrogen-rich compounds as asparagine, glutamine, or arginine; they may also contain saltsoluble proteins (globulins), as well as phenolic compounds and anthocyanins which contribute to their acidity and affinity for basic stains. When the organic content of vacuolar saps is high, the disparities between their inorganic anions and cations, which was regarded early as a feature of the living state rather than of dead cells (e.g., by Stiles in 1924), are apparent since the excess of cations over anions may then be balanced by organic anions. In fact, such observations contributed to the early recognition of the independent absorption of ions. There are, however, some very evident contrasts between typical animal and plant cells. The former tend to exist at (or near) osmotic equilibrium with their surroundings; they rarely develop large internal aqueous phases, for their soluble metabolic end products tend to be secreted externally, as into blood and urine. Moreover, in the heterotrophic nutrition of the animal body, the cells continually receive organic solutes already elaborated via the gastrointestinal tract from which they are absorbed into interstitial fluids and into blood and thence into cells. Thus, higher animals have so evolved that they have retained conspicuous interstitial fluids which bathe their living cells and which are at or near osmotic equilibrium with their cellular contents, even though individual solutes (e.g., potassium, sodium, or even urea) may be at very different concentrations across cellular boundaries. In effect, therefore, higher animals have enclosed their own complex internal environment for their cells, while plants, of necessity, allow them freer access to the outside world. Thus, a feature of animal cell physiology is the maintenance of an ambient medium to bathe the living cells which is relatively constant and which changes in internal ~ 0 1 1 1 position only within the relatively narrow limits of total osmotic concentration that animal cells tolerate. Also, animal cells have carried or developed through evolution a very great sensitivity at their surfaces to foreign substances, and this is seen in the antibodyantigen relations which have no obvious counterpart in plants. Thus, animal rells respond to the complex composition of the relatively concentrated ambient medium within which they occur. By contrast, plant cells seem often to preserve tenaciously, and relatively unrestricted by wide changes in the composition of their ambient media, the composition of the fluids that bathe their iiitemril surfaces. Being endowed with the more autotrophic nutrition of plants, the constituent cells manufacture internally, from very simple sources, the metabolites that animal cells receive preformed from their ambient media in their more heterotrophic nutrition. Moreover, plant cells often respond sensitively to many stimuli mediated by simple molecules, such as the array of plant growth regu-

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lators which they may encounter in, or receive via, the external medium and which even initiate important trends of behavior. The contrasted nature of plants and animals has, therefore, an important bearing on the problems now under discussion.

B. VACUOLAR CONTENTS IN

AN

AUTOTROPHIC NUTRITIONAL SYSTEM

The organizational consequences of the essentially autotrophic nutrition of plants, for carbon via photosynthesis and for nitrogen via nitrate reduction, while obvious are far reaching. In their static locations, higher plants greatly elaborate their surfaces of contact with air by a canopy of leaves, and with soil and dilute soil solutions via an elaborate system of roots which are continually being extended at their tips and in their laterals. This is all very obvious. It is not as obvious that the principle of extending the surface of contact between the living system and the external environment also operates at the cellular level, for even mature plant cells are in virtual contact with the external world. Being bounded by a cellulose wall, more or less elastic, such cells can accumulate the internal concentrations typical of plants, for they also utilize this osmotic value to maintain turgor. As the internal solute concentrations are built up during the growth of cells (by “accumulating” ions from a very dilute external medium or by secreting into vacuoles products which originate in cytoplasm from photosynthesis or from nitrate reduction) and as turgor is maintained, the living substance is “spread thin” to maintain a great area of contact between cytoplasm and the ambient media and with the external environment. This, of course, occurs after the main growth by division and by self-duplication of organelles is completed, and the extent of this phenomenon is not always appreciated. Figure l a shows how large an internal vacuolar space is enclosed within a parenchyma cell of a tobacco leaf and how its cytoplasm, with its enclosed plastids, is spread out thin against the cell wall and is thus in contact with the intercellular spaces and the outside air. (A curious and even unique example is that of the stellate pith of Juntas. In this situation, the cells achieve great external surface with minimal internal volume.) Figure I b shows the tenuous layer of cytoplasm within cultured cells of carrot, spread out so thin that in places it is little thicker than the combined plasmalemma and tonoplast, while the enclosed vacuolar space is enormous. The genesis of the vacuole systems during growth, i.e., in the origin and development of the mcziome, may be

FIG. 1. General views of highly vacuolated plant cells. a, A tobacco leaf cell, showing the very thin parietal layer of protoplasm, with its inclusions, and the very large central vacuolar space. Calibration: 5p. ( b ) , Mature cells of carrot, as grown in culture, showing the cell wall, intercellular spaces, very thin layers of cytoplasm, and very large vacuolaispaces. Calibration: 5p. (Electron microscopy by Dr. H. W. Israel.)

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traced to the origin of minute vacuoles in an otherwise highly vesiculate cytoplasm in which canals resembling endoplasmatic reticulum seem to inflate at their tips to form vesicles (Fig. 2 ) . Therefore, the means by which the composition of vacuolar saps is regulated should not be dissociated from the means by which vacuoles are created and grow. It is one problem to understand the responses of cells, after they have p o w i z (cf. Fig. l b ) , to such limited changes as may then be induced by changes in their environment; it is quite another problem to see how the system was created de izovo (cf. Fig. 2) and to recog nize that the later responses may differ greatly from the former, not only in magnitude but also in kind. In fact, this point of view prompted an early distinction (Steward, 1935) between the “primary” acts of solute accumulation and the alternative absorption phenomena that may be “induced” or superimposed upon the pattern established during growth. It is also characteristic of plants that so many cells remain alive to fulfill their functions, and these, having established in their vacuoles a storage capacity for solutes, become subject to the demands of other areas of the plant body. While more active regions of de ?iovo solute accumulation may draw off inorganic solutes from less active ones, others that actively accumulate sugars and simple nitrogen compounds (as in leaves) may in turn act as “sources” and donate them to “sinks” elsewhere. Thus, cells must be considered not only as they originate de izouo but also as they respond, by an ebb and flow of solutes, to physiological stimuli. OF C. THEGENESIS

VACUOLES:

THEd e

Ir‘OL’O C R E A T I O N O F

THE SOLUTE

CONTENT

All cells must originate from preexisting cells-the red blood cells of vertebrates which multiply in the bone niarrow or the parenchyma cells of higher plants which originate mainly in the division of cells cut off by, or laid down near, the various apical, secondary, or intercalary growing regions. No doubt, at cell division and cytokinesis some partitioning of existing solutes emirs along with the equational partitioning of cellular organelles of all kinds. I;r.oni that point onward, however, the acquisition of solutes and their fate i n the cells is as much a part of their development as the formation of their structures. When angiosperm cells divide rapidly, as in growing regions, they are coiiimonly small, without prominent vacuoles, although the vuci/oiiie may be represented by provacuolar organelles so that the development of vacuoles as reservoirs for the solutes they later contain is a very proniinent feature of the growth of cells as division subsides. The content of vacuoles, however, regulates the turgor of cells so that the control of turgor and cell size is a featiirc of such responses to external stimuli as in the familiar tropisms or i n the responses of various “motor” cells (e.g., guard cells) to light. There are then two crucial

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Frc. 2 . T h e cytoplasm of cultured carrot cells, showing the genesis of x'acuoles. The cytoplasm ( a and b ) of developing cells of carrot in culture, as fixed in glutaraldehydc, showing the plasmalemma ( p ) , the tonoplast ( t ) , and vesiculate enlargements of the reticulum with granules attached. T h e vesicles ( v ) as shown are the progenitors of the larger vacuoles of more mature cells. Calibrations: 0.2Jp. (Electron microscopy by Dr. H. W. Israel.)

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and obvious questions: ( 1 ) How are the initial solute concentrations built up de novo in plant cells as they grow? ( 2 ) How are the solute concentrations so produced regulated? Viewed in this light, the problem of solutes in cells is a developmental and a dynamic one; it is not to be seen merely in terms of passive penetration of any external solutes into preformed cells for, during growth, the internal vacuolar space is both created and occupied; the retention of stored solutes by a “semipermeable” barrier alone is not enough, because the solutes in question may be depleted by the demands of growth and metabolism in the same cells or even in those of other organs with which they are in ultimate contact (e.g., as in the relations between the parenchyma celIs of Jeaves and their ability to deplete root cells of salts). Moreover, having been so depleted, the cells may reversibly regain a specific complement of solutes. Thus, the problem of solutes in cells, their composition and regulation, should be viewed as but another example of the improbable feats that cells perform as they grow. By the creation of ordered morphology out of random molecules, entropy is reduced; by the secretion of solutes from within or without, cells achieve concentration levels higher than those of the ambient medium and work is done as free energy is increased within the vacuole. When these events occur de n o ~ i athey are surely connected, for it is the construction of the improbable large molecules and the organelles that development entails (of which reduced entropy is the thermodynamic expression) that creates the milieu in which, by internal secretion of solutes, cellular osmotic work is done.

D. CONCEPTS OF ACTIVETRANSPORT: THEIRORIGINAND D~~VIXLOPMENT Work on what is now known as active transport was performed on plant systems before it was much investigated in bacteria and in animal cells. The term used to describe the movements of ions against concentration gradients into cells was “ion” or “salt” accumulation. In fact, the observations that directed attention to nonequilibrium explanations of the concentrations that commonly obtain across cell boundaries were made on plants, and it was the study of plants that first directed attention to the cell as an osmotic machine dependent upon respiratory energy to do the physicochemical work involved in the de nova absorption (and later retention) of ions from dilute solution. [For convenient summaries see Hoagland (1944) and Steward and Sutcliffe (1959) and references there cited.] Most of this work and these concepts preceded the knowledge of ATP as the by-product of respiration and as the chemical means by which energy could be locally and specifically applied to do the work of the cell, even as it also preceded modern knowledge of the fine structure of plant cell membranes, ground protoplasm, and the organelles revealed by the electron microscope. The trends of investigation and interpretation of ion absorption and accumu-

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lation initiated about 1926 by the first experiments of Hoagland et ill. on Nitella through the period to 1958 were summarized by Steward and Sutcliffe (1959). Throughout this period and even later, there were many different schools of thought and many different experimental systems were exploited. A main theme of the 1959 review by Steward and Sutcliffe was that each type of experimental material had its own limitations and degrees of freedom and thus it yielded a part of the overall interpretations needed. The laudable attempts to isolate the problem from other features of cells and to treat it as though it were a discrete and separable phenomenon have not led to satisfying explanations. Thus, Donnan phenomena have been invoked in variously corn plicated but, necessarily, equilibrium systems; bioelectric phenomena (which in their turn are often difficult to explain) have been invoked to explain the causes of ion movements; following the recognition of the role of respiration in ion transport, the intervention of respiration has been variously regarded as the direct consequence of emerging HCO; and H+, of the flow of electrons, or of the indirect supply of usable energy via ATP to drive an osmotic work machine; also, individual organelles (mitochondria) have been regarded by some as the primary sites of the ion accumulation. Nevertheless, it is increasingly clear that in the interpretation of ion accumulation in plant cells, as in other problems in which the entire cellular organization is involved, the methods of dissecting and isolating the problem can only reveal or describe certain limited features of the whole mechanism; in short, they may demonstrate only what may be regarded as feasible parts of an overall machinery.

E. *‘DIVISION OF LABOR”IN

A

HIGHLY ORGANIZED SYSTEM

Thus, it is cells (not membranes, not organelles, not isolated sites) that absorb, accumulate, and retain the inorganic ions or which secrete the organic constituents (sugars, organic acids, amino acids, and so on) into cellular compartments where they are stored at high concentrations, even as it is cells, not only their component parts, that grow. Thus, while much has been learned in the last 10 or 1 5 years about metabolism and about cell membranes and cellular organelles in ways that tell more about their propensities and the resources that cells have at their disposal, nevertheless, the interpretation of the balanced working of the whole cellular machine still remains obscure. Progress toward the ultimate explanations of salt accumulation and of active transports, therefore, awaits the deeper understanding of cells and how they grow. Nevertheless, it is useful at this point to evaluate where the problem now stands, if only to guide future work and thought. The essential interpretations, following Hoagland and Davis (1929), which were developed by Steward (1935, 1937, and references there cited) and carried forward into the review of Steward and Sutcliffe (1959) still stand. This is

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true in the sense that active transports are not inere physicochemical equilibria to be interpreted by conventional equilibrium thermodynamics; they are, in fact, active events driven by metabolism and which are responsive to the forces that promote or suppress growth. These events are also essentially a part of the “division of labor” which exists in highly organized systems (cells or organisms) that allows them to perform their complex functions. In fact, as entropy i s locally reduced and organization and complexity is increased (whether morphologically or physicochemically) in one sphere, entropy is inevitably increased in another. Hence, the ideas of turnover, of reversible cyclical systems, which are so familiar in the interpretation of metabolism, now need to be extended to the means by which cells do work; cells are not only “molecular machines” but complex systems in which organelles and cellular compartments, membranes and granules, represent the working parts. As plant cells respond to external stimuli (over and above the exogenous supply of nutrients), their growth by division or enlargement, their differentiation, quiescence, dormancy, and later recrudescence into further growth are all controlled and determined and, predictably, these events have repercussions upon their water and solute relations. Much of this might be described, although not easily interpreted or recapitulated, in a model system. Such a complex working machine is not susceptible to equilibrium thermodynamic analysis, however, although it may ultimately be interpreted by newer concepts of nonequilibriuin therii~odynamics. Essentially the problem is one of cells, of cytology, as surely in 1969 as it was in 1932. At that time, it was pointed out that to interpret how potato hiber cells in thin aerated discs accumulated potassium and bromide ions siinulancously one also needed to know that in doing so the cells mist receive adequate oxygen, their aerobic respiration must increase, and their cytoplasm mas visibly activated (activation of protoplasmic streaming with the nuclei tending to occupy a central position in the cells) in ways that suggested that they m i s t also be able to grow. Now that so much more is experimentally possible with angiosperm cells and tissues in culture in the interpretation of niorphogeiiesis (Steward and Mohan Ram, 1961), they can also be reexamined from the standpoint of experimental and biochemical cytology and of their ability, as growing cells, to do the work. This is the purpose of this review. The theme, however, is that attention should not be focused exclusively tip011 coizcentmtion differences between inner and outer phases with respect only to specified ions or solutes-the total complement of solutes which collectively will determine the “activity” of the water and the gradients that exist within and without the cells should also be considered. The problem should not be studied solely in terms of events at the membrane surfaces, for the driving forces that maintain nonequilibrium movements originate in the cytoplasm. Cells, whether free or in organs, should be considered as maintaining a “division of labor”

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among their parts. This enables a localized free-energy increase to be “balanced off’ against a free-energy decrease elsewhere, or a local increase of structured complexity (i.e., reduced entropy) to furnish the driving force that, appropriately coupled, permits a nonequilibrium movement to occur and express itself as an unstructured increase in free energy of solute coizceiztration within the system in question.

11. T h e System Involved: Its Membranes and Fine Structure In the classic concept of the vacuolated plant cell as an osmotic system, it was necessary to define (1) an internal solution obviously homologous with the vacuole, ( 2 ) an outer solution obviously represented by the ambient medium, and ( 3 ) a membrane system. In Pfeffer’s concept, the living cytoplasm behaved as a membrane, but from the outset the concept of plasma membranes indicated the seat of the osmotic properties. Thus, any interpretation of the cell in relation to its solutes, or any interpretation of mechanisms that secrete solutes internally, inevitably involves concepts of the boundary surfaces of the cytoplasm, its membranes, and the internal structure of the ground cytoplasm. These topics have had a long history, but in this article some current concepts must be formulated which are necessarily based on more recent developments. To integrate these concepts with any changed interpretations of solute secretions or active transports, however, it is necessary to refer to earlier views of membranes and cytoplasm insofar as they bear upon the problem of salt and solute secretion in cells.

A. CLASSIC CONCEPTSOF CYTOPLASM:ITSMEMBRANES AND THEIR ROLE From the outset, several contrasted ideas were advocated. First, there was the possibility that the functional boundary surfaces of the cytoplasm are mere physicochemical surfaces of contact or interphases, between immiscible fluids. or alternatively, that they are morphological entities, even autonomous organelles of the cell, with a definite developmental history. Similarly, there were ideas on what has been termed “passive” membrane permeability in contrast to what Overton recognized even at the outset as the need for a more active or, as he termed it, “physiological” permeability. Initially, it was more feasible to approach the study of the protoplasmic membrane in terms of physicochemical interphases, and certainly the more feasible approach to their permeability prop. erties was through the concepts of passive permeability phenomena. Neverthe. less, the alternative concepts have finally proved to be more enduring and arc currently being developed in ways that contribute to the problem here ir question. On the evidence of electron microscopy and fine structure, membrane: emerge as morphological entities and as autonomous organelles in cells. Also on the evidence of movement of ions or solutes against concentration gradients

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cells need to use their vital machinery to bring about nonequilibriuni movements of solutes in ways that the passive permeability phenomena could not alone explain. Detailed concepts of membrane structure arose out of the need to explain the passive permeability properties. These properties required the incorporation into the membrane of lipids sufficient to account for the rapid penetration of nonpolar molecules. Similarly, the membrane properties needed to explain the penetration, albeit with some difficulty, of small charged polar particles which was conceived in terms of water-filled pores. All this resulted in the now familiar sieve-pore concepts, heavily based on the properties of such model membranes as the dried collodion membrane system. Faced with evidence of the obviously porous membrane of Beggiatoa, however, as studied by Ruhland, and of the obviously more fat-soluble membrane of cells of Rhoeo discolor, after Collander, the contrasted properties of “pore permeability” and “solubility permeability” were incorporated into the membrane in ways that were flexible enough to explain differences between organisms, on the one hand, or differences-in permeability in time or during development, on the other. It was, however, the investigation of the structure of oriented thin films, after Adam, Harkins, Langmuir, and others, that produced a model system, and the ideas of Davson and Danielli that enabled biologists to conceive of oriented thin films possessing solubility properties, on the one hand, or through their structure the equivalent of discrete pores for the entry of small molecules, on the other. Up to this point, there was little direct evidence of the molecular structure of membranes, and similarly very little had been learned about the detailed structure of the ground cytoplasm. Therefore, throughout a period (1925-1945) during which the ideas of active salt or solute accumulation involving metabolism were being developed, the emphasis was largely upon the membranes as boundary surfaces having properties necessary to keep the cytoplasm intact. From the outset, however, it had been recognized (even by vital staining techniques) that inner and outer surfaces had very different properties, and evidence compatible with this view came with the demonstration that vacuoles could be isolated with their mewbrane intact (Siefriz, 1927; Plowe, 1931; and later workers down to Gregory and Cocking, 1966a,b). Thus, concepts of the transfer of solutes from medium to vacuole were apt to be more concerned with various ways to generate zoithitz the cytoplasm appropriate carriers of ions which could be formed at one surface and discharge the transported ions at another. Hence, the membrane--whether plasmalemma or tonoplast-could be conceived as affecting rates of movement or even contributing qualitatively to the selectivity of the movements without providing per se the active machinery or the driving force that determined the final internal concentration. For this, metabolism was invoked to generate and

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regenerate the carriers. In any situation in which the internal concentration did not exceed the external, i.e., in which the movement was merely passive, physical diffusion would, of course, alone suffice; however, in those cases in which the internal concentrations exceeded the external, osmotic work was required. The ideas that cells do cellular or even osmotic work in accumulating solutes in vacuoles gained much credence from the understanding of ATP as the universal energy currency in cells, capable of being directed to a variety of processes in which energy is required. Furthermore, the knowledge of the origin of ATP as the useful by-product of the overall respiratory process made it apparent how chemically usable energy could be stored and later directed to useful purposes which were, therefore, not as closely tied to the total end product of respiration, namely carbon dioxide, as might have hitherto been supposed. In fact, at this point a dichotomy of views became clearly defined. Prior to this, LundegHrdh and Burstrom (1933) and Robertson and Wilkins (194%) had pointed to a salt or anion respiration as a definite respiratory moiety, stoichionietrically related to ion transfers. By contrast, Hoagland, Steward, and their co-workers had repeatedly implicated respiration more indirectly, had avoided direct stoichiometrical relations betweens ions transported and carbon dioxide produced, but had rather invoked cellular work related to metabolism in a more indirect fashion. An understanding of the role of ATP made it apparent how the general respiratory metabolism could produce ATP capable of being specifically directed to some useful work, even though the actual way in which it was harnessed still remained obscure. All this placed the emphasis on biochemical processes in the cytoplasm, on the involvement of respiration as the main source of useful energy (in the form of ATP) and, as the knowledge of it emerged, on the concept of the mitochondrion as the “power house” of the cell which generated the chemically usable energy. Therefore, ideas about the active participation of membranes in ways that communicated the driving force for active secretion were still undeveloped.

B. THECYTOPLASM: ITSFINESTRUCTURE AND BIOCHEMISTRY Modern concepts of protoplasmic membranes developed first from the morphological knowledge gained, approximately in the last decade, by the use of the electron microscope. The knowledge of structure became increasingly involved with biochemistry and, conversely, the investigation by biochemical methods of the enzymes, proteins, and lipids of cells has also become involved with their form. Therefore, current biochemistry is almost as concerned with fine structure as is work on the morphology of the cell per se. This union of biochemistry and submicroscopic morphology has, therefore, implications for the interpretation of the problem of active secretion. The useful concepts that have developed from several main lines of approach are as follows.

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1. Membranes and Bozlndary Surfaces

The development of permanganate and glutaraldehyde fixation and appropriate staining methods led to the electron microscope evidence of the unit membrane (Robertson, 1959; Elbers, 1964) ; this gave reality to the otherwise abstract notions of protoplasmic membranes. The even later techniques of freeze etching, which avoid the use of fixatives but also preserve the material in question by a combination of low temperature and such agents as glycerol, reveal membranes as surfaces seen in relief, and they permit the organelles of the ground cytoplasm to be seen in their three-dimensional perspective (FreyWyssling and Miihlethaler, 1965). Dramatically, however, these surfaces are neither smooth nor plain, for they have a terrain and a topography involving both pores, on the one hand, and granular accretions, on the other. 2. Cytoplasmic Vesicles

Meanwhile, the study of the ground cytoplasm, hitherto regarded as optically transparent and more or less fluid, produced a rich harvest of information. This ranged from the internal structure of such well-known organelles as the plastids and the nucleus to the knowledge of mitochondria, with their internal membranes or cristae, to the recognition of the dictyosomes (or the Golgi bodies) and to the understanding of the role of the endoplasmic reticulum and, in plant cells particularly, to the knowledge of the protoplasmic connections via plasmadesmata which unite the protoplasts of adjacent cells. The thread running through all this fine-structural work, however, is the idea of the vesiculate nature of protoplasm, its architecture, and the conipartiiieiitatioii of its organelles, with the obvious emphasis on internal surfaces or membranes upon which biochemical events can be spatially disposed to ensure their sequential operation.

3. Spatial Arrangement of Exzymes on Surfaces Whereas work with the electron microscope depends on the preservation of the organization intact, biochemical morphology began with attempts to isolate entities from cells which could be biochemically recognized. The first development in this direction was the obvious association of respiratory pathways and electron transport with mitochondria. As the association of enzymes with membranes became more familiar, other examples emerged, so that it is now a rather generalized concept to visualite the spatial arrangement of enzymes on nienibrane surfaces in ways that determine their ability to promote reaction sequences. Thus, enzymes may now be conceived as not merely being 012 the surfaces of membranes but even constituting a part of membranes and, hence, they may produce by their biochemical effect not merely the selective movements of solutes through membranes but also in part the necessary driving force.

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4. Isoluted illembranes While the work on enzymes and their relations to membranes was progressing, techniques were being developed to isolate membranes as physical entities by centrifugal and other means. Thus, membranes are no longer to be regarded as physical interphases, for they have a physical and biochemical reality in isolation. This has enabled their content of lipids and proteins to be studied, has permitted their different constituents to be removed and identified and their properties determined, and even the results of their recombination noted. In other words, this sort of work leads to more realistic model systems on which the properties of ion selectivity, electrochemical properties, and biochemical and enzymic properties may, in due course, be investigated itz vitro, and correlated with structure. j.

Artificial Mertibrums and Model Systems

Studies of biochemical function and submicroscopic morphology emphasize membranes as the basic organizational element within the cell. Implicit in this concept is the need to understand the physicochemical laws that apply at surfaces of molecular dimensions. This has been the subject of much work with model systems and with more abstract mathematical treatments. Currently, the bilayer lipid membrane structures of the Mueller-Rudin type (Mueller et ul.. 1962) show promise for the study of physical chemistry of ultrathin structures (near-100 A) and for study of the behavior in zhro of isolated membrane components (Maddy et uZ., 1966). Such structures supplied with suitable additives show many of the properties of biological membranes including similar electrical properties, excitability, response to calcium ion and ion selectivity (Izatt et al., 1969) in response to certain additives such as valinomycin and monactiii (Mueller and Rudin, 1967, 1968). While the utility of these structures as models for biological membranes remains to be seen, they may, neverthcless, provide an approach to the in vitro study of biological processes which utilize the properties of surfaces in their function (Whittam et uZ., 1968). From these various lines of work, some general concepts emerge of the structure of protoplasmic membranes and surfaces and of their role in the ground cytoillasln and in the cellular organelles. This general picture needs to be s u m marized and its implications for the interpretation of active solute movements visualized.

C. CURRENTCONCEPTSOF MEMBRANES, PROTOPLASMIC SURFACES, AND CYTOPLASMIC ORGANELLES-AN EPITOME These concepts need to be distilled out of the great volume of work and SpecLllation that has succeeded the Davsoii and Danielli hypothesis of membrane structure. While the basic information is presented in many review articles and

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journals, it is convenient to regard the salient papers and symposia presented in 1967 at the International Biochemistry Conference at Tokyo as a useful point of departure. At the outset, however, there is an evident gap to be bridged. To interpret active transport phenomena, the measurements and theories must eventually relate to cells and to movements over finite distances, as from ambient media into internal vacuoles. The observations and interpretations of the electron microscopist, however, focus attention upon an infinite array of surfaces that ramify throughout the cytoplasm and its organelles, and the dimensions involved are in angstroms. Also, the physiological observations on metabolism are made upon the intact system, whereas the observations of biochemists are on enzymatic mechanisms and are being made at the limit of their ability to resolve the particles and to see them with the electron microscope. Thus, the difficult transition is to pass from the necessarily static picture of electron microscopy to the more dynamic concepts demanded by the biochemical changes as they occur at the level of minute particles: to visualize what a given solute or ion would encounter as it passes from the ambient medium across a plasmalemma, through the dynamically working cytoplasm, finally encountering a tonoplast, to be secreted into an internal aqueous vacuole. This is, however, what must be done, and in the attempt to do so it should be recognized that the word “membrane” has taken on new connotations. It may now denote any surface of contact which, in the organized system, separates one region from another; it may also rep resent any site of activity in isolated preparations where chemical change may occur. On reexamination, the relatively static, somewhat flexible, membrane concept of Davson and Danielli is now being modified to take account of (I) knowledge of protein turnover and the need for a more dynamic membrane system, (2) ideas that proteins may be much more integral components than the “sandwich-like” leaflet structure that Davson and Danielli visualized, (3) thoughts that enzyme action must be a functional part of the membrane phenomena (especially ATPase and its activation by ions), (4) various ideas from electron microscopy which suggest that the membranes in sitzl may not always conform to the original Davson-Danielli hypothesis, and ( 5 ) results of freezeetching techniques which now permit many protoplasmic surfaces to be seen in relief without the use of chemical fixatives. The implications of these ideas for a current concept of membrane structure have been the subject of several recent reviews (Korn, 1966; Sjostrand, 1967; Robertson, 1967; Branton, 1969). Freeze-etching reveals the membrane surfaces as much more sculptured and with much more evidence of granular structure than had hitherto been suspected. Protoplasmic surfaces-whether of vacuoles, nuclei, dictyosomes, inner and outer membranes, or any organelles-possess a topography, an J the func-

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tional significance of this terrain is not yet adequately understood. The finer granules of which these entire structures may be composed (order of 50-200 A ) are of relatively constant dimensions, however, and they are similar in size to the minute particles that contain the enzyme functions in many biochemically isolated membrane preparations, as for example those obtained from disrupted mitochondria. In fact, these membrane subunits may now be dissociated and reassociated in vitro in ways that show that their enzyme activity is related to their composite form. According to Green’s general membrane concept based on studies of mitochondria (Green and Perdue, 1966), there is a structural membrane subunit composed of lipid and protein (in their linear arrangement, these subunits could be responsible under the electron microscope for the appearance of a unit membrane), and to each of these units are joined detachable “sectors” or “head pieces” closely associated with the enzyme activity. Similar particles have been isolated from mitochondria1 inner membranes which contain the enzymes of electron transport (ATPase and cytochrome oxidases) and in which “coupling factors” are supposed to hold the responsible functional structure together (Racker et ul., 1964; Racker, 1967). Other generalized interpretations of membrane surfaces interpolate a protein moiety throughout the membrane structure in ways that allow it to respond by changes of porosity and thickness in a manner that could determine its permeability properties interpreted in terms of pores, whether these are regarded as being of fixed dimensions or as a statistical property of a dynamic system (Solomon, 1961). These ideas are compatible with the theoretical approach of Kavanau (1965) and are summarized diagramatically by Kavanau (1966, cf. Fig. 3, p. 1099). Somewhat similar concepts seem to emerge from the attempts to interpret the external surface of the plant protoplast, i.e., the plasmalemma, as the surface at which the synthesis of cellulose occurs, and here one may refer to concepts formulated by Muhlethaler (1967). Although the properties of membranes are usually conceived in terms of the protein and lipid components, nevertheless, they may also be modified by agents which act through the physical properties of water as conceived by Paulitig (1961) and Miller (1961). The concept here is that such agents, e.g., those that act as anesthetics (Catchpool, 1966), may behave as inert nuclei around which “icelike” arrangements of water molecules of the membranes may be stabilized, with consequential effects upon the physical properties of the water and the permeability properties of the membrane. It is probably too soon, as it is obviously difficult, to decide definitely upon the relationships of these different concepts, except that they have in common the idea that membranes or functional surfaces are built up of subunits, the subunits comprise protein and may contain enzymes, and that they are put together in accordance with a definite although spontaneous architectural plan

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which may, nevertheless, be subject to modifications which may alter the functional properties of the composite whole. Any current interpretation of the interlocking sequences of biochemical reactions that occur in a controlled manner during metabolism (i.e,, as in respiration in mitochondria or in photosynthesis in chloroplasts, or in protein synthesis on the ribosomes or polysonies) conventionally invokes a spatial arrangement of the appropriate enzymic or cyclical systems by means of subunits or particles arranged as within the surfaces of a mitochondrion or a chloroplast. Just as the controlled, directed course of chemical reactions in metabolism is held to be a function of these architectural arrangements, so one must conceive of a similar situation with regard to the controlled, directed movements of solutes or ions. In fact, Lehninger (1964) has freely invoked these concepts to explain the relationships of the mitochondrion to its inorganic ions. The properties of such membranes may be altered by the addition of various substances (e.g., valinoniycin, nigericin) in ways that affect this coupling of ion transport and energy transfer and suggest an active participation of the membrane in these events (Moore and Pressman, 1964; Graven et ul., 1966; Cockerell et al., 1967). Both the energy transfers and ion movements of mitochondria are also affected by ineinbrane components such as phosphatidyl inositol, which is incorporated into the membrane system (Vignais et ul., 1963). Thus, ion movements or charge separations across membrane surfaces appear to be associated with energy conversions which also involve membrane surfaces. Isolated structures of the kind referred to above, and conventionally termed membranes, are regarded as dynamic in the sense that they negotiate energy changes (they may contain ATPase or negotiate electron transfers) and, as has been said, their subunit structures may be easily dissociated or reassociated. Thus, whereas formerly one spoke of the plasma membranes in the sense of Pfeffer, mainly in the context of plasmalemma and of tonoplast, one should now conceive of the entire cytoplasm, with its highly vesiculate inclusions, composed of membranes throughout, albeit with ribosomal particles both free and attached. Thus, earlier concepts of a uniform liquid sol-gel or fluid-solid cytoplasm have disappeared. In fact, there may well be an underlying structural matrix, perhaps contractile, which could mediate within the overall architectural plan the spatial distribution of the compartments and organelles which constitute the higher orders of complexity in the protoplasm. This notion is reminiscent of the idea of kinoplasin of Scarth which, as early as 1927 and long before our current knowledge of fine structure or of the endoplasmic reticulum, anticipated the need for some such structural matrix in protoplasm to explain its fluid properties (seen by its ability to flow), on the one hand, and its solid properties (evident by its elasticity), on the other, and to explain also the seemingly high viscocity it displayed en masse with the relatively low viscosity it shows on the

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more micro scale by Brownian movement. In short, Scarth prophetically visualized the ground cytoplasm as being permeated by a sort of random structural network, which he likened to a heap of faggots in the interstices of which the fluid properties reside. Even today one sees hints of even finer structure than the reticulum in the ground cytoplasm. This superfine structure might provide the basis, the underlying structural even contractile matrix, that could coordinate or tie together the varied activities of ground cytoplasm. [Parenthetically, one might emphasize that the only chemical hint of the existence of such an entity is in the metabolically inactive structural protein moiety, which incorporates proline and which forms combined hydroxyproline and seems to be so closely identified with growth and multiplication in growing cells (Steward and Pollard, 1958), even as it is distributed throughout the ground cytoplasm (Israel et ul., 1968).] Within this highly ordered cytoplasmic structure, however, plant cells create and fill an inclusion of a very different kind. This, the vacuole, is finally set apart as an unstructured volume into which water and solutes are secreted; and, of course, it is bounded by a membrane surface-the tonoplast. In their genesis, vacuoles seem to arise from swellings or outgrowths of the vesiculate cavity between otherwise continuous membrane surfaces [Fig. 2 ; cf. the work of POUX (1962) as confirmed and extended by Mesquita (1969)]. In this internal secretion of water and solutes which forms a vacuole and concomitantly stocks it with solutes, the essential dilemma is to understand how the energy changes incidental to these nonequilibrium movements are negotiated; and it is in this area that one needs to draw upon the concepts derived from work on mitochondria and chloroplasts. Earlier, respiration was visualized as producing the available energy, incorporating it into the so-called high-energy molecule of ATP, then transporting the energy in that form to the site where it could be used and, by appropriate chemical machinery, donating the energy by breakdown of ATP to useful work. It now seems, however, that dynamic biochemical changes are much more an integral part of the process, whether it occurs in a membrane, an organelle, or an enzyme protein system. In fact, the energy of the processes so activated, as in mitochondria and chloroplasts, is often held to involve, if it does not represent (as in the views of Green et ul., 1968), conformational changes in the immediate physical locale or in the molecule that is activated. Thus, Green seems to believe that the energy that results from electron transfer in the mitochondrion is not merely communicated to ATP as the stable end product capable of doing some useful work via some “high energy chemical intermediate” in the process, but rather that the electron transfer gives rise to a conformational or structural change which is itself the way the energy is communicated, Thus, membrane parts may in fact be the “intermediates,” and the transfer of energy

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may involve physicochemical principles of ion or charge separation (Mitchell, 1961). Thus, electron transfer may give rise, on the one hand, to ATP and, on the other, to an activated or energized surface. In this sense, the ATP would not be regarded as the immediate link between respiration and the useful work, but rather the way in which a pool of energy may be stored in a form available for later use. Many of the views expressed above now seem reminiscent of earlier ones designed to interpret the apparent involvement of protein synthesis and turnover in the intake of solutes, especially of potassium and halides by vacuolated plant cells. These views invoked the concept that large molecules, e.g., proteins, can negotiate energy transfers by their changing states of order and disorder as they pass reversibly from surface films to globules and, in the more extreme cases, as they are synthesized, broken down, and turned over. After having seen in this manner the trends in the understanding of the system involved, an attempt should now be made to see other trends in the experimental investigation of solute uptake by cells and in the various views that have been formulated by different workers and schools of thought.

111. Physiological Studies on Salt Accumulation in Plant Cells: Past and Recent Trends

A. PERIOD1925-1945 As already shown, Hoagland’s work on Nitella moved the subject farward in a. major way, because it showed clearly that the mechanism was an active one

and not the expression of a physicochemical equilibrium or a passive permeability process. These observations then unleashed a number of investigations made with thin discs of storage organs, with excised roots and other systems, which demonstrated the active accumulation of nonmetabolizable salts, of which potassium bromide was the then convenient experimental examp!e. While it was the role of light that led Hoagland to postulate an active mechanism of salt accumulation in Nit&, not because the light was directly involved per se but rather because it furnished suitable metabolic substrates, it was the effects of oxygen on the uptake of ions by storage organs and by roots that directed attention to respiration as the ultimate source of the energy required to drive the process in question. These observations, together with the effects of temperature, which indicated a metabolic rather than a physical basis for the entr) of ions, and the effects of mobile ions of one sign (e.g., cations) on the niow ment of another (e.g., an accompanying anion), were all part of the earl) physiological background of the problem in question. By the early or mid-1930’s, however, it had become quite apparent that the decisive problem was to identify the metabolic machinery that motivated cells

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to accumulate their salts de nova, recognizing that these metabolic events were linked to respiration that furnished the necessary energy, not only for the ion intake per se but also for the other anabolic events that occurred in cells simultaneously. Experiments were made to describe the biochemical backgrounds of the system capable of accumulating salts in thin discs cut from plant storage organs. This was done even in a period when analyses of inorganic ions by the quantitative methods of the day were arduous, when knowledge of intermediary metabolites was limited, and prior to techniques that became available through the use of radioisotopes or chromatography. In retrospect, these investigations were well ahead of their time, for they arrived at the clear concept that protein synthesis in cells coupled respiratory energy to the act of synthesis and also to the concomitant ion uptake, which occurred in cells manifesting some endogenous capacity to grow. While this capacity to grow was not evidenced by great change during the course of the experiments, it was nevertheless demonstrable by the behavior of cells able to divide internally when placed in contact with moist air. Thus, while Hoagland’s original work on Nitellu moved the subject forward into new concepts of its physical chemistry, requiring it to be conceived as a nonequilibrium process, the work in the period 1933-1940 as clearly moved it forward in a biological sense, for it became identified with the operation of biological machinery, characteristic of cells able to grow and actively metabolize. In fact, Hoagland’s work on salt-depleted root systems emphasized, even at this early date (Hoagland and Broyer, 1936), that entry of salt (anion and cation) was not an isolated event, was not only a function of respiration, but was also attended by massive conversion of stored carbohydrate in the salt-depleted roots and that the formation of organic acids was a function of the unequal uptake of anion and cation. Nevertheless, throughout this period, others sought mechanisms built on equilibria (e.g., variants of the Donnan equilibrium), or on established steady states which simulate equilibria, and their views were inevitably at variance with those which recognized that the cells must be able to deviate from equilibria and to harness their metabolic energy to do physicochemical work. It is interesting to recall that views on active salt accumulation were freely current in the plant physiological literature well prior to modern knowledge of phosphate bond energy or of ATP as the energy currency that is expendable in cells that do work. While these concepts were developing late in the period in question, it was not until about 1941 that they became applicable to problems of salt uptake or ion transport. In retrospect, therefore, it is somewhat remarkable that so much experimental evidence still current or valuable today had emerged prior to the recognition that phosphate bond energy might be coupled to proteins to do work (as for example in the phosphorylation and dephosphorylation of myosin, which became known about 1944), for phosphate bond en-

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ergy was only considered to be the link between respiratory energy, on the one hand, and physicochemical work in ion accumulation, on the other, about 1945.

B. PERIOD1945-1960 This section aims to present an overall view of the types of research and the interpretations made in a period which really began with the end of World War 11. This not only permitted much otherwise dormant investigation to be resumed, but new tools and concepts were available. The ready use and availability of isotopes permitted individual ions and molecules to be followed; the usage of chromatography with its related techniques of radioautography permitted much more sophisticated investigations of metabolism than hitherto; the electron microscope permitted cells to be more intimately known at the level of their membranes and organelles; the ultracentrifuge permitted isolations of subcellular components, and already existing bioelectric techniques became still more refined. Along with the increased knowledge of intermediary metabolism (glycolysis, Krebs cyde, the pentose phosphate pathway), ideas emerged which involved phosphorylated compounds in a variety of syntheses and metabolic reactions made possible by the recognition of ATP as a universal chemical energy currency linking respiration to useful work. During this period, a steadily increasing array of specific metabolic inhibitors enabled the metabolism associated with transport to be partitioned. Concurrently, new approaches to the study of growth of cells and tissues in aseptic culture, together with the newly recognized growth factors that stimulate or regulate growth, provided new and controllable experimental systems. It is against this background that the trends of research on solutes in cells needs to be seen. In appraising the trends since 1945, it is convenient to divide the vast amount of work that has been done and published into two parts. The first part is conveniently marked by the extensive review and appraisal of the subject published by Steward and Sutcliffe in 1959. Up to this point, the main experiments fall into categories, summarized in the review in question, and which may be recognized as follows. ( 1 ) Investigations and interpretations in which, granted the existence of the system in vim, it is nevertheless conceived to operate through recognizable and predictable physicochemical principles. ( 2 ) Experiments designed around the central idea that movements of solutes involve carrier complexes, reversibly formed and appropriately dissociated and capable of movement, either actively or passively. ( 3 ) Attempts to involve electron transport, implicit in respiration and reversible oxidation of the cytochromes, by which the oxidative metabolism is mediated directly in the events of ion transport.

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( 4 ) Further investigations on the involvement of ion and solute secretions with concomitant events of metabolism, respiration, and growth. C. MORE RECENTTRENDS Through the period 1945-1959, virtually all of the current conceptual approaches to ion accumulation in plants had to some degree been mentioned or explored. Nevertheless, the period 1959-1969 produced a great spate of papers. While many of these add miscellaneous detail, too few bring new insight or understanding, and their number (at least several hundred) clearly precludes their detailed citation. Many are repetitive in the sense that they carried out with radioactive isotopes kinds of investigation which had already been done, more arduously, with unlabeled solutes, and few have been interpreted against a more adequate understanding of the participating cellular machinery than the earlier investigations invoked. Nevertheless, one may broadly classify the avenues of approach applied in this period as follows. 1. Kinetic Interpretatiom

The analogy between directed ion movements and the kinctics of enzyme action has been explored. These ideas imply the combination of an entering ion with a carrier molecule, which plays the role equivalent to the enzyme, but instead of altering the nature of the substrate as in the enzyme system the ion is finally transported from place to place (i.e., across the membrane), The salient example here is Epstein and Hagen’s original study (1952) of uptake of ions by excised barley roots and the competition, or otherwise, between different cations. Concepts of carriers having specific binding sites are useful if the sites can be defined. Sites in different parts of organs (e.g., roots) need to be distinguished from sites within cells, and the more it is necessary to proliferate sites for the same ion (i.e., Epstein, 1966; Luttge and Laties, 1966) to account for the behavior at different external concentrations the less useful the concept becomes. When in competition for the same sites, the relations between different cations (e.g., K and Na) vary with concentration in ways that do not follow from the theory (Sutcliffe, 1957) or in ways that could be described by simple diffusion (Briggs, 1963) ; furthermore, the kinetic data admit of several different forms of kinetic treatment (Oertli, 1967). The first postulated binding sites were for cations (Epstein and Hagen, 1952), and some recognition of sites for anions developed later (Epstein, 1953; Elzam et al., 1964). Other than the concept of “permeases” (Cohen and Monod, 1957) which had a genetic origin but also permits kinetic interpretation (Stein, 1967), no such specific carrier sites for nonelectrolytes were demonstrated as part of the enzyme kinetic analogy.

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2. T h e Suggested Role of Specific Binding Sites

Reversible binding or association of ions to specific sites was postulated by Epstein and Hagen (1952) to account for the apparent competition in the uptake of different ions. Danielli (1954a) and Solomon (1961) and others have invoked concepts of ionic double layers and the spheres of influence of charged sites as described by zeta potentials and as these could be applied to the selective passage of ions through water-filled pores in membranes. In the extreme case, it has even been proposed by some (Troshin, 1958, 1961; Ling, 1962, 1966) that all of the ion content of animal cells is either bound or complexed at niacromolecular sites. While this might account for a first step in plant cytoplasm, other events must surely occur in fully vacuolated plant cells. 3 . Ion Fluxes and Membrane Potentials

The term flux denotes the simultaneous entry and exit of solutes in a dynamically maintained system. Demonstrations compatible with this concept required the use of radioactive isotopes which permitted the movement of a radioactive ion to be distinguished from its nonradioactive counterpart. Thus, when radioactive chloride was presented to cells (e.g., of Vallisueria leaves) it became obvious that it could be readily exchanged with the nonradioactive chloride present within, and this exchange was related to the condition of the cell membranes (Arisz, 1964). From studies on this sort of interchange, the idea emerges that the ions of cells are in compartments with different degrees of accessibility to the entering ion. Usually the most easily exchangeable ions are those supposed to be bound in cell walls or present in interstitial spaces, the ions in cytoplasm being less easily exchangeable, and those in vacuoles exchange at the slowest rates (MacRobbie and Dainty, 1958). Granted the above, ions may move under some circumstances passively, under others actively. The relations between the electrical potential differences across cellular membranes and the associated movements of the ions distinguish whether the movements are active or passive. The basic mathematical relations that enable this to be done have been formulated by Ussing (1949), Teorell (19@), and Goldman (1943), and these relationships, when applied to the ion movements into plant cells, have been variously reviewed (Briggs et al., 1961; Dainty, 1962). If cells are established in a steady state with reference to a specified salt, il given ion can be exchanged for another isotope and its influx and net movement into the cell can be measured; these movements may then be correlated with the measurement of an EMF between cellular compartments and the efflux of the comparable isotope. The distribution and the movements of ions observed in this way distinguish between ions that are in apparent equilibrium with the measured

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EMF (these ions move passively) and other ions that are not in equilibrium with the EMF (and these, therefore, move actively). These activated movements, likened to specific ion pumps (e.g., for K or Na), have been variously defined and localized, even if tentatively, at the plasmaleinma or the tonoplast in papers dealing with coenocytic vesicles by MacRobbie and Dainty (1958) for Nitella obtusa; Hope and Walker (1960) for Churu austvalis; MacRobbie (1962, 1964), Spanswick and Williams (1964), and Spanswick et al. (1967) for Nitella traifrlucens; Blouiit and Levedahl (1960) for Halicystis ovalis; Gutknecht (1966) for Valonia ventricosa; and with higher plants by Etherton (1963) and Higgenbotham et al. (1967) for oat and pea seedling tissue; Bowling et al. (1966) for Ricinur comunis roots; Poole (1966) for discs of beet root tissue; Macklon and MacDonald (1966) for discs of potato tuber; and with fungi by Slaynim (1965) for Nezrospora crassa; and by Miller et al. (1968) for slime inold plasmodia. In any given situation which is approached along these lines, substantial siinplifying assumptions are necessary to make the interpretation of the data feasible. Such assumptions are that ions move freely and independently, that the boundary membranes and the intervening ground cytoplasm must be individually homogeneous and have no net charge, and that the permeability properties are the same for entry as for exit of ions. Acceptance of such drastic assumptions is only justifiable if they lead to a really plausible and useful interpretation. The dilemma is that the observed relationships between membrane potentials and ion fluxes are neither as coiisistent nor as direct as the equations seem to require. This is shown by the effects of varying external potassium concentration, which did not produce the expected change in membrane potential in Nitella, as in the work of Hope and Walker (1961); by the electrical consequences of changes in external pH (Kitsato, 1968) of CO,/HCO_ effects (Hope, 1965; Poole, 1966) ; and of the presence or absence of calcium (Higginbotham et al., 1964) which were not predictable from the basic theory. In such circumstances, investigators have adopted arbitrary devices peculiar to each case to explain their data. It cannot, of course, be denied that ion movements and electrochemical gradients will ultimately be correlated, but the difficulties inherent in doing this constitute evidence enough that other properties of the system are involved. A feature of the study of ion movements related to EMF’S is their dependence upon near steady-state conditions, and this automatically precludes any treatment of situations in which there is a definite change in ion content with time, or of systems in which there are massive changes (because of metabolism and growth) in composition. Most of the work on ion fluxes has, in fact, been done with the large internodal cells of Nitella or similar organisms which are relatively inert when investigated and so fulfill the above conditions at the time and under the circumstances of the measurements.

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Attempted applications of flux studies to systems which in varying degree are capable of some constructive metabolism and growth, i.e., ranging from discs of storage organs (Briggs and Pitman, 1959; Van Steveninck, 1962) to the growing tips of roots (Pitman and Sadler, 1967; Scott et al., 1968), have encountered the obvious difficulty that these systems are more or less weighted toward net influx so that the interpretations in terms of fluxes and of membrane potential differences should not be expected to be applicable (Briggs et al., 1961, cf. Chapter 13). In short, despite much work on ion fluxes and membrane potentials, no unequivocal statements have been forthcoming to describe the situation as it applies to so apparently simple a system as Nitella. Thus, from the standpoint of this review, this approach has not yet reached any finality; this comment would apply even more forcibly to those actively growing systems that so obviously depart from equilibrium or even steady state.

4. Involvement of Carriers and Perrnedses Carrier concepts are almost as old as speculations on the mechanism of ion accumulations in cells. They were first invoked to provide a gradient of a moving molecule distinctive from the one stored, At first, the carrier complex was thought to be reversibly synthesized at one part (e.g., the plasmalemma) and released at the other (e.g., the tonoplast) so that a positive concentration gradient facilitated its movement in the cytoplasm. An early example of this thinking was Osterhouts’ idea that cations entered in neutral complexes with a hypothetical acid H A and, much later, Lundegardh’s concept that anions moved in combination with a positively charged entity “X,” later homologized with oxidized cytochrome. From time to time, there have been many variants on the carrier molecule theme but modern biochemical and genetical studies and work on membranes have given it a greater degree of sophistication. It has been generally recognized that cells display a range of passive permeability types, interpretable in terms of existing lipid-sieve ideas, but it had long been obvious that metabolites (i.e., amino acids, sugars, and so on), as well as ions, could exist at internal concentrations greater than the external. The active transport of salt was early recognized in plants, but much of the evidence for active transport of organic metabolites came from work on microorganisms and animal cells. After 1942, increasing information on metabolic pathways and the increasing availability of nonmetabolizable chemical analogs of naturally occurring metabolites, specific metabolic inhibitors and, most important, specifically labeled radioactive metabolites, led to more definitive concepts of the proposed carriers. Such carriers were conceived to be in the membrane and could there mediate the rate of even passive movement of molecules into the cells. The passive trans-

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port could have stereospecific properties, i.e., one stereoisomer could enter more rapidly while another might be excluded, thus conveying an impression of “facilitated diffusion.” This “facilitated diffusion” has been interpreted in terms of fixed-membrane structures (Danielli, 1954b) and also in terms of mobile carrier mechanisms (Widdas, 1952; Rosenberg and Wilbrandt, 1955; Browyer,l957). The stereospecificity has been regarded as a feature in support of the idea that the carriers were proteins or even enzymes which, therefore, had a transport function. Thus, in addition to “pore permeability” or “lipoid” solubility pernieability, there appeared to be carrier mechanisms that could also function in passive permeation. Later, “counter-transport” or “counterflow” was demonstrated (Rosenberg and Wilbrandt, 1957; Wilbrandt and Rosenberg, 1961) which was also stereospecific and could couple the passive movement of one molecule down its concentration gradient to the reciprocal active movement of another molecule against its concentration gradient. To explain this, a protein that could serve a carrier function was located within the membrane. This concept permits two solutes such as chemical analogs, which might share the same carrier, to influence the transport and permeation of each other, e.g., the glycosides isomaltose and aethylthioglucoside (Halvorson et al., 1964), lactose and radioactive thioniethylgalactoside (Kepes, 1960), or glucose and 3-0-methylglucose (Morgan et al., 1964). This influence may appear as mutual competition for passive uptake under some circumstances or, if one member had entered the cell to high concentration, its subsequent efflux into a dilute solution of its counterpart would drive the uptake of the second member against its concentration gradient. Furthermore, the uptake of the one compound should bear a stoichiometrical relationship to the efflux of the other. (See Stein, 1967, Chapter 4, for a concise summary of these concepts.) The evidence that locates the carrier in a membrane is genetic. This utilizes evidence of the separate genetic control of entry into the cell and of the metabolic use within the cytoplasm of the cells (cf. Cohen and Monod, 1957). Specifically, the entry of galactoside into Escherichia coli could be under separate genetic control from its metabolism via galactosidase. This is the genesis of the concept of genetically determined protein carriers termed permeases, in this case “0-galactoside permease.” In several cases (Burger et d.,1959; Okada and Halvorson, 1964; Rothstein and VanSteveninck, 1966), the genetically demonstrable permeases also participate in “facilitated diffusion” and also function in a “counter-current” uphill transport system, such as that illustrated above. All the above events are essentially passive. However, metabolic energy (e.g., ATP) may also be coupled to the permease in such systems to render the transport of a given solute truly active, inasmuch as the metabolic energy now furnishes the sole driving force to

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accumulation against a concentration gradient (Okada and Halvorson, 1964; Rothstein and Van Steveninck, 1966). Thus, while the permease may constitute a “cog in the machine,” it does not furnish the driving force. Evidence for genetically determined retention of ions (e.g., K) has been obtained from E. coli (Lubin, 1962). A given mutant was found to be deficient in its ability to accumulate potassium, although the membrane did not appear to be damaged since other solutes were retained. This led to the study of potassium transport and also of the metabolic effects of internal concentration on such potassium-dependent cellular processes as protein synthesis (Lubin and Ennis, 1964; Lubin, 1967). The requirement for K ion in protein synthesis has also been shown in vitro (Spyrides, 1964). Similarly, Pardee and Prestidge (1966) associated sulfate transport in Sulmonelb with a genetically controlled protein, and there are also cation transport mutants in Nezlrospora which have been linked to genetically determined deficiencies in a membrane protein (Slayman and Tatum, 1965). Attempts that have been made to isolate permeases or “carrier proteins” have met with varying degrees of success. Substances implicated as permeases for Bgalactosides, sulfate, calcium, neutral amino acids, glucose, and potassium-sodium transport have been isolated from bacterial and animal sources (see a recent review by Pardee, 1968). The criteria of success in such isolations depend upon reversible binding of the molecule transported to the proposed carrier. Such reversible binding capacity does not per se constitute proof of a carrier function, however, nor does it reveal the precise mode of transport. The carrier function follows from the isolation of appropriate proteins from membrane preparations and the simultaneous genetic determination of a carrier capacity and of a binding capacity. hi fact, partial restoration of transport capacity may follow when the crude isolated carrier is restored to a genetically deficient strain (Anraku, 1968). Although a genetically controlled binding capacity coincident with an active transport capacity is presumptive evidence of a molecular carrier (as in the six or more well-documented cases cited by Pardee, 1968), it is still an open question whether all these are simply properties of a carrier molecule resident in an external membrane or, alternatively, they might equally involve mechanisms or structures which reside in the cytoplasm, especially in the highly organized cells of higher plants. Metabolic energy of the cytoplasm may be coupled to carriers in several ways. One such example concerns a high-energy protein factor (HF) regarded by Roseman and co-workers (Anderson et al., 1968) as necessary for active transport of certain sugars in some bacteria. This factor constitutes an alternative to ATP for channeling metabolic energy directly to transport of a solute.

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5. The Role of ATPases

Direct evidence for the involvement of ATP in potassium and sodium transport across cell membranes derives from the work with red blood cell “ghosts.” Red blood cells lyse when placed in dilute saline media, and hemoglobin and other soluble contents of the cells leak out, leaving the membranes or “cell ghosts.” The cells then may be “restored” by adding sufficient salt to bring the tonicity of the medium to its original level, and these restored ghosts demonstrate permeability and transport properties much the same as those of the intact red blood cells. Gardos (1954) and Hoffman (1962) have shown that active influx of potassium can be supported by ATP hydrolysis in red blood cell “ghosts” that have been “loaded” with ATP during the lysing procedure. Similarly, Caldwell et ul. (1960) and Baker et al. (1961) have shown that nerve axons in which the internal axoplasm has been replaced with a defined artificial solution can actively extrude sodium and accumulate potassium, provided ATP is present in the internal solution. Experiments such as these support the concept of a ATP-activated carrier located in the cell membrane. Attempts to define activated carrier systems by virtue of the ATPase activity they exhibit in isolated membrane preparations have been successful in certain animal systems. Skou (1957) described a “K-Na-activated ATPase” from the microsomal fraction of crab nerve which is sensitive to ouabain and which appears to be of nearly universal occurrence in animal cells (Skou, 1965; Albers, 1967). This enzyme is held to mediate the coupled influx of potassium and efflux of sodium in animal cells, but it has not been conclusively demonstrated in plant cells (Bonting and Caravaggio, 1966; Leggett, 1968), although MacRobbie (1965) described evidence for ouabain-sensitive potassium transport driven by ATP in Nitellu. This protein, having the characteristics of an ATPase, has tentatively been isolated from animal sources, and its properties are still under investigation (Ruoho et ul., 1968). It is significant that this mechanism is present only in cells bathed by a relatively strong solution which is rigorously controlled in its composition. By contrast, organisms such as higher plants may be bathed by very dilute and less defined solutions and, as yet, they do not seem to possess such a mechanism. Hokin and Hokin (1963) have identified an ATPase function in animal membrane preparations which is also intimately involved with inositol turnover (Le., in “inositol phosphatides”) . In this system, the ATPase activity is sensitive to cation (K/Na) concentrations, and lipid components of the membranes are presumptively involved in ion transport. Direct application of ideas of phospholipid carriers is rendered difficult by problems of stoichiometry between transport and rates of lipid turnover (Tarlov and Kennedy, 1965).

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6 . Chdrge Separation and Ion Tramport in S p a t i d y Oriented Systems The essence of all these ideas is the attempt to harness the known formation of charged entities in metabolism (e.g., cytochromes with their positive charges, or metabolites oxidized by loss of electrons) to the events of ion transport. To have any bearing on a directed flow of ions, the metabolic events must also be spatially oriented, and this was the view promulgated by Lundegbrdh. Lundegdrdh’s concept depended upon oxidation-reductions at separate sites, namely, oxidations (involving molecular oxygen) at the external surface and subsequent reductions by oxidizable metabolites at the internal surface. These reversible events could produce a positively charged carrier capable of transporting anions, driven by the pace of oxidation-reduction, and directed by the spatial separation of the oxidized and reduced molecules. Lundegbrdh initiated and utilized these ideas in ways that required that plants invoke additional respiration as they absorb anions and eventually regarded positively charged cytochromes as the actual carrier of anions in the cytoplasm. These concepts required more strict stoichiometric relationships between ions absorbed and sugar and oxygen used than was substantiated by the data; moreover, they did not provide as explicitly for cation absorption, they left the problems of anaerobes in doubt and, to the extent that entry of ions was related to exit of CO, (Hi- and HC0,-) the situation of green cells in the light was unspecified. For these and other reasons, the Lundegbrdh concept as enunciated about 1940 left much to be desired (see Steward and Sutcliffe, 1959, pp. 373-388). Nevertheless, Robertson and Wilkins (1948a) seemed to give more precision to the Lundegdrdh hypothesis by equating the ions that could be moved by respiring a given amount of sugar to the tiuinber of electrons that simultaneously passed over the electron chain. As the events of electron transport in respiration became increasingly localized in mitochondria, attention was directed to this organelle as a priinarp orgaii of ion accumulation (Robertson et al., 1955); in Robertson’s first vicws, rhe plant mitochondria were conceived to absorb ions at one surface (plasmalemma) and to discharge them at another (tonoplast). Alternatively to these views of direct involvement of mitochondria as primary sites of ion accumulation and even movement, the mitochondria were involved as sources of the ATP required to drive processes needing energy (see Steward and Sutcliffe, 1953, 13. 261, 13. 388 et seq.). Nevertheless, although the mitochondria may not be the sole, or even primary, sites of initial salt accumulation in cells, their study has been useful as an example of a means to associate the biochemical eveiits of oxidation on oriented membrane surfaces with the physical chemistry of imbalmces of charged ions across membranes. After the similarities between the energy-yielding oxidative events in mitochondria and the light-mediated cnergystoring events of chloroplasts became clearer, the salt relations of these other-

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wise distinctive organelles aroused similar interest. Interest in these organelles is in two broad directions: first, their actual composition with respect to ions and their behavior in vitro as absorbing organelles and, second, the association of these events with their fine-structured membrane surfaces. Studies of the fine structure (e.g., Green and Perdue, 1966) and of the biochemical activity of isolated mitochondria1 preparations (e.g., Racker, 1967) have provided the concept that major acttiities of oxidative phosphorylation are closely associated with, or even a part of, the membrane structure. Further, it has been demonstrated that in vitro ion transport is closely associated with the energy conversions occurring in these organelles (Cockerel1 et ul., 1967). Calcium accumulation by mitochondria is seen as an alternative to ATP synthesis (Lehninger, 1964), and pH changes in the external media in vitro may lead to ATP formation within chloroplasts (Jagendorf and Hind, 1965; Jagendorf and Uribe, 1966) and under certain conditions within mitochondria (Reid et d., 1966). These various sources of information support a general theory of membranebound enzymes deemed capable of spatially directed reactions and of separating products from substrates across membranes (Mitchell, 1961) . This physical separation of products and charges represents, in these ideas, a step in the translation of metabolic energy into useful work, since the charge separation may be used per se or it may be subsequently involved in ATP formation. The latest statement along these lines by Robertson (1968) reiterates the charge separation idea, formulates ion transport inechatiisms as electrochemical phenomena, ignores accumulations of nonelectrolytes, still regards mitochondria as primary sites of accumulation and ion transport, but places the ideas essentially inherited from the concepts of Lundegdrdh in the context of current trends toward orienting energy-generating enzymes and metabolic systems on m e n i b r a i ~ surfaces, as in any of the cytoplasmic vesicles. In the final analysis, the objective is to regard ion movements as responses to metabolically created differences between charges across membrane surfaces. The views of Robertson, following upon those of Lundegirdh (1939) and Mitchell (1961), focus the entire attention upon mitochondria or plastids, i.e., upon organelles noted for their production of ATP by either oxidative or lightmediated reactions. To the extent that these views illuminate the unequal ionic concentrations that obtain between the organelles and the ground cytoplasm, they are important; this, however, should not obscure the fact that the primary site of the osmotically active solutes, internally secreted in plant cells, is in vacuoles. It is, therefore, still a question whether or not the supposed relations between the ion content of ground cytoplasm and its pH and the internal reactions and content of the organelles can equally apply to the solute relations of the whole cell. These involve the contrast between the ambient medium and the unstructured interior of cells, represented by the vacuole, with its intervening

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complex layer of cytoplasm. It is an obvious dilemma, still not satisfactorily resolved, that if mitochondria accumulate ions autonomously they must still release them for secretion into vacuoles. If mitochondria manipulate their metabolism to regulate either ATP turnover or the charges across their membranes and the consequential ion movements, how does this apply to movements de novo into the ground cytoplasm and subsequently into vacuoles ? Hypotheses of directed movements of mitochondria across cytoplasm and their reversible filling and emptying of ions, all based on in vitro studies (Robertson, 1968, pp. 727 3 ) , are not yet convincing. The basic idea is that mitochondria in vivo alternatively swell and shrink. As they swell, anions with accompanying hydrogen ions are secreted (this seems to be an alternative way of saying that they release organic acids) ; as they shrink, hydroxyl ions with accompanying cations are said to be released. As the mitochondria move in the cell, these alternating processes can occur in association with different vesicles (e.g., parts of the reticulum). The Robertson concept is that in one phase the mitochondrion may secrete anions, and in another cations, into the reticulum; in each of these areas, there are in consequence gradients of H+ or OH- across the membrane of the reticulum. Where there is a local excess of H+ this is exchangeable for other base (e.g., potassium) ; where there is a local excess of OH- this is exchangeable for other anion (e.g., Cl), and the exchanged H + and OH- can reform water, thus furnishing energy. Because the reticulum is a continuum, it obviates the need for a carrier throughout the cytoplasm or for the mitochondria to transport directly, as in earlier ideas. It seems, however, fair to say that the new views are the older ones in a slightly different guise. They are not simplified by the new biochemical concepts -they are rendered more circuitous and less easy to pin down. Lundegbrdh postulated that cells use as carriers of ions in cytoplasm the same catalysts that undergo oxidation (by loss of electron and acquiring a positive charge) and subsequent reduction that, reversibly, provide for a flow of H+ from reduced substrates to external oxygen with its concomitant flow of electrons along the electron chain. These ideas were so manipulated as to achieve “charge separation” at the outer cell surface and its disappearance at the inner one. The separated charges in question appeared upon the heme catalysts of the oxidation system. Essentially, the current ideas make the same mechanism operate acros the membrane of an organelle instead of across the whole cytoplasm. The actual mechanism of ion transport is conceived as follows. Metabolism releases energy which appears as a charge across a membrane. As in any Donnan system, that asymmetry of charge can account for differential uptake of o u t ion. Alternatively, the metabolic energy may appear as ATP when, instead of an unequal distribution of ions, the electric charge is dissipated by driving a reversible ATPase to forin ATP. Therefore, when ions are moved ATP does not appear; when ions are not moved ATP may appear. Thus, a system in which

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the balance is shifted away from ATP should promote ion movements; a system that allows it to accumulate should not. This is the essence of the doctrine. It has even been suggested that an observed drop in ATP content of carrot storage tissue when active transport was induced (Atkinson et al., 1966) was a result neither of the direct use of ATP in the transport per se, nor of the concomitant use of ATP in other events, but rather because the ion transport occurred in lieu of the further synthesis of ATP. This seems to be a “topsy-turvy” world. W e are trying to visualize how a cell does work in salt uptake. Normally cells produce and expend ATP to do their work. Here, however, the reverse is argued, for the cells may either do the work or produce the ATP. Great significance is attached to observations which suggest that when drastic pH changes are superimposed upon the external mitochondrial or chloroplast surfaces, thus modifying the otherwise metabolically maintained charges upon it, there are consequential effects upon the ATP balance within. Both the formation, or otherwise, of ATP and the ion movements to which it is reciprocally related should, therefore, become a function of hydrogen ion gradients across the mitochondrial membrane. This is circuitously reminiscent of Osterhout in the 1930’s-even to the supposition that the membranes are impermeable to ions so that all entry and exit of ions must be by some form of exchange (Robertson, 1968, p. 37), which is conveniently made to conform to the requirements of a given experiment. If the appropriate experiments could be done with really viable mitochondria, however, it could be tested by obliterating, or even reversing, the pH gradient across their membrane and by studying all the consequential ion movements. In short, the new Robertson-Mitchell ideas, involved as they also are with phosphorylations and mitochondria, still leave the problems of ion transport essentially where they were. They fail to face the biological realities of cells in contrast to organelles, of growing versus passive systems, of selective accumulation of ions into organelles in contrast to the overall ion absorption from the ambient medium and secretion into vacuoles. They place all the emphasis on charged particles and neglect the fact that so much osmotically active solutes are nonelectrolytes or weak electrolytes. In their latest form, they seem to regard the mitochondrial membrane with its ATP as a condenser which can be charged or discharged to take up or store electrical energy temporarily. By contrast, the flow of ATP had been earlier visualized as more analogous to the expendable flow of current originating from a metabolic dynamo (the mitochondrion or chloroplast) and driving, externally to it, the nonequilibrium machinery that does work. Since all these views do not yet explain the secretory activities of growing plant cells, new ideas based on new approaches, to be summarized in Section V, must still be sought. In fact, this is the essential dilemma. Vacuoles and internal solute concen-

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trations are created de nova as a concomitant of growth under essentially nonequilibrium conditions to which conventional physicocheinical interpretations based on steady states or equilibria do not apply. Attention, therefore, now needs to be turned to nonequilibrium processes and mechanisms and, for this reason, recent developments of nonequilibriuni thermodynamics are of interest (Katchalsky and Curran, 1965). 7 . Nonequilibrium Thermodynamics

Specifically, one can here conceive of each component in a complex system being subject to its own gradients of activity or potential. Nevertheless, changes that affect one such component part of a complex system or matrix will have repercussions on its other members. While these relationships may not be intuitively recognizable, the mathematical formulations of irreversible thermodynamics now permit them to be manipulated. Some examples are the irreversible conversion of energy to work in iiiuscle (Caplan, 1966; Wilkie and Woledge, 1968), the passage of molecules and water through biological membranes (Goldstein and Solomon, 1960; Dainty and Ginsburg, 1 9 6 4 ) , and the changes in apparent permeability of membranes in some plant tissues involving interactions with metabolic inhibitors (Glinka and Reinhold, 1964) . More specifically, there are recognizable reciprocal relationships not otherwise obvious between ion movements, electrical potentials, and water movements in model systems (Kedem, 1961; Kedem and Katchalsky, 1961). The dilemma in the further application of these ideas to cells will be recognition of the appropriate parameters to be chosen and the assigning of biological meaning to any reciprocal relations that emerge. Two characteristics of this theoretical framework show promise, however. First, there is now the evident possibility of manipulating and testing relationships between separate cellular processes even when there is no previous reason to expect that such relationships exist. Second, entropy changes are now recognized as fundamental to the operation of nonequilibrium systems, and the growing cell is here a prime example. It may well be that the further elaboration of these concepts will bridge the gap between present detailed knowledge of biochemical events and the overall, integrated operation of the cell system as a whole, including its solute accumulation. 8. Solute Uptake and Growth: T h e First Use of Cultiired Tissiies

A different kind of experimental system was needed which would give full play to the ability of the cells to grow, in order that their metabolic behavior and their salt intake might be contrasted with similar cells in a more quiescent state or with the limited endogenous capacity to grow, as previously shown in

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the thin tissue disc experiments. It was for this reason that the special techniques of tissue culture under controlled environmental conditions, using carrot or artichoke tissue or other suitable materials in liquid culture, were developed. Under aseptic conditions, a full range of nutrients could now be supplied to the cells, as well as the special stimuli, as in coconut milk or other fluids or other substances, which were able to make the cells grow at a maximum rate. From the outset of this work, the clear intention was to deal with systems which really do undergo net change as they grow, to utilize these processes in the experimental design, and not, as so many had hitherto done, to endeavor to eliminate them altogether. This approach also required that more attention should be paid to kinetic changes and to amounts absorbed with time than merely to the final states or internal concentration levels in contrast to those of the external solution. It is also significant that the then new investigations on cultured tissue began with the avowed objective of solving the mechanism of solute uptake, but they were quickly diverted by the many other unexpected gains this approach revealed. These gains now prove applicable to the original problem under investigation, as the later parts of this review will show. For example, the attempt to learn more about protein synthesis in the growing cells, utilizing the new techniques of amino acid chromatography, led to a reevaluation of amino acid metabolism in plants, all of which was incidental to studies of protein synthesis in the discs and in the cultured cells. This work also led to ideas of compartmentation in cells, metabolic turnover, and compartments in which solutes could be isolated from, or closely available to, centers of active metabolism and synthesis. Links between respiration, on the one hand, and protein synthesis and turnover, on the other (previously indicated), were shown to be more conspicuous features of growing than of quiescent cells. Furthermore, specific growth factors, other than indoleacetic acid, came to light and their role in cell growth and cell division now constitute new tools for the investigation of accumulating cells. Above all, the seeming distraction with the study of the morphogenesis and the totipotency of the growing tissue cultured cells now proves to be useful, for these systems can now be studied with the assurance that the cells in question can utilize their full genetic information in growth and, if need be, one can proceed from cells to whole plants. The subject again needs a new look to give it new degrees of freedom, however. The claim is that this will emerge as the earlier trend toward studying growing systems comes into its own, as the problem is extended from the study of electrolytes to embrace also the nonelectrolytes, and especially as it is concerned not merely with stable concentration levels or with the isothermal work done to move ions from one level to another in systems at or near equilibrium, but becomes frankly concerned with rates of uptake of salts and solutes and water in the massive changes that cells undergo as they grow rapidly.

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IV. The Growth Requirement of Cells: Their Implications for Solute Uptake

A. ENDOGENOUS CAPACITIES AND EXOGENOUS REQUIREMENTS OF CUT DISCS It was earlier regarded, and still is by some, to be an asset to utilize for experiments on the mechanism of salt absorption plant tissues or materials that are as free as possible from all the complications of growth and metabolism. To this end, investigators turned to slices cut from storage organs, i t . , storage roots (of carrot or beet), tubers (of potato or Jerusalem artichoke). No living tissue so isolated from an intact storage organ of secondary origin is really wholly free from a residual endogenous capacity to grow, however. In many cases, this may be shown by their behavior in contact with moist air when, depending upon the anatomy of the organ and of the slice so exposed, cell divisions may occur in the surface cells and may show by their intensity and distribution some relations to, or dependence upon, adjacent vascular tissue. Steward (Steward et al., 1932) recognized and exploited these properties, showing how they endowed the cells, especially the surface cells, with a renewed ability to absorb and to accumulate ions (at least for periods of 100-200 hours) which varied with the tissue and the conditions. Other authors, however, either overlooked these factors altogether or, consciously or unconsciously, allowed their effect to elapse by long exposure of the cut slices to water or dilute, nutrient-free, salt solutions. Such pretreatments, inappropriately termed “aging” the slices or discs, not onIy allowed the evanescent growth of the tissue to expire but also permitted the cells to absorb salts from the ambient medium. It is interesting, though strange, that three classically favored storage organs for use in tissue slice experiments, namely, the potato tuber, the carrot root, and the JerusaIem artichoke tuber, actually exhibit all the range of responses needed to alert investigators to the principles here involved. Moreover, isolated roots, indefinitely cultured in the form of subcultured root tips, were soon to testify to the continuing powers of growth in isolated plant parts, especially when they are subjected to the appropriate nutrient conditions (White, 1943). The potato tuber had been a favorite material in which to study wound healing (Priestly and Woffenden, 1923); it was the material that inspired Haberlandt’s concept of the cell division hormone and his dictum that it originated in the phloem of the very infrequent vascular bundles the tuber tissue contained. Nevertheless, this tissue was not brought into rapid continuous proliferating growth until this was accomplished by Steward and Caplin (1951). Meanwhile, carrot root tissue was among the first of the tissues of dicotyledonous plants to furnish cultured masses seen to be capable of indefinite and external proliferation. The tissue as so cultivated, albeit rather slowly on relatively simple media, probably owed its initial start to the proximity of cambium and

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its continued ability to grow to the somewhat nebulous events of “habituation.” By contrast, when tissue explants were removed from a site remote from the cambium (Caplin and Steward, 1949; Steward et al., 1956), relatively small carrot explants gave their best growth when they were exposed to a complete basal nutrient medium which was enriched by the nutrients and organic stimuli of coconut milk, i.e., the liquid endosperm from the coconut. Surprisingly, the potato tuber tissue could only grow rapidly in a medium that contained both the coconut milk and a synergist [first, 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) and, last, an array of other growth substances or synergists.] The artichoke tuber tissue, which responds well by growth and cell division when exposed to moist air, strangely exhibited a rapidly declining time drift even when placed in aerated water under conditions of oxygen and temperature under which potato tissue showed a long maintained and high respiratory rate, while the tissue reaccumulated salts. When this initial burst of activity, lasting for the artichoke tissue about 70 hours, expired the submerged aerated tissue slices defied all further attempts to stimulate their respiration and their salt uptake to its earlier level. This situation persisted until the tissue could be caused to grow again by the same agents (coconut milk) that first induced the carrot root and the potato tuber to proliferate. Some angiosperm storage organs (e.g., tissue of certain pome fruits) never embarked on a high and continuing rate of solute uptake (Berry and Steward, 1934), whereas others (e.g., tissue of the potato tuber) lost their inherent ability to accumulate and retain solutes after treatments (protracted storage at 1O-2OC) that also impaired both the ability of the cells to divide and to synthesize protein (Steward et al., 1943). The relevant points in the metabolism of tissue slices are as follows. Respiration and its correlated protein synthesis are at first high in the surfaces of thin discs of the potato, carrot, and artichoke; this is demonstrable at 23°C and in air-saturated water. This metabolism is very responsive to oxygen pressure, temperature, and the thickness of the discs. In thicker discs, some cells are too far from the surface to be influenced by the cutting of a slice or by contact with entering dissolved oxygen. The metabolic events stimulated by cutting are long maintained in the potato tuber while starch is hydrolyzed and protein progressively synthesized but, in artichoke or in carrot, some endogenous limitations quickly decree that the initial activity subsides in single salt solutions. Not even nutrients suffice to restore the initial level of activity in carrot or artichoke tissue unless they are also accompanied by suitable growth factors; these growth factors were, in fact, discovered by trying to ascertain the conditions that could cause very minute, aseptic, secondary phloem explants of carrot (free from all contact with, or close proximity to, the cambium) to grow again at their maximum rate.

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B. ENDOGENOUS CAPACITIES AND EXOGENOUS REQUIREMENTS O P CULTURED TISSUE EXPLANTS The main events of the induction of growth in otherwise quiescent tissue of angiosperms that are relevant to this review and which are drawn from such sources as carrot root, artichoke tuber, potato tuber, and so on, are as follows: (1) In an otherwise complete liquid nutrient medium, explants of the sec ondary phloem of carrot grow at a very great rate if they are supplemented bp 5-10% by volume of the liquid endosperm of the coconut, by an extract of immature corn grains, or by the liquid from the vesicular embryo sac of im mature Aesczllzls fruits. All these sources represent the naturaI nutrition of immature embryos. ( 2 ) The rates of growth so achieved vary somewhat with the carrot root, but from 2.5 mg of initial tissue, containing approximately 25,000 cells, they often produce 200 mg or more of tissue containing of the order of 2-3 million cells after 18-20 days of growth at 7Oocin continuous diffuse light. (3) Little of this response is induced by single substances, but it is the result, on the contrary, of interactions between several factors which produce the effects outlined below : (a) A source of reduced nitrogen is beneficial, often replaceable by casein hydrolyzate, btit sometimes requiring the use of ammonium nitrate in the solution for its full effect, (b) A part of the growth response involves a moiety of the complete growth complex which triggers off growth by any one of a series of growth factors, collectively called AF,, which characteristically interact with myoinositol to comprise what has been termed growth-promoting system I (Shantz et ul., 1967, Shantz and Steward, 1968). (c) A part of the growth response involves a moiety of the total growthpromoting complex which triggers off the growth by any one of a system of factors, collectively called AF,, which characteristically interact with indoleacetic acid (IAA) to comprise growthpromoting system I1 (Degani and Steward, 1969; Steward et al., 196%; Steward and Degani, 1969). ( d ) In varying degree, the standard tissue explants isolated from different carrot roots, even of the same variety of the same source and from the same stock, may still exhibit idiosyncracies in their responses to these growth-promoting systems; nevertheless, ull respond, in greater or lesser degree, to the three natural sources mentioned. These natural fluids owe their effectiveness, wholly or in large part, to both system I and 11, but not necessarily to the same substances acting as the components of AF, and AF, in each case. (e) The prime example of AF, (here termed AF,,,,) is an isolate from

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Aescu1u.r (Shantz and Steward, 1968) that proved to be a rhamnose-glucoseIAA (1 :1 :1) complex, while the prime example of AF, (here designated zeat.) is the substance zeatin isolated from corn ( Z e d ) by Letham (1963) and synthesized by Shaw and Wilson (1964). There are now very many synthetic analogs of AF,, all of which interact with IAA (e.g., the kinetin of Miller et dl., 1955) and many compounds synthesized by Shaw (and tested in this laboratory), but the analogs of AF,,,, that interact with inositol are still to be found in the very active fractions of coconut milk, corn, or even Aesculz/s fruits. (Some of these active preparations were demonstrably free of teatin as a type of AF,.) ( f ) Although carrot explants exhibit a large part of their ability to grow when stimulated by either system I or system 11, over and above ordinary nutrients, it is nevertheless apparent that the maximum effect is somehow produced by the balanced action of both systems and that the linkage between them operates through the reduced nitrogen compounds of casein hydrolyzate via inositol. Moreover, whole coconut milk still produces a total growth response not yet fully attributable to combinations of its known parts (Fig. 3 ) ; in fact, certain active phenolic substances (leucoanthocyanins) have not been related to the growth-promoting systems named. Nevertheless, one may designate some clones of carrot explants as those that are particularly responsive to system I; these can be detected by their responses to inositol, and these clones commonly give a high degree of response to whole coconut milk and produce many cells which also enlarge. Other clones can be identified as particularly responsive to system I1 and these can be recognized by their response to IAA. Commonly, these carrot clones respond well to IAA plus zeatin but their growth in coconut milk may be less in terms of weight because they produce more cells which habitually remain small (Degani and Steward, 1969; Steward and Degani, 1969). (g) In addition to the interactions of the component parts of the exogenous growth factor systems of coconut milk, it is clear that the whole complex also requires, and interacts with, the nutrient element iron and, after iron, one can also detect further marked effects attributable to other trace elements (manganese, zinc, copper, molybdenum) added singly or in combination with each other. These complex interactions have been studied with respect to growth and nitrogen metabolism (Neumann and Steward, 1968, with respect to iron; Steward et ul., 1968b, with respect to iron, manganese, and molybdenum; unpublished studies of K. V. N. Rao in this laboratory on nitrogen compounds and on organic acids with G. Norton as these metabolites are affected by trace elements); but it is also very clear that their effects could also be extended to the study of the other solutes, organic and inorganic, in the cultured cells. (h) Briefly, the cultured carrot tissue behaves as thou8h its cells are highly

E+CM

W C M Casein in presence

of I and II

SystemII

Basal

Basal

S stamE

Casein B+CM in presence of X and 11

B tCM

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compartmented; they contain storage pools of metabolites not readily accessible for the protein synthesis that the growth factors induce. Thus, the carbon for this synthesis is drawn most readily from the sugar of the external medium which, together with available nitrogen that may be drawn from endogenous sources (e.g., glutamine or alanine), synthesizes protein. It is also very clear that potassium is most essential for all these activities and in varying degrees iron and the other essential trace elements play contributory but regulatory roles. ( i ) The metabolic behavior of the cultured carrot tissue only becomes intelligible insofar as the protein synthesized, or at least a large metabolically active part of it, is regarded as being in a state of turnover, releasing part of its carbon to respiration as the immediate products of breakdown are reworked to form the nitrogen-rich storage compounds (especially glutamine) that are secreted into aqueous inclusions in the cell. In fact, the cultured tissue may be caused to focus its metabolism about alanine as the principle nitrogenous solute (as in the coconut milk-supplemented medium) or about glutaniine (as in the medium supplemented with the partial system 11). Furthermore, it has been seen that the role of the coconut milk complex is not only to stimulate the protein synthesis of the now growing cells de novo but also to accentuate the pace of its turnover. ( j ) Complex as the metabolism and maximum growth of carrot tissue may seem to be, that of the potato tuber tissue presents the further complications that its maximum proliferated growth requires, in addition to the entire complex of coconut milk, one of the many now known synergists of which 2,4-D or NAA are the prime examples (see Steward, 1968, pp. 182-191). (k) This list of salient points derived from the aseptic culture of cells and tissues would be incomplete without the following. As long as cells remain attached to the original tissue explant, their behavior is severely restricted and circumscribed. If, however, they are freed and remain suspended in the same medium, they may respond to the growth-promoting complex in a very different way, for they may now grow in an orderly fashion similar to zygotes. Whether or not embryonic growth, rather than random proliferation, is to occur may be a response, not merely to the simultaneous, i.e., synergistic, combinations

FIG. 3. Diagram attributing the overall growth of carrot explants to the basal medium, casein hydrolyzate, the coconut milk complex, and its various component parts. The numbered scale represents the typical growth response in milligrams fresh weight per explant after 18 days. B, Basal medium; CM, coconut milk (10% by volume); CH, casein hydrolyzate (200 ppm); AF,, cell division factor from Aesculus (1 ppm) linked to inositol ( 2 5 p p m ) ; AF,,, cell division factor linked to IAA, e.g., zeatin (0.1 ppm); IAA, indolaceatic acid (0.5 ppm). From Degani and Steward (1969).

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of the growth factors, but also to certain prescribed time sequences in which they should be individually applied (Steward et al., 1967). This has been termed the “sequential effect,” and when all these factors are appreciated a now surprising number of angiosperm cells may be grown rapidly and may be developed into plants (Steward et al., 1969). (1) It is relevant, however, that when free cells grow in this way they seem to have lost entirely their visible characteristics as mature cells of the plant body, for they now resemble each other much more than the cells of the ddferent tissues and plants of their origin (Steward et al., 1966, pp. 125-128). (m) It is, however, of some interest that the technique that made these many advances possible was developed to furnish an alternative experimental systcni to the aerated tissue slices of storage organs for the study of salt accumulation. This was done in the implicit belief that it was necessary to control and investigate inore effectively the role of growth, metabolism, and protein synthesis of the cells and to relate their salt absorption thereto. The fact that the tissue system in question has led so fast and far into other channels of biochemistry and inorphogenesis makes a belated, but nevertheless illuminating, reconsideration of the concomitant effects of these treatments on the solutes of the cells all the more rewarding. The principle features of the tubes in which small numbers (1 to 3) of tissue explants are commonly grown and also of the flasks in which up to 50 to 100 aseptic tissue explants, or the free cells derived from them, may be grown and investigated are described in an article by Steward (1963) treating the general topic. The purpose of this extensive excursion into current knowledge of the techniques and possibilities of cell and tissue culture will become clear. It is briefly that one can now create, under culture conditions and for relatively large amounts of growing material, the several states that intervene between cell division, as in meristematic cells, and full enlargement at maturity, as in differentiated cells, and compare these with the quiescent cells that may exist in mature organs. The point of view to be developed is outlined as follows. Any enlightened outlook on the solute relations of cells should be able to comprehend what they do severally in each of their ontogenetic stages; it should also permit one to visualize their responses in sitz~to the factors that influence them during their growth. Cells in some parts of the plant body make inevitable demands in order to store solutes, while cells in other parts release them to support the synthesis and growth elsewhere. The experiments to be described (Section V) will shed new light on the ways in which these solute movements may occur in response to (1) the prior nutrition of cells and ( 2 ) their ability to grow.

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V. Absorption Studies with Cultured Cells and Tissue Explants A. THERESPONSES OF DIFFERENT CARROTCLONESTO DIFFERENT STIMULI The first survey made use of the idiosyncracies of different clones of otherwise standard (2.5-mg) carrot phloem explants grown for 18 days under standard conditions in rotated culture tubes, in liquid media at 70*F, and in constant light (Caplin and Steward, 1949; Steward and Shantz, 1956). Individual clones may respond somewhat differently to the various media and exogenous stimuli and so provide a wide range of growth responses, but what may seem to be a difficulty in this sort of work was here turned to useful ends. In order to present many data concisely and visually, the polygonal diagrams of Fig. 4 are adopted. In these diagrams, each clone of explants is represented by one of many axes which radiate from a common origin and diverge at angles which subdivide 360' equally in the manner previously adopted by Steward and Degani (1969). The numbers 1 to 19 were assigned to the clones in accordance with their descending order of growth in fresh weight in response to the basal medium; these data [Fig. 4a(i)] reflect the endogenous capacity to grow of the various clones which received only nutrients but no exogenous stimuli. The growth as affected by different stimuli, e.g., inositol, IAA, and wholc coconut milk, is shown in Fig. 4a(i)-(iv). In these diagrams, the order of the clones is retained throughout (as indeed in the rest of the figure) and only the scale for growth in coconut milk [Fig. 4a(iv)] is changed (reduced to 1/6) to accommodate the great growth so stimulated. Following the same general pattern, the content of potassium, sodium, and their sum are shown in Fig. 4b(i)(iv). The concentrations of these constituents are shown in Fig. 4c(i)-(iv), all drawn to the same scale. If all the growth and all the treatments affected all the parameters proportionally to the endogenous responses to basal medium, all these figures would have the shape of Fig. 4a(i). By scanning the series 4a(i)-(iv), one sees the level of responses of different clones to the treatments and that they affected the clones differentially; by scanning the series from 4a-c, one also sees how compatible the responses were with respect to the different parameters (e.g., fresh weight, potassium and sodium content, and concentration). The general relationship is that the tissue explants acquired salts as they grew, cf. Fig. 4b(i) and (iv). During these events, however, the concentrations of ions were very far from constant, the general effect being that the tissue explants that grew only in limited amount in response to their endogenous capacity [Fig. 4c(i)], attained much higher concentrations of ions (I( and Na) than the explants that were greatly stimulated by coconut milk [Fig. 4c(iv) 1. The shapes of the polygonal diagrams, therefore, show that the different growth

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stimuli (ii)-(iv) act selectively upon the relative intake and retention of water and of ions by the different clones. The broad picture is that the explants that grew to the maximum extent and that produced many new cells [Fig. 4a(iv), GROWTH, FRESH WT.

(mg /explan?)

._._.-.-.-._. FRESH

-

SALT CONTENT

SALT CONCENTRATION

( p m o l e s /explant)

( p n o l e s / grn fresh wt.)

WT.

TOTAL CATION ( K + N d

........... "

POTASSIUM ( K ) SODIUM ( N a )

FIG. 4 . Salt content and growth of carrot explants. Idiosyncracies of 19 clones. Abbreviations as in legend for Fig. 3, for other descriptions see text.

b(iv), and c(iv)] absorbed relatively more water than salts, whereas by comparison, the explants with limited growth on the basal medium [Fig. 4a(i), b(i), and c(i)] absorbed relatively more salts than water. The presumption here is (later confirmed, cf. Table IV) that the new active cells built their

CELLS, SOLUTES, AND GROWTH

319

osmotic value largely upon organic solutes [cf. Fig. 4c(iv) 1, whereas the explants with fewer and older cells [Fig. 4c(i)] built their osmotic value much more upon inorganic salts. This idea will recur, but the data already point to the view that the first content of vacuoles in rapidly growing cells is largely organic (e.g., sugars) and that it is later replaced by salts. Within this general framework, however, the different stimuli (inositol, IAA, coconut milk) affect not only the number and size of cells, for they also affect the metabolism of the several clones in ways that also determine the mutual relations between potassium, sodium, water and, no doubt, other solutes. The shapes of the diagrams in Fig. 4a(ii)-(iv) show the responses of the explants by water uptake and retention; by contrast, the diagrams in Fig. 4b(ii)-(iv) show their responses in terms of salt content, and Fig. 4c(ii)-(iv) compare, in terms of concentration, the responses to salts and to water. It is, therefore, very hard to visualize these discriminatory effects as anything less than the properties of whole cells. The general lesson to be drawn from these diagrams is the rather obvious one that the factors inherent in the de n o w uptake of solutes by cells can only be seen and interpreted concomitantly with their growth. Thus, many alternative studies, made often with large carrot discs (even from the xylem) in single salt solutions, deprived of nutrients, washed free of their endogenous growth capacity (e.g., the technique inappropriately called “ageing”), unstimulated by exogenous growth factors, and allowed by prior treatment to become saturated with salts, cannot possibly show the features and the problems revealed in Fig. 4. While some would justify such procedures by their simplicity it would seem more appropriate here to emphasize their biological unreality. The data of Fig. 4 bear the obvious interpretation that the tissue explants with maximum growth, as in the coconut milk medium, had a strong preference for potassium over sodium (K/Na ratios of the order of S ) , whereas the explants with minimal growth, as in basal medium, had a weaker preference for potassium and a stronger preference for sodium (K/Na ratios of the order of 2). Table I contains a representative set of data all obtained from one clone in one experiment; this usefully supplements Fig. 4. The interpretation with which the data of Table I are compatible is as follows. Carrot explants consistently absorb potassium to a minimum level of about 100 pmoles per gram fresh weight despite a wide range in the external conditions. This amount may, or may not, be accompanied by chloride but it represents the potassium the cells need to maintain them. Over and above this minimal amount of potassium, inorganic cations (K and N a ) may be taken in completely [Table I, (a)] or partially [Table I, (b) and ( c ) ] balanced with chloride; this second moiety of salt content in the cells is a flexible function

320

F. C . STEWARD A N D R. L. MOTT

of the growth of the cells and of the coinposition of the external medium, and it makes an appropriate contribution to the osmotic value of the cells. Cells that no longer grow [Table I, (a)] can rely to the maximum extent upon salts (KCI and NaCl) for the increment of their osmotic value above the medium (about 7 atm), but cells in rapid growth [Table I, ( b ) ] utilize salts TABLE I

THESALT CONTENT(K, Na,

C1) OF CARROTEXPLANTSSUBJECTED TO DIFFERENT

AND

GROWTH STIMULIAND

A

RANGEOF EXTEKNAL SAL'r CONCENTKATIONS ~~

Analysis of the tissue Analysis of the medium

Treatment

Ion concentrations ( pmoles/ml) K Na C1

Osmotic value (milliosmole)

Osmotic value due Ion concentrations to salts (!Jmoles/gm fresh wt.1 (milliosmole K Na CI calc.)a

~

( a ) CaCI:, ( 0 5

mtnole/liter) with KCI f NaCl

( b ) Basal medium

+ coconut milk with KC1 +

1 10

1 1

3

5

122

40

164

299

12

24

178

12

186

344

22 21

43 43

209

8

226

406

100

117

217

398

116

11

15

23 1

282 243

20

1

10

10

8

6 6 16

? 29

105 143

150

5

11

l?

124

98

35

21

3

1 21

31

60

21

130 92

11

13

75 113 113

87

3

84 110

28

8

NaCl (c) Basal medium 2 with KCl 22 NaCl 12

+

68

215 258 293

a Where inorganic cations and anions balance, e.g., as at ( a ) , this figure was calculated from the total salt (KCl NaCl) ; where these ions did not balance, the estimate was made from the total cation ( K Na), thus assuming an accompanying organic anion to complete the balance ( 1 milliosmole = 0.0224 atm) .

+

+

to a lesser degree for this purpose (approximately 3 atm), even when the salt content of the medium is high. To obtain carrot explants that had adjusted their salt content to a wide range of external potassium, sodium, and chloride concentrations under circumstances which, as nearly as possible, eliminated all growth by cell division, they were merely exposed to a dilute CaCl, solution (0.5 inmole/liter) with such added KCl and NaCl as would produce the range of potassium-sodium conditions seen in Table I, (a). Under these circumstances, the tissue behaved very differently from the rapidly growing tissue [cf. Table I, (a) and (b), also Fig. 4c (i) and

321

CELLS, SOLUTES, AND GROWTH

+

Na) with (iv) ], in that it achieved a virtual balance of inorganic cation (K anion (Cl). Even so, the tissue only responded to a 20-fold range of extern'd 1K) by a 2-fold change in its internal concentration and this was, in fact, the response of cells which maintained, in terms of inorganic salts, their normal increment of osmotic value (about 300 milliosmole or approximately 7 atm) above the external solution. Thus, these nongrowing cells had built up, preferentially and irrespective of the external [K], approximately 100 pmoles per grain fresh weight of potassium in their cells. With added external salt, however, such cells could also take in more salt indiscriminately in the form of potassium with chloride, or sodium with chloride according to the conditions and presumably at the expense of other solutes (organic) in the cells. At the other extreme, the data of Table I, (b) show the responses of tissue that had grown rapidly under the stimulus of coconut milk and in contact with and [CI]. The outstanding feature is that the a similar range of [K], "a], [K N a ] vastly exceeded [Cl] (order of 1 5 pnioles per gram fresh weight) and was, therefore, in this case, balanced by organic anions. Thus, the growing cells of Table I, (b) again absorbed potassium to about 100 p o l e s per gram fresh weight, whatever the external solution to which they were subjected. In addition, and to the extent that some cells had departed from or had not entcred the actively dividing state they, like those of Table I, (a), absorbed some sodium with chloride, but to a very much smaller extent. Thus, the concentration figures and the concentration ratios in Table I, (a) and (b) should not conceal the fact that the cells absorbed potassium, unaccompanied by anion (Cl), to a certain fixed content, compatible with and determined by their growth; over and above this they may take in either potassiuiii with chloride, OY sodium with chloride, OY extra potassium balanced by organic anions, although even here there was a preference for K ion in the more active explants. The tissue explants exposed only to the basal medium [Table I ( c ) ] , depcndent only upon their endogenous growth factors, but again exposed to a similar range of potassium-sodium concentrations, displayed behavior intermediate between the results shown in Table I(a) and (b) but in a manner generally compatible with this interpretation and predictable from Fig. 4c(i). Therefore, the conclusion is that the coconut milk stimulated the tissue explants to accumulate potassium preferentially, whereas those in the basal incNa) of dium, which contained more large cells, accumulated cation (K which the sodium may be a more-or-less conspicuous part (Table I ) . Only when anabolism and cell division were reduced to a minimum was there a substantial and balanced absorption of potassium and sodium along with chloride. coiiiparisons have been drawn in the literature (see Steward and Sutcliffe, 1759; pp. 348, 385, 396 and references there cited) between the ability of

+

+

322

F. C. STEWARD AND R . L. MOTT

potato, carrot, and beet discs to absorb potassium in preference to sodium (the K/Na ratios being in the order potato > carrot beet). These ratios have been interpreted as features of tissue drawn from the organ in question. The carrot explants exposed to basal medium (data of Fig. 4) clearly fall within the accepted range for carrot tissue, whereas the data from the tissue made to grow by coconut milk as clearly do not. Therefore, the K/Na absorption ratios of the discs are more likely to reflect the ability of the cells to support growth than any inherent distinction between the plant organs in question; this is especially so because the ability of the cells to grow (visibly as in moist air and also by protein synthesis) follows the same order.

>

B. THETIMECOURSEOF GROWTH,METABOLISM, AND ABSORPTION IN CARROT EXPLANTS:THETYPICAL BEHAVIOR The standard carrot explants (2.5-mg) for these experiments were removed from the storage organ at a distance of 2-3 mm from the cambium. These explants, therefore, contained cells which had differentiated into storage phloem parenchyma, which had acquired a content and concentration of solutes typical of their storage role, and which would not normally divide again in the intact organ. AIthough these cells had some residual capacity to grow, which is evident by internal cell divisions that occur to some extent in moist air (as in wound healing), and which is evident in varying degrees when cultured in a basal medium [Fig. 4a(i) 1, rapid growth depended on a supply of exogenous stimuli, as in coconut milk. As a basis for later interpretations, the onset of growth with time, as shown by different criteria, is illustrated by typical data in Fig. 5. It is apparent that the composition of the initial explants was in sharp contrast to that of the cells that had encountered a recrudescence of growth, as at 6-8 days in culture. It is now known that the principal changes that occurred during the induction of this growth were as follows. In situ in the root, the tissue had a modest salt (K Na) concentration (order of 50-70 pmoles per gram fresh weight), in which sodium was relatively conspicuous (K/Na ratio of the order of 4) and this cation content was only partially balanced by chloride (order of 20%). The osmotic value of this tissue far exceeded that of its measured salt content so that the quiescent storage tissue, consistent with its function, was rich in organic solutes. (Subject to the manipulations of removing explants from the root and rinsing them, they may first lose some salts so that the true absorption by the experimental explants really starts from an even lower level, as at 12 hours, cf. Fig. 7.) On the other hand, the very rapidly growing tissue in culture had acquired much more potassium (order of 120-140 pnoles per gram fresh weight), had become preferentially richer in potassium with respect to sodium (K/Na ratio of the order of 4-10), and it was very low in chloride ([Cl]/[K] "a] of the order of

+

+

323

CELLS, SOLUTES, AND GROWTH

0.20). Thus, the events of growth induction reversed, under the conditions

imposed by the nutrient medium and its growth regulators, the sequence of changes in the maturation of the quiescent cells in the root. Figure 5 shows the time relations of various responses which accompany growth with time. ( 0 1 G r o w t h : C e l l s ithousonds/explont) F r e s h wecght Img/e*plant)

-

( b l Growth Cell s i i e ( u q / c e I l r

o--C

m

0040

-2 0

~ _ _ ~ l

4

12

28

Time (doysl i c ) Growth' C e l l s / u g f r e s h weiqht

1irne (ooysi ( d l P r o t e l n c o n l e n t : P r o t e i n N i p q N,/erplanti F r e s h weight i m g / e r p l a n t i

-

( e l ProlPin c o n l e n l ' Protean N (mq N/qm f r e s h u

o---

' f ) T y p i c a l doily increments of Diotein and K/exolont Z protein N increment ( p g N/doy/explont) 2 K content iiicrerneiit (pmoles/doy/explontl

-

T i m e (days) I

Y

T i m e (days)

( g ) Typic01 chonges 01 K concentrotion, f r e s h weight, a n d cell n u m b e r with t i m e 2001

-

P

-

3

Time C

m

-

Cells altoin a potassium concentration in new dividing cells svpermposed upon any uptake by nondividing cells ,osmot~c volue here exceeds thot of totol salt concentration

-

Kand H200bsorbed port possu, CI uptake 1s minimal hul the osmolic value agoin requires storage of nonelectrolytes.

-

2

A s growth suhstdes. the balanced increment of water. salts. and organic solutes 1s chonged 0s cells return lo storage of nonelectrolytes 0s they mature In the nulrient medium

.-.

FIG. 5 . Time courses in the growth and composition of cultured carrot phloem explants at 23°C in diffuse light. a, Growth, cells (thousands) and fresh weight -0 per explant; b, growth, cell size (pg/cell); c, growth, cells per pg fresh (mg) 0 and fresh weight (mg) weight; d, protein content, protein nitrogen ( p g N ) 0-0 0-0 per explant; e, protein content, protein nitrogen (mg N per gm fresh weight); f, typical daily increments of protein ( N g N ) and K (NmoIes) per day per explant; g, typical changes of K concentration, fresh weight, and cell number with time.

324

F . C . STEWARD A N D R. L. MOT"

The figures show that the onset of growth by fresh weight occurred after a short lag period of about 4 days (Fig. 5a) and, thereafter, it followed a typical sigmoid time curve. The onset of cell division starts prior to the major uptake of total water since preformed cells can divide and, in fact, the rise of cells in number was faster than the uptake of water (Fig. 5c) because after 2 days cells divide faster than they enlarge and their average size falls to a minimum at or about 8-10 days; thereafter, cell enlargement became more prominent so that their average size increased (Fig. 5b). Meanwhile, the growth was supported by protein synthesis at the expense of existing soluble nitrogen which was, however, replaced from the external solution. In fact, the time course of protein-nitrogen per explant is a reflection of the growth in terms of cell number (cf. Fig. 5a and d ) . In the phase of very rapid cell division, i.e., up to about 8 days, protein increased in the explants faster than water was absorbed (this is a consequence of the formation of many small, less vacuolatcd cells); this is shown by the comparison drawn in Fig. 5e. At any point along these time curves, the first derivative indicates the rates of change, but of particular interest is the change in the rate of increase of the total protein with time (Fig. 5f), especially as this is related to the similar rate of increase of potassium content. Thus, while cells are multiplying and growing fastest, they also in, their potassium; these crease their protein content and, pdri ~ ~ E S E S Z Lincrease events reached their peak at or about 8-12 days. The time relations of potassium and water uptake and of cell growth and cell division may be represented as in the diagram in Fig. 5g. Thus, the growth-stimulating effects of coconut milk can be compared against the endogenous growth capacity, as expressed in the basal medium; variants resulting from the differences between clones can also be recognized, as indicated in Fig. 4 ; and the salt relations can be interpreted on a cellular as well as a fresh weight basis. The behavior of the tissue explants in simple salt solutions devoid of all other nutrients reveals the salt accuiiiulation responses under conditions that restrict growth to a minimum. Figure 6 shows the behavior of three contrasted clones of carrot explants (cf. Fig. ba, b, and c) ; it also contrasts the behavior of the fully growing explants in basal medium plus coconut milk (B CM) with controls in a basal medium (B) only [cf. Fig. 6a(i) and (ii) ; b(i) and (ii) 1. The comparison between the growing and relatively nongrowing explants is made between media which compare the basal medium plus coconut milk [Fig. 6c(ii)] with an adjusted basal medium having the same total potassium and chloride content [Fig. 6c(i) 1. Figure 6a(i) and b(i) contrasts clones with different potassium-sodium response in the basal medium; the former acquired a lower total cation content (K Na I98 p o l e s per gram fresh weight) and potassium was very promnent (K/Na ratio at 18 days == 2.3), while the latter acquired more cation

+

+

325

CELLS, SOLUTES, AND GROWTH

+

(K N a = 222 p o l e s per grain fresh weight) and sodium was more conspicuous (K/Na ratio = 1.1). The clone of Fig. 6a was similar to clone no. 8 of Fig. 4 ; the clone of Fig. 6b was similar to clone no. 16 of Fig. 4. Growth on the basal medium alone was virtually confined to cell enlargement but during Responses in basal medium

(B) 160

Responses in basal medium+ coconut milk, 10% (B+CM)

z

ccn

Days

Days

Days

Note. Bosal medium ot clil adjusted t o salt Content o f B + C M

-K (,urnoles@rn ,,....,..,..#....

fresh wt.) Na (prnoies/gm fresh wt.)

0s (It

- .-. -----

-I

CIIII

Days

C l (pnoles/grn fresh wt.) Growth in fresh wt. (mg/explant)

FIG. 6. Time courses of salt uptake in relation to growth. Behavior of three selected clones (a, b, and c ) . The maximum observed deviation from the mean of at least two replicate samples are shown.

this growth the tissue also displayed properties that may be related to its growth when cell division was involved. The clone with a high storage capacity for sodium (Fig. 6b) exhibited a slower growth [Fig. 6b(ii)] and the longest lag period; the tissue with a higher capacity for storage of potassium (Fig. 6a) showed a more rapid growth and a shorter lag period [Fig. 6a (ii) 1. Similarly,

326

F. C . STEWARD A N D R. L. M O T T

the tissue with a longer lag period [Fig. 6b (ii)] contrasted with that of Fig. ba(ii) in its slower buildup of potassium content. Thus, in some as yet obscure fashion the ability to respond to the complete growth factors of coconut milk is related to the avidity with which the tissue acquires potassium and sodium; a high potentiality for potassium uptake in the basal medium can usually be associated with a large response by growth in coconut milk [cf. clones no. 13, 14, and 15, of Fig. 4c(i) and a(iv)]. Conversely, a rich preference for sodium [cf. clones no. 9, 10, 16, 17, and 18 in Fig. 4c(i)] was associated with relatively poor growth in response to coconut milk [cf, Fig. 4a(iv)]. This is yet another indication that the salt relations of these tissues should be interpreted in terms of their ability to grow, and vice versa. The implication is that a rich content of potassium is associated with the greatest ability to grow by cell division. This alone will not induce cell division, however; this is shown in the comparison of Fig. 6c(i) and (ii). Figure bc(i) shows the potassium, sodium, and chloride concentrations of explants adjusted to a basal medium with the same high potassium content as the medium enriched with coconut milk. Although these explants did not grow appreciably, their cells quickly acquired a high potassium concentration. In their counterparts stimulated to grow [ Fig. 6c( ii) ], cells multiplied rapidly, remained small up to about 8 days but, nevertheless, built up their potassium content to about the same level ([K] order of 120 p o l e s per grain fresh weight). Thus, a high potassium content is contributory to, but is not the cause of, rapid growth by cell division and, conversely, when cells do divide rapidly they can acquire a high potassium content even from low external pofussizm concentrations. By contrast, in the later phases when cell enlargement overtakes division, the different clones may either maintain their potassium concentration, as in Fig. 6a(ii), or sustain their water uptake by greater use of other solutes, e.g., nonelectrolytes I Fig. bc(ii) 1. All these data are compatible with the more general treatment shown in Fig. 5g. C. THEBEHAVIORO F CARROTEXPLANTSI N SIMPLESALT SOLIJTIONS

Figures 4-6 summarize the behavior of the tissue in its most actively growing condition (as in B CM) or in its very slowly growing condition (as in B). To investigate tissue under conditions that restrict its growth to a minimum, the explants were observed in contact with a dilute CaC12 solution (0.50 mniole: liter) which, as in previous practice (Steward and Millar, 1954), was deemed adequate to preserve the viability of the cells in the absence of other nutrients. The absorption of other neutral salts (halides of the alkali metals) was then measured. These experiments permit comparisons to be drawn with other work on carrot and similar tissue in which the role of growth was minimal, i.e., Davies and Wilkins (1953), Van Steveninck (1962), Laties et ul. (1964),

+

327

CELLS, SOLUTES, AND GROWTH

concerning ion fluxes; Atkinson et al. (1966), MacDonald et a/. (1966), Reed and Kolattukudy (1966), Morohashi et al. (1967), concerning respiration and metabolism; Thimann et d. (1960), Laties (1964), Splittstoesser and Beevers (1964), Osmond and Laties (1969), concerning organic solutes, and Pitnian (1963, 1964), Poole and Poel (1965), Van Steveninck (1966) concerning the effects of various external conditions. Comparisons may also be made with ear-

a

C

Explants Day 0

Medium

O K

@No

U C l

d

b

5 Days

20Days

T i m e (days) l

l

l

~

Sodium (Na) ~

.

~

~

~

~

~

-

~

Potassium

(K)

-#-*-a

Chloride (CI)

FIG 7. The absorption of salts by carrot explants from non-nutritional solutions. Time course of the uptake. a, Behavior of endogenous K and N a in tissue in contact with dilute CaCI2 solution (0.5 mmole/liter). b, Behavior of K and N a in tissue in contact with dilute (0.5 mmoles/liter) CaC1, solution with added KCI and NaCl each at 1 mmole/litrr. c, Cation and anion balances in the tissue at day 0 and in the initial medium. d, Cation and anion balances in the tissue after 5 and 20 days of the treatment as in b.

lier studies on tissue discs in aerated solutions in which, for a brief period up to 72-100 hours, the role of growth was significant (for summary see Steward and Sutcliffe, 1959, pp. 335-363). However, under aseptic conditions experiments may be long continued (up to 2 0 days) and, after having investigated the role of neutral salts, the subsequent effects of progressively restoring the full nutrient system can be explored. Figure 7a shows the behavior of the potassium and sodium already present in the cells when they are exposed to a dilute CaC1, solution. After a very brief loss of ions and a speedy reabsorption I analogous to similar occurrence in blotted discs (Steward, 1932)], the potassium and sodium concentration in the

328

F. C . STEWARD AND R. L. MOTT

tissue remained steady throughout 20 days, affected only by the minimal water uptake that occurred under these conditions (percent fresh weight gain of the order of 10%). Figure 76 describes what occurred when the external soIution was enriched with 1 mmole/liter each of KC1 and NaC1. Under these conditions, there was no significant increase of cells in number but they increased in size from 0.065 to 0.073 pg per cell. Thus, the endogenous ability of these cells to grow by division was strictly confined by lack of nutrients and lack of stimuli, but cell expansion did occur and, with it, absorption of salts. [Parenthetically it should be noted that these small explants had about the same specific surface as in the work on thin discs already referred to and, being aerated to equilibrium with air, they shoud also have synthesized some protein in response to potassium; for summary see Steward and Sutcliffe (1959).] The carrot explants, similar to the discs, absorbed potassium and chloride simultaneously and reached a steady level after 5 days. Although the simultaneous uptake of sodium, along with chloride, was lower, this continued progressively for 20 days and throughout this period a balance between [K Na] and [Cl] was maintained (cf. Fig. 7d). Two relevant points should be stressed. First, the data again show simultaneous uptake of anion and cation, in stoichiometrically equivalent amounts, to relatively high accumulation ratios (K order of 150). Second, although the balanced (K Na = Cl) uptake of carrot explants (Fig. 7d) resembles that of carrot and other discs in single salt solutions it, nevertheIess, contrasts sharply with the work on growing tissue explants in which potassium is absorbed far in excess of sodium and virtually unaccompanied by chloride [cf. Fig. 7b and d with Fig. 6c(ii) 1. Figure 8 shows the behavior of aseptic carrot explants maintained viable in dilute CaC1, after 8 and 19 days of contact with 1 mmole/liter of the chlorides of each of the alkali metals. Although the chloride determinations were made, they are omitted from the figures since, after the pattern of Fig. 7d, the balance between halide and total alkali metal cations was almost exact within a few percent and was limited only by the accuracy of all the techniques. The conclusions from Fig. 8a-f are:

+

+

(1) The relative order of uptake from solutions of a common anion was K > N a > Rb > Cs > Li; this sequence was, however, not unexpected [Stiles and Kidd (1919); Hoagland et ul. (1928); Robertson and Wilkins (194813); Collander (1941); Epstein and Hagen (1952); Menzel and Heald (1955)]. ( 2 ) When sodium was absorbed, the initial potassium was retained (Fig. 8c) ; when potassium, rubidium, or cesium was absorbed, the initial sodium, although low, declined perceptibly (Pig. sb, d, and e ) ; lithium, could not, however, displace sodium (Fig. 8 f ) .

327

CELLS, SOLUTES, AND GROWTH

The data of Fig. 8b and c show that the uptake of alkali metal and halide in the expanding nondividing cells of these explants could occur almost equally whether sodium or potassium was the external solute. This result is in sharp contrast to the role of these cations in the growing, dividing explants, for in these circumstances [cf. Fig. 8b and c with Table I, (b)], only potassium would suffice. Even in Fig. 8 there is a suggestion that the sodium and potassium are CaCI,

C a CI, + R b CI

O'? *a

0

Day

8 Day

19

O

L OL L d8 l

Day

e

b

Day

Day

CaCI,+CsCI

gii 0

Day

8 Day

19 Day

CaCI,+NaCI

C

CaCI,+LiCI

f c

0.5

'

0

Day No

8 Day

Rb

FIG. 8 . The absorption of salts with a common anion by carrot explants from nonnutrient solutions. ( a ) The behavior of endogenous potassium ( K ) and sodium (Na) in tissue in contact with dilute CaClz solution (0.5 mmoles/liter). (b-f) The behavior of the various ions in tissue in contact with dilute CaCI2 solution plus the salt supplied at 1 mmole/liter. The analyses represent explants at day 0 and after 8 and 19 days under the designated treatments (inorganic cations and chlorides invariably balance).

alternatives as osmotically active solutes but that potassium, which in contrast to sodium is retained, performs some essential function for the growth by extension which occurred and which, therefore, allowed the cells to absorb sodium or other cations. The conclusion is that there are two distinct aspects of the absorbing system and the absorption process. The one concerns cells that can grow and divide, with all that this implies. In this system, potassium plays an essential role as well as being an absorbable cation. (This is comparable to what was termed, in 1959, phase I of ion uptake.) The other can operate in cells that need only to enlarge. In this system, potassium and sodium may be osmotically active

330

F. C. STEWARD A N D R. L. M O T T

alternatives although here also there is room for some specific role of potassium. (This is comparable to phase I1 in the earlier terminology.) The key point, however, is that in phase I1 the osmotic role is not confined to the salts of sodium, potassium, or even other alkali metals; in fact, in normal ontogeny it is attributable first and preferentially to the organic solutes (cf. Figs. 5 and 6). The useful role of other alkali halides as osmotically active solutes is minimal TABLE I1 EFFECTS OF VARIOUSSALTA N D NUTRIENT TREATMENTS ON FRESHW E I G H T OF CARROT E X P L A N T S ~ Fresh weigh@ (mg/explant ) Treatment

8 days

CaC12(0.5 mmole/liter) ( b ) CaCI2(0.5 mmole/liter) KCI( 1.0 mniole/liter) NaCl (1.0 mmole/liter) ( c ) CaCI2(0.5 mmale/liter) KCI( 1.0 mrnole/liter) NaCl(1.0 mmole/liter) sucrose ( 2 % ) KCI( 1.0 mmole/liter) ( d ) Ca(N03)2(0.5 mmole/liter) NaCI( 1.0 mmole/liter) sucrose (2R>) ( e ) Basal medium ( f ) Basal medium supplemented with 10% coconut milk ( B CM) (a)

+ +

19 days

2.52

2.71

2.6s

2.74

3.20

3.61

3.23

3.97

4.16

5.53

9.56

58.30

+

+

+

+

+ + +

The treatments as described also apply to Fig. 9. Data at To = 2.43 mg per explant. Although not recorded in detail, the disparity between the highest and lowest estimate of fresh weight per explant, determined on replicate batches of six, represented 2-3% of their mean weight. a

t~

(Fig. 8d, e, and f ) ; to the limited extent that they resemble potassium and may even interfere with it, whether in a basal solution or in a complete solution (B CM), they may even reduce slightly the growth and absorption responses that would otherwise occur.

+

D. THE BEHAVIOR OF POTASSIUM AND SODIUMHALIDES IN TISSUE EXPOSED TO THE PROGRESSIVELY RECONSTITUTED NUTRIENT SYSTEM The data of Table I and Fig. 8 sharply contrast the absorption behavior of the alkali halides in the rapidly growing and relatively nongrowing explants as regulated by the nature of the external solution. The data of Fig. 9 and of Table I1 show how the absorption of ions was affected as the external solutions were progressively replenished by essential nutrients and growth stimuli. The device adopted was to show the effects after 8 and 19 days; these times were selected in light of the time course experiments.

331

CELLS, SOLUTES, AND GROWTH

Figure 9a,b,e, and f recapitulates behavior now to be expected (cf. Figs. 6 and 7 ) , namely: ( 1 ) In Fig. 9a, the tissue made no growth and merely retained its existing

ions.

..-

&

.+F -

zoo]

a

Day 0

i

8 Days

19Days

Day 0

%Days

19Days

i

3 200-1

n Day 0

i

8 Days

19Days

i

i L

P

2:;h Day0

8Days

19Days

~

0 Q)

-

i

E

-

Day 0

8 Days

LZl Sodium (No)

19Days

0 Potassium

O

i (K)

Day 0

8 Days

0 Chloride

19Days (CI)

FIG. 9. The absorption of salts from progressively enriched solutions. Analysis of tissue explants at day 0 and after 8 and 19 days of designated treatments. a, The behavior of endogenous K, Na, and C1 of tissue in contact with dilute CaCI, solution (0.5 mmole/ liter). b-f, The behavior of K, N a and C1: b, in tissue exposed to dilute CaCI2 solution (0.5 mmoles/liter) plus salts (KCI and NaCl each at 1 mmole/liter); c, in tissue exposed to dilute CaClz solution (0.5 mmole/liter) plus salts, plus sucrose ( 2 % ) ; d, in tissue exposed to nitrate as dilute C a ( N 0 3 ) , (0.5 mrnole/liter) plus salts, plus sucrose (2%); e, in tissue exposed to the basal medium; and f, in tissue exposed to the basal medium plus coconut milk (10%).

( 2 ) In Fig. 9f, the tissue made large amounts of growth (Table 11) and its concentrations of ions were heavily directed to potassium and their halide did not balance potassium and sodium. ( 3 ) Under the treatments of Fig. 9e, the tissue grew to the limit of its endogenous capacity, largely by cell enlargement (Table 11), there was a more

332

F. C. STEWARD A N D R. L. MOTT

nearly complete balance of cation (K a much more conspicuous component.

+ Na)

by anion (Cl), and sodium was

Whereas Fig. 4 shows the role of certain parts (IAA and inositol) of the growth-promoting systems added to an otherwise complete nutrient medium, Fig. 9 shows the effect of supplying the tissue progressively with its needed nutrients and stimuli to absorb the salts furnished as in Fig. 9b. In Fig. 9c, the supplement was sucrose; in Fig. gd, the supplement was sucrose plus nitrate; in Fig. 9e, the supplement included all the basal nutrients and in Fig. gd, all the nutrients and growth stimuli. The results show: (1) Supplied with salts alone the tissue used its existing endogenous capacities to absorb salts with cation and anion in balance (Fig. 9b). (2) Supplied with the same salts plus suger, there was more growth in fresh weight, as cell enlargement was mediated by potassium and sugar, yet the sa!t concentrutions were much lower than at (1) (cf. Fig. 9c with b) and the cat'ionanion balance here was less obvious at first, but was attained by 19 days. Thus, the first preference here was for sugar uptake, but any salt absorption that did occur balanced cation (K Na) with anion (CI) in these nondividing cells. ( 3 ) Although the weight gain in Fig. 9c was greater than in Fig. 9b (Table 11) and the salt gains were less, nevertheless, the tissue did acquire osmotically active solutes as osmotic pressure determinations showed (osmotic pressure z 270 milliosmole in the tissue of Fig. 9b and this was even greater in tissue that also received sugar). Therefore, the tissue with low internal salt concentration as in Fig. 9c utilized nonelectrolytes to achieve its high osmotic value and to support its cell enlargement. If the tissue adapted to salt alone or to sugar alone, the final osmotic values were virtually the same (370 milliosmole) , this shows that this tissue could utilize either salts or sugar indiscriminately for its osmotic pressure (cf. Table IV). (4) With the further addition of nitrate, with its attendant promotion of synthesis and growth, the tissue finally (Fig. 9d as at 19 days) grew more (Table 11) and also absorbed more potassium but without the stoichionietric Na = Cl) of cells which only enlarged. (By actual determitiabalance (K tion this lack of balance was not accounted for by internal nitrate, which was virtually zero.) This response of the tissue to added nitrate (depending upon the synthesis brought about by potassium, sucrose, and nitrate) takes time, so that at 8 days the salt relations of the explants were similar to those when sugar and salts were alone supplied (cf. Fig. 9c and d at 8 days). Later, between 8 and 19 days, and when new growth occurred, the salt accumulation was increased and approached more nearly in kind that of the fully growing tissue (cf. Fig. 9f and d as at 19 days).

+

+

333

CELLS, SOLUTES, AND GROWTH

Therefore, the results of Fig. 9 reveal a sequence of events. 1,acking all I ~ U trients and stimuli, the tissue absorbs salts; furnished with sugar and salts, it preferentially absorbs sugars while its growth is still severely restricted; furnisl~cd with salts and sugar the tissue responds by its salt intake according as its further growth is fostered by nitrate, by all basal nutrients without exogenous groMTth stimuli, or by the complete system (B CM).

+

E. THEBEHAVIOR OF POTASSIUM AND SODIUM HALIDES I N TISSUE FI R S1 STIMULATED TO GROW,THEN DEPRIVED OF NUTRIENTS AND STIMULI,AND SUBSEQUENTLY RESTOREDTO GROWTH This experiment, using the cultured tissue explants, is somewhat atlalogo~~s to Hoaglaiid’s use of salt-depleted roots. In Hoagland’s barley root experiinents (Hoagland and Broyer, 1936), the absorptive potential resulted from cells that had grown, had been deprived of salts, and had stored sugars in lieu of salts. In the first period of these experiments, carrot tissue was caused to grow for 6 days with full nutrient and growth stimuli (€3 CM) ; in the second pa-iod, it was restricted for 4 days to external nitrate and sugar. This allowed any prcvious growth potential to be expressed and organic solutes to be built up in cells with restricted access to salts. In the third period (day 0 to l o ) , various treatments (nos. 1 to 5 ) were applied. One such treatment (no. 2 ) merely rcstol-ed neutral (but not nutrient) salts, another (no. 3 ) restored neutral salts and nutrients, and a third (no. 4 ) restored salts and the full system (B CM). Tm7o further treatnients (nos. 1 and 5 ) exposed the tissue throughout (no. 5 ) to the full nutrient (B CM) and (no. 1) to the depletion medium through thc second and third periods. Under all these final treatments (except no. 1). the external concentrations of potassium, sodium, and chloride were the same (K = 11 mmoIes/liter; Na = 5 mmoles/liter; C1 = 9-1 5 mmoles/liter) . O n the samples, collected in accordance with the above plan, determinations were made of the average fresh weight per explant, of the number of cells pcr explant (from which the average cell size was derived), and of the potassium, sodium, and chloride content. The time course of the observed response is shown in Fig. 1Oa-d. After 6 days in the full medium (B CM), the carrot explants weighed S mg, were undergoing very rapid growth by cell division, and had acquired their characteristic high content of potassium accompanied by low sodium and chloride. If the explants remained in this full nutrient, they followed the typical growth curve shown in Fig. 1Oa (no. 5 ) and their salt content would have followed the time course of Fig. 6c(ii). When deprived of full nutrient and exogenous growth stimuli at 6 days, the tissue, furnished only with nitrate and Sllcrose (second period), continued to grow but at a slower rate (Fig. 10a) and this residual growth resulted in more but smaller cells (Addendum to Table 111) ;

+

+

+

+

334

F. C. STEWARD A N D R. L. MOTT

_1

2nd Period I

a

i

100

4

3 r d Period

c

I

150-1

B + salts

2nd Period

I

b

J

0

0

2

4

6

8

1

OJ-

I

0

Time (days)

Time (days) 2nd Period Ca(N0,)2+ sucrose+ salts

b .K

5

100

r

E

P

2

50 ,-,-,-*--a 0

2

4

6

6

8

10

Time (days)

.....,.,.......*.". No

-K

4

2

0

8 1 0

Time (days)

,-,-,-.-,

CI

FIG. 10. The absorption of salts (third period) at different levels of nutrient and growth stimuli by tissue depleted of salts (second period) after it had been induced to grow (first period). a, Data of fresh weight; effects of time and treatments nos. 1-5. b, Data of salt (K, Na, and Cl) concentrations; effects of time and treatment no. 4. c, Data of salt (K, Na, and Cl) concentrations; effects of time and treatment no. 3. d, Data of salt (K, Na, and CI) concentrations; effects of time and treatment no. 2. For further details see text. Key to treatments: Treatment no.

1st period

2nd period

3rd period

~~

+ sucrose

1

B+CM

Ca(N03)2

2

B+CM

Ca (NO,)

+ sucrose

3 4

B+CM B+CM

Ca(N03)2 Ca(N03)2

+ sucrose + sucrose

5

B+CM

BfCM

Ca (NO,,) salta

+

+ sucrose + sucrose

+

B salts B+CM B+CM

(K, Na, and Cl) levels adjusted to those of basal medium plus coconut milk ( B + CM) by addition of KCI and NaCl. a Salt

335

CELLS, SOLUTES, AND GROWTH

meanwhile, the potassium concentration declined because, in the absence of external potassium, it could not keep pace with the growth and uptake of water. While this was occurring, the osmotic value, which was not maintained by other salts, was sustained or even increased by organic solutes (cf. a similar set of TABLE 111 THE GROWTH(FRESHWEIGHT,CELLNUMBER, CELLSIZE) AT DAY 10, OF TISSUE SUBJECTED TO TREATMENTS NOS. 1-5 IN FIG. 10" Thousands of cells per explant

Fresh weight (mg/explant)

Treatment

Cell size ( pdcell)

No. 3.

+ Sucrose + Sucrose Salt Basal medium + Salt

89.1

945.0

,095

No. 4.

B

+ CM with Salt

256.5

2411.0

.I 04

No. 5 .

B + CM (continuous)

269.4

3383.0

,095

No. 1. CaClz

31.1

637.0

,049

No. 2.

36.4

626.0

,059

CaC12

a Explants (18.9 mgm) at Day 0 contained 350 X 10:' cells of size 0.046 pgm/cell. Explants (8.2 mgm), cultured ( B CM) 6 days contained 119 X 103 cells of size 0.057 pgm/cell. Explants (28.4 mgm) cultured ( B CM) 10 days contained 591 x 103 cells of size 0.051 pgm/cell. Explants (3.1 mgm) freshly obtained from the carrot root, contained 47 x 109 cells of size 0.065 wgm/cell.

+

+

data, Table IV, treatments no. 3 and 4 ) . Therefore, at day 0 when the subsequent treatments commenced, the explants comprised cells that had grown but which were partially depleted of their normal salt content. This population of explants was the starting material for various observations on renewed salt uptake as influenced by different levels of renewed growth, a few of which are shown in Fig. 10. The various growth responses from time 0 to 10 days, resulting from the different nutrient and growth factor supplies, are shown in Fig. IOa and they are sucrose salts < B salts in the order Ca(N0,) sucrose < Ca(N0,) < B CM. The levels of external salt (i.e., K, Na, and Cl) were adjusted to be the same throughout these treatments, except for treatment no. 1. It will be recognized that the pretreatment was the same in Fig. 1Ob,c, and d and the subsequent absorption behavior is that now to be anticipated in the depleted cells in response to the nutrient levels and to the growth so induced. The immediate response by renewed growth resulting from a full range of growth factors (CM) restored the high level of potassium concentration, unaccompanied by sodium or chloride, and this was maintained steady at the levels now seen to

+

+

+

+

+

336

F. C. STEWARD A N D R. L. MOTT

be characteristic of the growing cells (cf., Fig. lob) as the explants continued to grow and as cells multiplied (Fig. 1Oa and Table 111). The basal medium without the growth stimulus provided for smaller but still substantial growth (Fig. IOa) as it built up the potassium concentration again to the level of the growing cells; however, this regime permitted more sodium and chloride to be TABLE IV THE OSMOTIC VALUEOF TISSUE SUBJECTEDTO VARIOUSTREATMENTS~ Treatment no.

Osmotic valuea Treatment First period

Second period

Third period

niilliosmole

Atm

Explants To

-

247

5.5

-

273

6.1

3

+ CM 7 Days, B + CM

7 Days CaCI, sucrose

368

5.3

4

7 Days, €3

+ CM

7 Days CaCI,

152

8.6

5

7 Days, B $- CM

7 Days CaC1,

7 Days CaCI2 KCI, NaCl

276

6.2

G

7 Dnys, R

7 Days CaClz KCI, NaCl

7 Days CaCl,

394

85

391

88

1 2

7

7 Days, B

+ CM

-

-

+

+ KCI, NaCl

+ sucrose +

-

+

f sucrose 21 Days,

B+CM

a Tissue was induced to grow (first period), subjected to treatments no. 3 and 4 with sugar or salt (second period), and successively (second and third periods) to sugar then salt (treatment no. 5 ) or to salt then sugar (treatment no. 6 ) . b Osmotic value determined by freezing point depression of the sap of tissue sampled at the end of the period indicated.

absorbed (Fig. 1Oc) and the total content of potassium in the ex1ilants was limited by their total size. When the explants remained on the “depletion medium” but received the added salts, they increased their potassium concentration more slowly and potassium was now accompanied by chloride and sodium to approach, by 10 days, a balance of cation and anion (K Na C1, as in Fig. lad). This response is different from that seen at 19 days in Fig. 9d only because the cells of Fig. l o d had previously grown and stored organic solutes. The lessons to be learned from such experiments as those of Pig. 10 are:

+

(1) When the cells are in full growth and in full organic media they accumulate, preferentially, potassium and organic solutes. ( 2 ) When growth (e.g., of new cells) subsides they can still absorb salts at

CELLS, SOLUTES, AND GROWTH

337

the expense of organic solutes and sodium and chloride can then become prominent. (3) When growth is restricted to enlargement of preformed cells, salts may replace organic solutes in a more stoichiometric balance between cations and anions. ( 4 ) In the alternative storage and use of lionelectrolytes and in the alternative exclusion or storage of neutral salts, the vacuolated cells in their different states, despite their differences in size, seem to preserve approximately the same osmotic value (G8 atm, cf. Table IV) . The above concepts relate to the absorption and accumulation of charged ions, on the one hand, and nonelectrolytes, on the other. The nonelectrolyte manipulated has been sugar. However, many nonelectrolytes or weak electrolytes may be absorbed and accumulated in amounts that are, no doubt, as much affected by the conditions of growth, nutrition, and salt supply as is the case for sugars. In this laboratory, other studies have dealt with storage in separate pools, or compartments, of 1“-labeled sugars and their derived metabolites (Bidwell et ul., 1964; Steward and Bidwell, 1966) ; even the weak electrolyte, proline-14C, could also be accumulated as such (Pollard and Steward, 1959) ; i n ~ s i t o l - ~when ~ C accumulated, existed in a condition of “flux” with the ambient solution (Shantz et al., 1967) ; organic acids (such as malic) and amino acids (such as alanine and glutamine) could accumulate in the cells according to the nature of the growth stimulus and the nutrition and examples of this sort could now be multiplied (unpublished observations of K. V. N. Rao and of G. Norton in this laboratory). Similar conclusions could be drawn from the work of Beevers and co-workers (Grant and Beevers, 1964; MacLennan et ul., 1963; Lips and Beevers, 1966a,b; Beevers et ul., 1966) with reference to sugars and organic acids, from the work of Osmond and Laties (1969) and of Osmond (1967) with reference to organic acids, and other observations by other workers on certain amino acids (Birt and Hird, 1958; Holleman and Key, 1967). Hence, the problem of accumulation of solutes is not confined to electrolytes, for the contribution of the nonelectrolytes to sap composition and osmotic value may be very apparent especially when the cells that are studied are in most active growth, and vice versa.

F. GENERALIZED APPLICATIONS OF THE CONCEPTS OF SOLUTEACCUMULATION IN RELATIONTO GROWTH The central theme thus far has allocated the driving force for solute accumulation in cells to the consequences of their growth, i.e., their self-duplication, their synthesis of complex molecules such as protein, their formation of vacuoles, and the need to accommodate therein a required level of osmotic pressure. While

338

P. C. STEWARD A N D R. L. MOTT

these concepts flow from work on the cultured tissue explants, they obviously need to be related to a more general schema. Such general schemata were constructed to accommodate the concepts extant in 1935, were modified in the light of knowledge as at 1959 and, as again modified in Fig. 11, are adaptable to the present point of view.

("

Relatively nonraeuoiated small cells of meristems and tissue cultures; bacteria, yeast, etc.

Actively dividing cells; absorption tn eyeloplasm and

\.,

Cell expansion, increasing capacity to absorb kn

\

___--____

-------------+Seedling roots, depleted by s h w t s , e.g., barley r m t s in unchanged depleted s o b dons; growing carrot expiants transferred to LOW Salt SOlutions

Cells with low salt content: organic solutes accumulated at limiting salt supply

Barley r w t s in replenished so1ution: growing explants in full nutrient solution; young attached leaves withaccess tonutients

Cells accumulate depending on further growth by enlargement cations (Kl often bal-

(b)

~-

1 Decreasing capacity to accumulate

Renewed rapid accumulation triggered by the environment conducive to renewed growth --

High salt, attached barley roots, maturing internodal cells of Nitella and C h a m ; ~ e s l e l e of s Valoma ; m a t ~ r l n gleaves

Further vital activity

at

Z'-3'C

--_--

c-------------Cells Of Intact, ",atuW Storage OTga"6; discs at low temperature, pox, etc.

metabolism m d slow growth high salt content often replacing

( e . ~ , ,protein synthe.IS) suppressed

Parenchyma cells of fruits, bulb Scales, potato tubers stored

Restoration of full growth and cell dwi s m n by balanced \ n u t r m t s and exog\ enous growth factors

Fully mature cells incapable of further growth and absorption

Accumulation from nonlimlting salt supply

steady state: salt saturated, will absorb more only if depleted

D i m s of storage organs. salt saturated, unable to grow; N i t d l n in steady state; mature va1amn vesicles

FIG. 11. Solute accumulation in relation to growth, metabolism, and salt supply. a, Cells dividing, complexity created, self-duplication of sites and organelles; b, rapidly metabolizing and expanding cells, salt content often potassium dependent; c, further growth and expansion strictly limited, net accumulation suspended.

In the carrot root, the cells that engage in the most active cell division are those of the cambium, and the storage parenchyma cells of the secondary phloem progressively differentiate from them. As they do this, they store salts and nonelectrolytes in ways conditioned by their environment in situ and, eventually, their further physiological activity is arrested. When removed from the dormant storage organ, different alternatives are feasible according as the conditions permit the renewed growth of which the otherwise dormant mature cells (cf. Fig. 11) are capable or as they are forced to absorb or maintain their salts without further growth (cf. Fig. 11).

CELLS, SOLUTES, AND GROWTH

339

The work with the aseptically cultured carrot explants has dealt, in various ways, with their recrudescence of growth, culminating in a return to active cell division, in contrast to responses by which the cells achieved salt saturation in media (simple salt solutions) that neither support nor stimulate growth. Only the latter work with the cultured explants is comparable to the bulk of the published experiments dealing with absorption phenomena in other plant systems, for there is little published counterpart for the work with tissue explants in renewed and active growth. The theme of the schema of Fig. 11 is the great intensity of salt and solute accumulation achieved in the most active systems and that it subsides during development until it can no longer be observed in cells which are fully mature and expanded, which do not grow again, and which have already had full access to salts or solutes. These ideas describe the normal ontogeny of cells in terms of their cell physiological activity as seen in the left panel of the figure. The criteria of such physiological activity which have been, and still are, useful at different levels are: (a) for growth; cell division, the onset of vacuolation (cf. Fig. 2), cell enlargement more than cell division leading to full cellular expansion (cf. Fig. I b ) ; (b) for metabolism; protein synthesis as evidence of anabolism and the creation of complexity, and a high intensity of respiration (in part linked to protein synthesis and turnover) as the overall measure of energy directable to cellular work; (c) the preexisting levels of salt and/or solute concentration which are determined by the prior environment of the cells up to the point at which the new observations begin by the use of whatever indicator ion (K, Rb, Cl, Br, and so on) is adopted. The second panel, to the right of the figure, again describes the subsequent fate of cells, mainly of dicotyledonous plants, which, having become mature as they developed in sitzl yet retain either some endogenous capacity for further growth (as in the surface cells of potato discs) or the ability to respond to exogenous growth factors so that they may again reexpress their genetically determined ability for growth and development (totipotency), as in the case of carrot cells. Such cells may, therefore, achieve full salt saturation from single salt solutions, may exchange their preabsorbed salts in different saline environments, or progressively reembark upon renewed growth and salt or solute uptake according to the level of growth and metabolism that their ambient medium supports. The same criteria of physiological activity that are applied in the downward direction (to the left) apply equally in the upward direction (to the right) as the events of growth induction restore the quiescent cells to their actively growing state. However, when cells embark on this renewed phase of growth and development, under aseptic conditions and with a full nutrient (organic and inorganic) supply, one may now distinguish more clearly than hitherto between ( 1 ) the role of potassium as the essential ion absorbed because it helps to “keep the

340

F. C . STEWARD AND R. L. MOTT

synthetic machinery running,” especially in the phase of cell multiplication when division is more important than vacuolation, and ( 2 ) the essential need of solutes to supply the osmotic value compatible with cell enlargement, with the form of the vacuolated cells, and with the obvious fact that the solute content increases with volume while the cytoplasm is only “spread out” over the cell surface. Here, the first solutes secreted internally seem to be organic; the first cation (unbalanced with respect to inorganic anion) is potassium, and the later intake (with a closer balance of inorganic anions and cations) may involve any neutral salts which, partially or totally, replace the organic solutes. Thus, as the result of the work on cultured tissue explants or cells, the chart takes on new meaning at its most crucial point, i.e., where new cells form and develop. It was these features that the work on (1) whole root systems or even that done with single roots, or ( 2 ) on shoots, even if done with a sequence of developing leaves, found it most difficult to identify because they involve so few cells at one time and, even in these, the events are so transient. There is neither use nor intent to recapitulate the previous background that led to the earlier forms of the chart in question; this can be seen by reference to the cited sources (Steward, 1935; Steward and Sutcliffe, 1959). In retrospect, however, some features of the earlier work are particularly relevant in the current context; these are:

(I) The work on potato discs (and also on excised roots) paid particular attention to potassium as the ion that was most important in regulating the metabolic activity conducive to active intake of other ions (e.g., Br). Here it is relevant that the metabolic effects of potassium and calcium were shdryly and reversibly (see Fig. 33 of Steward and Sutcliffe, 1959, and references cited there) contrasted in ways that affected not only the overall respiration but the onset of the protein synthesis which was used as the measure of that recrudescence of growth and vital activity to which attention is now being redirected. ( 2 ) The emphasis on the metabolic effects of potassium and their ability to affect ion (Br) uptake was not attributed solely to the K ion already in the cells but more to the K ion being absorbed. Even in 1941, Steward and Preston (1941, p. 111) stated: “In fact, in so far as the metabolism is regulated by salts, it seems that during entry these have access to more potent spheres of influence than the ions already stored in the cells since their effects are disproportionately large relative to the amounts actually absorbed.” Thus, even then there was the concept that potassium ion in the cytoplasm, or close to the seat of vital activity, was more important in the overall mechanism than that already stored in the vacuoles. (3) The analogy between capacity to accumulate solutes and levels of protein synthesis persisted throughout, but it was dramatically shown by two unexpected

CELLS, SOLUTES, AND GROWTH

341

observations on potato discs. These effects were (a) the contrasted effects attributable to the presence, or absence, of free carbon dioxide in the system; and (b) the peculiar aftereffects of low-temperature (2°C) storage of potato tubers. The latter conditions produced in the tubers circumstances in which the aerated thin discs cut from them respired rapidly but, nevertheless, lost ions to the outer solution; they also failed to accumulate Br ions while simultaneously they increased their water content greatly, but could not undergo cell division. These events (summarized in Figs. 8-14 of Steward et al., 1943) are now very significant. The dislocation of protein synthesis so caused precluded the de novu accumulation characteristic of growth in potentially dividing cells. The production endogenously of high sugar concentrations in the cells enabled them to absorb water and to increase their vacuoles withozlt the need uf external solates and, significantly, when the endogenously released sugars performed this function they displaced existing inorganic ions that “leaked” out of the cells. The following quotation (Steward et al., 1943, pp. 255-256) is especially significant in the light of more recent results here reported. In the case of potato there is clear evidence that the ability of the cells to absorb and accumulate salt, as well as to retain their electrolytes and non-electrolytes for long periods against distilled water, is associated with their ability to synthesize protein, to grow and to divide. . . . Low temperature storage inactivates protein synthesis which, linked to salt uptake and at least a part of the aerobic respiration of normal potato discs, constitutes both an essential feature of growing cells and of the ‘dynamic machinery’ whereby salt is accumulated and solutes are retained in the vacuole.

All these ideas are compatible with concepts that are applicable to the plant body as a whole. Sites of primary solute accumulation are those where cells are formed and where solute concentrations are developed in vacuoles de novo. In the usually heterotrophic nutrition of most plant cells, they normally derive their sugars exogenously, reduce nitrate for protein, and require potassium. Cells of roots derive their sugars from shoots, but having obsorbed to the limit of their capacity along gradients established during development from the apex (Prevot and Steward, 1936; Steward et al., 1942), nevertheless, may reversibly empty and refill with externally absorbed salts. They “empty” as shoots deplete roots and, in so doing, the active “sinks” in shoots, where cells and organs may pass through a “Sad’s Grand Period of Growth,” acquire salts from roots, but in order to do so the shoot must supply the cells of roots with organic solutes. These organic solutes, furnished by shoots to roots, first displace the salts to be translocated and subsequently the salt concentration is restored by absorption. It is this interplay between shoot and root, or to use the expression “the division of labor” between them, which is the basis of the balanced internal nutrition of higher plants which gives to them as a whole their autotrophic functions.

342

F. C. STEWARD AND R. L. MOTT

G. SALT AND SOLUTERELATIONSVIEWEDON A CELLULAR BASIS: EFFECTSATTRIBUTABLE TO ENDOGENOUS AND EXOGENOUS STIMULI The schema of Fig. 11, insofar as it is supported by the new data, places the emphasis upon the concepts here summarized : (1) The de novo accumulation of solutes in cells is an intimate part of the events that occur at cell multiplication and during the ensuing development as cells enlarge. ( 2 ) The emphasis is upon initial secretion of solutes internally into cells, whereas the emphasis to be placed upon the absorption of neutral salts is a later feature, with the prominent exception that the early uptake of potassium, unbalanced by inorganic anions, is essential from the outset for the whole cellular machinery. (3) The activity of cells or organs in the absorption of inorganic ions is a function of their capability for further growth and of the prior conditions to which they have been exposed-a marked deficit of salts may, therefore, cause an internal accumulation of other solutes and vice versa. ( 4 ) What is generally true of cells is in a wider context also true of organs since each may pass through an absorptive phase related to its “Grand Period of Growth.” Growing organs which can act as “sinks” can deplete others which function as “sources” in accordance with physiological gradients that have been observed (summarized in Steward, 1968, pp. 289-296). ( 5 ) While, as the chart shows, any cells may be expected to pass in their ontogeny through similar phases, nevertheless, they will be modified by the circumstances imposed by their location and their ultimate role (even though genetically they are all identical). Thus, the cells of some mature storage organs have stored sugars, organic acids, nitrogen compounds, or even starch, and all these specifically imposed characteristics will have their repercussions upon the extent to which cells utilize neutral salts within their vacuoles. The summary article by Laties (1969), dealing with recent literature under the title “Dual Mechanisms of Salt Uptake,” appeared after this review was first written and submitted. This section is inserted here to permit the concepts of Laties to be seen in the perspective of this review and of Fig. 11. First, what is meant by “duality” and how does it relate, as seen by Laties, to the stages of uptake in cellular ontogeny as here presented? In one form or another, duality of mechanism has been with us over many years. Some absorption events were seen as “primary” in the sense that they involved the de novo uptake by the system as it grows, whereas others were regarded as “induced’ in the sense that superimposed changes gave to the cells a renewed ability to absorb (Steward, 1935). The “duality” that describes different sequences in the absorp

CELLS, SOLUTES, A N D GROWTH

343

tion into cytoplasm and vacuole during development was the basis of phase I and phase I1 (Steward and Sutcliffe, 1959) and it still appears in the chart in Fig. 11. The “duality” discussed by Laties, however, arises solely from the kinetic treatment after Epstein (1966) and the observation that, under the enzyme analogy, one is faced with two (or more) distinct rates of uptake of the same ion which operate at distinct external concentration levels. Epstein and co-workers invoked two or more separate ion carriers to account for these different rates of uptake. Laties, on the other hand, has reduced these observations into two mechanisms. One mechanism (system 1 ) 2 is regarded as the “filling” of the cytoplasm via the plasmalemma, whereas the other (system 2 ) 2 is interpreted as the “filling” of the vacuole, from the cytoplasm, via the tonoplast. Younger developing cells (e.g., as in root tips) emphasize system 1, whereas in older, more vacuolated cells (e.g., cells further from the tip, or cut discs of storage tissue) both systems 1 and 2 occur. Thus, Laties has abandoned the earlier concept of two or more distinct carriers or sites with supposed concentration specificity in favor of distinct mechanisms characteristic of cytoplasm and vacuole. Thus, the kinetic analogy has perforce returned to ideas more consistent with the structure and development of cells and of which the earlier concepts of phase I and I1 took cognizance. According to Laties, system 1 is concentration saturated at low levels (e.g., 2 mmoles/liter of potassium), is independent of inorganic anions, and is very potassium selective; system 2 is only concentration saturated at much greater levels (e.g., order of 50 mmoles/liter of potassium), is very dependent upon inorganic anions (e.g., Cl) but it can involve other cations for it may even specifically favor sodium over potassium. In the outcome, these concepts are not very different from the interpretations of cytoplasmic and vacuolar uptake as outlined in Fig. 11. However, Laties adheres to ( 1 ) ideas dependent upon specific pumps which operate only with ions at specific concentration levels and not at others and which, in their operation, do not invoke the total solute concentration of the cells, (2) the concept that system 2 is entirely a function of high external concentration via the cytoplasm, and ( 3 ) in its operation, system 2 has not been related either to the de nuvu formation of the vacuole, its total solute concentration, or its content of organic solutes as determined by the prior history of the cell. In short, the interesting analysis of Laties fails, in our view, to make adequate contact with the observations that can be made on cultured cells and which lead to the concepts of Fig. 11 and Section V, F. Viewed against this broad background, the kinetic curves and absorption iso-

<

2 The terminology of systems 1 and 2 after Laties has no direct relationship to the growth-promoting systems I and I1 as referred to on pp. 312 and in Fig. 13.

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F. C. STEWARD AND R. L. MOTT

thernis of Laties seem to fall into place. In our view, the so-called absorption system 2 is only revealed by the kinetic analysis when the preformed cells are subjected to abnormally high total external concentrations. These concentrations have implications for the withdrawal and eventual replacement of water, on the one hand, and require consideration in terms of the changed balance between the internal and external ion concentrations due to a 10-fold increment of a specific ion supplied externally. Within this broad pattern of behavior, the ideal would be to trace all events during development, ub initio, on an individual cellular or even organ basis-an obviously impossible task. The virtue of the cellular studies on growth induction, however, is that they furnish a feasible approach to the crucial and early events of cell multiplication and of development as these relate to solutes, more so than any work on cells in situ can at present achieve. For this reason, Figs. 1 2 and 13 return to this problem in more detail. The further detaiI presented in these figures flows from, and was anticipated by, considerations presented earlier in this review and, indeed, even from earlier publications; it was also a direct sequel to the new experimental results summarized in Figs. 4-10. Figure 1 2 places the data on a more cellular basis with respect to the various parameters shown. Figure 13 also faces the evident fact that the overall stimulus to growth in cells is highly composite, so that one may now dissect this into its component parts and so see their respective implications for the solutes of cells. Figure 12 presents the behavior of 10 clones in a nianner analogous to Fig. 4. they are arranged [Fig. 12a(i) ] in a similar sequence determined, as previously, by the order of their growth in fresh weight in the basal nutrient medium alone. For these 10 clones, the effects of supplements (inositol, IAA, and the full complex of coconut milk) are shown in the horizontal series of diagrams designated (ii), (iii), and (iv). The vertical series a, deals with effects on fresh weight and average cell size in the tissue explants, series b with the number of cells, series c with the average salt (potassium, sodium) content per cell, and series d with the average concentration of salts (potassium, sodium) in the cells. Direct comparisons between members of the vertical series are possible because the scales are the same throughout each series, with the sole exception of the scales of fresh weight and ceII number of Fig. 12a(iv) and b(iv) which were reduced to 1//5 to accommodate the great growth that occurred. The data of Fig. 12a(i) and (iv) and of d(i) and (iv) essentially recapitulate, for these clones, features already embodied in Fig. 4 and which are again evident, namely, that the explants caused to grow most in fresh weight [cf. Fig. 12a(iv)] and in cell number [cf. Fig. 12b(iv)] had the strongest preference for potassium over sodium [cf. Fig. 12d(iv) with d ( i ) ] , although not the highest total salt concentration. The endogenous ability to grow in fresh weight,

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CELLS, SOLUTES, AND GROWTH

in the basal medium alone, invoked cell division and cell enlargement to different degrees in the different clones [cf. Fig. 12a(i) and b ( i ) ] . For example, in clones no. 1, 3 , and 6 cell divisions were more prominent in the basal medium alone, whereas in nos. 2, 4, and 8 there was more emphasis on cell enlargement. GROWTH (FRESH W T , CELL SIZE)

GROWTH (CELL NUMBER)

- -

FRESH WT (rng/explant) CELL SIZE (pg/cell)

SALT CONTENT

( p o l e s xlo'e/cell)

------- CELL NO.XlO'/eXplant

-

W T CONCENTRATION

(pmotes/gm fresh w t )

TOTAL CATION (K+N.,) (Kl SODIUM (Na)

- WTASSIUM

PIG. 12. Growth and salt content of 10 clones of carrot explants. Increase of cells in number, size, and salt content.

In some cases, cells were larger after 18 days than initiaHy, despite the cell divisions that may have occurred in the interim (e.g., nos. 2, 3, 4, 7 , 8, and 9 ) ; in these, the total salt (K Na) and the total potassiuin per cell likewise increased [ cf. Fig. 12a(i) and c(i) 3. The diagrams of series c give full weight to effects o f cell size, but the diagrams of series d, being based on concentrations, relate the intake of salts to water as the cells expanded, From Fig. 12a(i) and d ( i ) ,

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F. C. STEWARD AND R. L. MOTT

it is clear that the increment of fresh weight was not precisely paralleled by the changes in salt concentration; therefore, the disparity between growth by cell division or by cell enlargement in the different clones affected the intake of water and salts differentially. By contrast, the explants subjected to the conditions of Fig. 12a(i) -( iv) had their endogenous limitations variously satisfied by access, exogenously, to the required nutrients and growth stimuli; when these were fully supplied, as in 12a (iv) , the idiosyncrasies of the cells of the original tissue were submerged and all the clones behaved in a much more similar manner [cf. Fig. 12a(i) with (iv); Fig. 12d(i) with (iv)]. Between these extremes (shown, on the one hand, by tissue explants nourished only by a basal medium and confined to the growth that their initial endogenous characteristics support or, on the other, by explants fully stimulated to grow by nutrients and a full complement of growth factors) partial growth factor stimuli, or various nutrient limitations, may be associated with variable patterns of salt (potassium, sodium, or chloride) or solute concentrations. With respect to potassium and sodium, as affected by inositol and IAA, these effects are very evident in Figure 1 2 [cf. horizontal series (ii) and (iii)]. Notably, there were selective differences in uptake of salts and water per cell as shown by the form of Fig. 12a(ii) and (iii) and 12c(ii) and (iii), respectively, with clear implications for the organic content of the cells. Certain clones, e.g., nos. 5 and 10, achieved the same growth in average cell size [Fig. 12a(ii)] as clones no. 2 and 8, but this enlargement was more supported by salts (K Na) in clones no. 2 and 8 than in nos. 5 and 10, which presumably utilized other solutes obtained from the medium. Since so many studies have been made with scant attention to the factors, endogenous or exogenous, that modify the behavior of the initial cells of the tissue used, it is not surprising that they have produced such variable and often inexplicable results. The range of behavior encompassed by different carrot clones, as here portrayed, and the range of activity they show from single salt solutions to full nutrient, presents ample room to include virtually all the published results that have been reported. Without a rationale such as that here developed, however, few or isolated observations are rarely useful, Great disparities in behavior may be achieved in the following ways. One may repress virtually all biological activity to the point at which the ion responses, although now reproducible, have little meaning (e.g., by long washing, by loading with salts, by lack of nutrients and stimuli, by the use of much tissue in the form of “thick’ discs in relatively little fluid, and by observations reduced to short periods of time). At the other extreme, one may so induce cells to display their maximum growth and metabolism (e.g., as in full nutrient and with exogenous growth factors) that their various responses are again uniform.

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CELLS, SOLUTES, AND GROWTH

Between these two extremes there is a demonstrable array of controlling factors that could condition the behavior of the cells to conform to the circumstances they encounter in normal development. The progressive responses of the cells to the component parts of the complete growth factor systems are, therefore, now relevant.

B I

B

1

BIlAA

B BIINOS

SYSTEM 1 SYSTEM I *II (a) GROWTH

-"--- - - FRESH W T

(rng /explant1 CELL SIZE ( F g l c e l l )

PROTEIN NITROGEN ( p q x IO-'/C~III

GROWTH CELL NO XICa/explant

I

(C) SALT CONCENTRATION

(prnoles/gm fresh w t ) K*Na ND -K CI

-

........

SYSTEM I *!3

BICM

(el

\SYSTEM SYSTEM 1 + U

(b)

BtCM

(d)

BtlNOS

SYSTEM 11

SYSTEM I*U

-----

I

BtlAA

SOLUBLE NITROGEN (pg x 166/ceiI)

(f) SALT CONTENT +males x I O ~ C ~ I I I

-K+Na -

...............Na

FIG. 13. Growth and salt content of carrot explants. Effects of various growth factors. B, basal medium; inos., myoinositol (250 ppm) ; IAA, indoleacetic acid (0.5 ppm) ; AF,,,,., Aesctrlus growth factor (2.0 ppm); system I, inositol AF,,,,,; system 11, IAA + zeatin; teat., teatin (0.1 ppm); CM, coconut milk ( 1 0 % ) .

+

The experiment of Fig. 13 utilized the existing knowledge of the partial growth factor systems I and I1 (Section IV, B) which are included in the overall stimulus of coconut milk. Although the experiments as performed involved several clones, various growth factor stimuli, and various levels of trace element nutrition, the important points can be made by reference to the data shown. The measurements made were of fresh weight and cell size (Fig. 13a), cell number (Fig. 13b), salt (potassium, sodium, and chloride) concentration (Fig. 13c),

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F. C. STEWARD AND R. L. hfOT1’

protein nitrogen per cell (Fig. 13d), soluble nitrogen per cell (Fig. 13e), and total salt (K Na) per cell (Fig. 13f). The figure shows the effects on these parameters of the component parts of growth-promoting system I (i.e., inositol and the AF,,,,), of system I1 (i.e., IAA and zeatin), and of the two systems combined. The right half of each diagram shows the effects of progressive enrichment of the basal medium with the component parts of system I, and the left half refers to system 11; the effects of the two systems in combination are also shown. The component parts of the growth factor systems may cause the cells to increase in number, or in size, in very different ways (cf. Fig. 13a and b) even to achieve the same total weight. These treatments also affected the balance of cation (K Na) with anion (Cl) (Fig. 13c) and the nitrogenous composition of the cells (cf. Fig. 13d and e) and, as the composite Fig. 13 shows, the kinds of metabolism superimposed upon the explants were more important for their composition than the number or size of their cells. There is a trend (previously noted) for potassium and cell size (water per cell) to vary concomitantly (6. Figs. 13a and f ) . Nevertheless, the total salt content per cell ( K N a ) and the ratio K/Na reflected the influence of the partial growth-promoting systems (6. effects of IAA and zeatin) in ways that were not attributable solely to their effect on cell number and on cell size (cf. Fig. 13f with a and b with reference to IAA and zeatin) . These effects of growth factors, interacting with the development of the cells, were not confined to neutral salts; this is shown by the comparison of Fig. 13e and f. In other words, the carrot cells could achieve rather similar sizes (cf. Fig. 13a) which were compatible with very different salt content (Fig. 13f) and soluble nitrogen (Fig. 13e) per cell. Consistently, however, whenever the cells grew and mutiplied best (i.e., with systems I and 11 combined) they were the smallest, had the least total salt as well as soluble and protein nitrogen per cell, although they had clearly established concentrations of potassium and sodium (Fig. 1%) typical of the growing cells. Thus, the ion transport phenomena and the final salt and solute composition of cells were related to the ways the cells grew and the means by which they were nourished. These relationships could not have been seen without investigating growing cells, nor without the control of their growth that the complete and partial growth systems made feasible. The further understanding of these problems should, therefore, make increasing demands on our knowledge of the biochemistry and physiology of the growing cells and, by contrast, it may profit less from interpretations based solely upon quiescent cells that have ceased to grow.

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H.

INDICATORS OF SALT U P T A K E IN CELLS AND IN THE PLANT

BODY

This review first took notice of the history of solute accumulation in plants (Section 111, A and B ) ; it then recognized some more recent trends (Section

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349

111, C ) . The main new ideas emerge from data obtained in the study of aseptically cultured tissue explants. A retrospective view at this juncture can, therefore, be informative and especially so before any attempt is made to formulate a general framework of interpretation. First, the work done on thin (0.7-mm) tissue discs (especially of potato tuber) in dilute (1 mg equivalent per liter), well-aerated (in equilibrium with air at 23 "C) , carbon dioxide-free solutions, exploited the existing endogenous capacity of these cells to undergo the recrudescence of growth and metabolic activity which, in moist air, culminates in internal divisions. In potato discs briefly washed after cutting, these events may be protracted, even in the absence of external nutrients or growth stimuli (e.g., protein synthesis at the expense of existing soluble nitrogen continues unabated even up to 500 hours and the stored reserves of organic nitrogen can maintain it much longer at this rate). Carrot root tissue, artichoke tuber tissue, and that of other storage organs, although inherently usable in the same way, are much less uniform, are less tractable in the form of thin discs, and their initial endogenously maintained activity is much more transient. For this reason, much of the early work on tissue discs was done with potato tuber. When externally proliferating, rapidly growing cells were invoked in the study, however, it proved simpler to achieve these ends using carrot root phloem rather than potato tuber tissue, for the latter required more complex stimuli, such as synergistic interactions with 2,4-D or NAA, to bring it into equally active growth. In retrospect, the conclusions from the two systems (the thin potato discs exploiting their limited endogenous capacity to grow and the small carrot phloem explants exogenously stimulated to grow) have a surprising amount in common. Both stress that the progressive events of solute intake invoke a complex background of metabolic events which have been variously described both for the potato discs (in papers by Steward and Preston, 1940, 1941) and for the cultured carrot tissue (for summary see Steward, 1968, pp. 261-271). Both systems require concepts that invoke the catabolic events of metabolism and the anabolic events of cells able to grow in the interpretation of the solute relations of the cells and, while each system has its particular advantages, each presents its peculiar problems. Some of the difficulties of interpretation are traceable to problems of analysis and the way they have been overcome. The early use of an analytically determinable nontoxic, nonmetabolized anion not initially present in the cells (Br) was effective, but it could only be so in systems conducive to the accumulation of such ions. In the light of present knowledge, the most actively growing cells (e.g., carrot) could have acquired their initial solutes (including potassium) while they virtually ignored bromide. The bromide uptake of potato discs, or of excised roots, should be seen as uptake by cells with a limited endogenous capacity for growth and a maintained endogenous supply of organic solutes

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available for anabolism by the cells and to be replaced by neutral inorganic salts. Thus, the stoichiometry of the uptake of potassium with bromide, or of rubidium with bromide in the earlier experiments with potato discs, still serves to relate kinds of metabolic activity and events in the absorbing cells with which such uptake is Concerned; it should not, however, be held to describe the very first stages of solute accumulation in newly formed cells insofar as these are now understood. These very early events seem to concern potassium ion in a unique and imbalanced way (so far as accompanying anions are concerned) and they also concern the organic solutes on which the osmotic values of angiosperm cells largely depend. It is significant that papers (published between 1930 and 1943) which developed the biochemistry conducive to salt accumulation in potato discs stressed the overriding role of potassium (often counterbalanced by calcium). The potassium in the cells maintained the necessary levels of respiration and the concomitant conversion of soluble nitrogen to protein. As this potassium-mediated metabolism occurred, salt entered. Moreover, there were progressive changes, motivated by contrasting levels of potassium and calcium which extended to the amino acids and the organic acids which buffered the aqueous extracts of cells (Steward and Preston, 1940). All this gave to potassium a regulatory role over the metabolism needed for anion uptake (e.g., Br); this was recognized even in a period when “anion-induced respiration” held sway. When attempting to measure the salt-absorbing potentialities of a given tissue or cell system, however, the useful choices of anion to be analyzed require a foreknowledge of the stage at which the measurenient is desired. At the outset, and for the study of solute accumulation de novo, there is no substitzlte for the zlniyzie role of potassium,and a halide indicator could have given a false picture; later, when neutral salts can replace organic solutes as they are used in mctabolisni and growth, potassium and other alkali halides have their uses and the cation may often reflect the absorption of the anion. By the time the cells absorb inorganic ions ubiquitously, however, they least illustrate the driving forces of growth, for the system has by then been created. To understand the driving forces toward solute and salt accumulated in cells, which derive from their ability to grow, two broad avenues were available and tried. The first exploited the gradients, previously observed and summarized, of activity that normal development presents; another utilized the cultured tissue systems in which the experimental tissue is caused to display very different levels of growth and metabolic activity so that its solute-absorbing properties could be identified. In both cases, it was convenient to have a sensitively determinable ion for study and 137Cs seemed suitable because even then (1 948) it was available in carrier-free form. The problems that then arose may be inei1tioned with respect to studies made with 137Cs on NLWC~JJZ~J and on carrot explants.

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CELLS, SOLUTES, AND GROWTH

The first, and still useful, result obtained with 137Cs defined the broad regions of active ion intake in the plant body insofar as these could be perceived by radioautographic means. These results were unequivocal and entirely intelligible, for they localized in Narcissus the centers of de E O Y O solute accumulation (revealed by the presence of 1 3 7 C s ) in the intercalary growing regions of leaf bases, in young leaves of shoots, and especially in the apical growing region itself (for summary see Steward, 1968, pp. 280-299). In roots, the general

10 20 30 40 50 60 70 80 90100

i

ii

FIG. 14. Absorption of 1 3 i C s by roots of Narcirsus. a, Absorption of 1 8 i C s by attached roots grown with (ii) and without ( i ) external nutrient supply. Radioautographs of paired longitudinal slices of roots. b, Graded absorption of 1 3 i C s in segments along the axis of roots grown with access to external nutrients.

longitudinal gradient (previously observed for potassium, rubidium, and bromide) still obtained, and it could be shown that new accumulation of ion was more confined to the extreme root tip (cf. Fig. 14a) when the roots had developed with full access to external salts than when they were limited to the salts and solutes of the bulb. While individual roots could be studied in this way, the techniques were not yet sensitive enough to obtain 1 3 i C s concentrations in cells, as in the root apex, but they were able to show that the concentrations of 137Cs there attained were in fact lower than in segments even a very short distance (3-4 mm) behind the tip (cf. Fig. 14b). This result clearly demonstrated a difference between the meristematic region of the root and that region where the onset of cell enlargement occurs. To the extent that the cells at the tip were growing, they obviously had need of potassium and this need was satisfied, to the required level, from the bulb; of this, the general absorption of 1 3 7 C s as a nonessential ion is no measure whatsoever. To the extent that the cells that had developed from the tip enlarged and exhibited a more ubiquitous

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F. C. STEWARD AND R. L. MOTT

requirement for solutes than the cells of the apex, the 137Cs uptake, similar to that of bromide and rubidium (Prevot and Steward, 1936; Steward et d., 1942), provides an indication. I.

ION U P T A K E BY

CULTURED TISSUES: A RESUME

The decision (made about 1946-1947) to develop a tissue culture system on which to base studies of the salt and solute relations of cells in different states of growth and metabolism has been amply justified by even the results here presented (cf. Figs. 4-13). It is now clear that cells discriminate between water and salts, between solutes and salts, between cations and anions, and between potassium and all other alkali metals in ways that become meaningful only if one attempts to develop, along lines long ago foreseen (Steward, 1935), a cellular ontogeny of salt accumulation. This concept (cf. Fig. 11) now extends earlier views based on the different needs and properties of cells (phases I and 11) in successive stages of their growth. To the extent that these still useful ideas emerged from the first use of the cultured tissue explants, using 137Cs as the ion absorbed, the experiments were helpful and justified, the views of other authors notwithstanding (Smithers and Sutcliffe, 1967a,b, and other papers cited there). The measure of importance then attached to further understanding of the growth induction in the carrot cells, however, and its bearing on the problems of their solutes, is the time that has been taken to amass the body of information here reviewed and that, except for two papers designed to illustrate emerging and provocative ideas (Steward and Millar, 1954; Lyndon and Steward, 1965), publication has been deferred. Others who claim to have used and emulated the system in question, despite suggestive points revealing that it was not in fact so duplicated, have not been so deterred. Indeed, the data of all the published papers could well be comprehended within the scope of the data here reported. This being true, little further reference will be made to either the tone or the content of the papers in question. Attention, however, should be directed to ideas that link earlier statements (Steward, 1935; Steward and Sutcliffe, 1959) with the present analysis. These concern two important points : (1) Do the tissue culture experiments discriminate, with respect to ion transport phenomena, between the cells at two contrasted levels represented by their status prior to, and subsquent to, growth induction, and do the cells behave toward ions differently when they are in a predominantly dividing as contrasted with an enlarging (or enlarged) state? ( 2 ) Do the tissue culture experiments, amplifying those based on the tissue slices, implicate protein synthesis as the indicator, probably the best indicator, of the anabolic activity that at once indicates a capacity for growth and an ability of cells to accumulate ions?

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Even though the prior papers (Steward and Millar, 1954; Lyndon and Steward, 1965) had not been published, the short answer to both questions, based on the data here presented, must be yes. The first use of carrier-free 137Cs as the ion to be absorbed and measured was motivated by its analytical convenience, and it was justified as a cation entirely free from the important physiological roles of potassium. While the 137Cs content of cells may depend on acts that potassium performs, the level of I3?Cs so attained is unrelated to the level of potassium required in the cells to maintain their activity. Thus, 137Cs was useful when comparisons were made of variables (eg., concentration) acting on the cells (e.g., growing or nongrowing) under otherwise comparable conditions. Under the conditions of maximum growth and cell division (i.e., the complete medium plus the growth factors), the cesium content of explants kept pace with their growth but, unexpectedly, the absorption of 137Cs did not vary with the external concentration in the manner expected of cells which, when exposed to single salt solutions, compensate by greater relative uptake (i.e., accumulation) at the lower dilutions. Most carrot phloem explants display some, and often substantial, endogenous ability to grow when in contact with a basal nutrient medium [Fig. 4a(i)], and this may involve some cell division and enlargement [Fig. 12a(i) and b(i)]. To reduce this growth to a minimum, comparable explants were exposed to similar ranges of 137Cs with only enough calcium salt to maintain the cells viable. Under these circumstances, any increment of weight that occurred was to the maximum extent determined by the enlargement of cells and to the minimum extent by their division. Under these circumstances, accumulation ratios were not only high, but were a logarithmic function of the external concentration. It is consistent with what has been presented in this review that the tissue explants that were virtually in a single salt solution and which were aerated and metabolically active but dependent only upon their endogenous properties understandably behaved like the tissue discs previously studied and, insofar as the data go, they did so. They accumulated to high accumulation ratios and their effectiveness in absorbing ions increased with dilution. It is also consistent with what has been presented here that the tissue explants that grew and developed in a full nutrient solution maintained cell division and did not, within the period of the experiment, become so restricted by internal solutes that they invoked the full ability of the cells to maintain their osmotic value even in the face of a very low external salt concentration. These cells, therefore, did not regulate their ion concentrations, as they grew, so much as content, and, because of its essential role in growing, dividing cells, that content would predoniinantly be made up of potassium. The fact that the internal concentrations of absorbed 137Cs were so low should not be solely attributed to mere competition with potassium, for even when such cells were

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placed in contact with approximately equal external [K] and “a], the internal concentration of the former far outweighed the latter (cf. Figs. 6 and l o ) . The fact that the two series (i.e., growing versus nongrowing cells) were so different in their relations to external concentration still indicates a difference between cells in these contrasted states. All interpretations of de novo accumulation of ions against concentration gradients seek a tangible measure of the absorbing intensity of the cells. This may be, variously, the overall metabolic activity and release of energy in usable form (ATP) for all useful purposes; it may be the reversible production and destruction of a carrier complex which is itself a function of metabolism; it may be the balance of oxidation-reduction at localized sites in cells; or it may be a manifestation of genetically determined or inducible permeases, and so on. Whatever the detail, the fact remains that there must be some measure, some criterion of activity, which lends itself to the comparison of cells in the different states of activity that promote or suppress the absorption of solutes. The usefulness of protein synthesis as a criterion of the potentiality for growth, of the anabolic increase of complexity, which requires the harnessing of energy to many useful purposes, has been stressed. At the same time, the fact of synthesis was regarded as a characteristic of the system which, being operative, could take the salt intake “in stride,” but it was not a stoichiometric measure of the salt that could be transported. Knowing that the exogenous factors that greatly stimulate the carrot cells to grow also promote both synthesis and turnover (Steward et al., 1956; Bidwell et ul., 1964), it was reasonable to determine whether or not measurements of that synthesis reflected the simultaneous absorption of ions. The absorption of proline-14C and its conversion to hydroxyproline [now known to be present throughout the cytoplasm, not in the wall, and a feature first of the nucleus and nucleolus (Israel et dl., 196S)l seemed to involve an essential structural moiety of growing cells. It seemed instructive, therefore, to compare the simultaneous uptake of 137Cs and the incorporation of proline-14C into cells. The relations that emerged suggested that cells in which metabolic activity is impaired by inhibitors of protein synthesis are apt to have reduced intake of 137Cs, but the relation between these two events was indirect. However, if there is to be an index of the ability of a growing, dividing, enlarging cell to utilize its metabolic energy (respiratory in origin, and mediated by ATP) in ways that drive the cellular machinery so that it niay do osmotic work, then it still seems that this index will, in some way, invoke the ability of the cells to create complexity by synthesizing protein. If the earlier work on ion accumulation by cultured cells had actually achieved this objective (cf. Lyndon and Steward, 1965), this review need not have been written. No explanations of the ways in which vacuolated plant cells regulate their

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solute content and concentrations can be effective if they ignore the driving forces of growth, whether in cell division or enlargement. This drive is not supplied by nutrients alone, but by the complex of growth factors that puts the machinery of growth in gear. To the extent that this drive is furnished to quiescent carrot cells by naturally occurring fluids from the coconut or from Aesculus fruits, or by extracts of immature corn grains, the generality of the overall effect is clear and the actions of its component parts evident (Figs. 4, 12, and 13). The growth factor systems operate at sites or in ways that also require exogenous iron (Neumann and Steward, 1968; Steward et al., 1968b). Carbon monoxide and cyanide act as inhibitors of the growth induction and less so of the growth so induced; this is consistent with the involvement of iron, but not necessarily via cytochrome. Tests made using a range of inhibitory substances have suggested that after growth is stimulated by coconut milk it is more sensitive to those inhibitors that uncouple metabolic energy from its use than to those that act directly upon respiration as mediated by cytochrome (Steward et al., 1961). This is only another way of saying the obvious, namely, that growing cells are different from nongrowing ones, if only because in the one case their energy is canalized to useful purposes (including salt accumulation and ion transport), while in the other it largely runs to waste.

VI. Salient Features of Solute Accumulations in Cells : Perspectives and Prospects The experimental observations presented lead to very different interpretations, or points of view, than those commonly brought to bear upon these problems. This is true because the design of the experiments recognized and sought to utilize the obvious fact that cells pass through evident phases during their development and that their salt and water relations are a function of their exogenous requirements and endogenous capacities for further growth and development. Such work contrasts sharply with other studies which seem to assume that cells are merely “ready-made” with the attributes that they finally possess when all further growth has ceased. Therefore, to utilize observations that may be made upon the behavior of actively metabolizing, dividing, and enlarging cells the problem should be analyzed in a very different way. Attention should be directed, first, to the total solute concentratious in cells -not only to the concentration of specific ions or solutes. This approach minimizes the importance of the even dramatically large accumulation (or absorption) ratios of specific ions; it diverts attention from the “isothermal work,” i.e., W = RT I n C,/C,, required to transport 1 gram mole of a given ion from the outer to the inner and greater concentration; but it focuses attention,

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instead, upon the t o t d solute concentration that predetermines the “activity” of the internal cellular water. In these terms, cells do not harness specific ion or solute pumps to specific solute concentrations; they rather function as “water pumps” which control the local activity of water, create an internal aqueous volume, and necessarily regulate “solute concentrations,” which are in turn partitioned between different available substances (cf. Section V, D) . Thus, by focusing attention upon the total osmotic value of cellular fluids (and its derived term the “diffusion pressure deficit” ot the “suction pressure of the cell”) which, in comparison with the ambient medium, determines whether or not water will be absorbed or expelled, the form of analysis is diverted from one in which electrolytes play the sole, or even the overriding, tole. This in turn places limitations upon concepts that a priori assume that ions are the chief solutes “accumulated” and that the problem is, therefore, inevitably and solely one of EMF’S, of specific ion pumps, and so on. By contrast, the behavior of those cells of plants that simultaneously attract water and conserve solutes, especially those solutes stored for use in metabolism, should be interpreted in terms of factors that regulate their metabolism and growth. Finally, by recognizing that the problem begins with cells that can and do divide, only to end with cells which, having enlarged, may have either lost these proclivities or can regain them, the interpretations necessarily diverge from those that are modeled on equilibria, or even on steady states which simulate equilibrium and to which equilibrium thermodynamics are held to be applicable. By contrast, the growing system should be recognized as one that requires analysis from the standpoints of nonequilibrium thermodynamics in a system in which energy considerations furnish the unifying principles or conimon denominator. Conceived or approached in these terms, the problem becomes one of “balancing-off ’ various energy components or moieties in cells, namely, the “unstructured free energy of solute concentration” in a vacuole against some energy moiety by which it was created. In part, this may require the use or “outlay” of metabolic energy, i.e., energy that may be temporarily stored or available as ATP, but it may require the overall, seemingly wasteful, consumption by the cells of far greater amounts of metabolic energy than that which ever reappears as the “free energy” of the accumulated solutes and which is cd~culablein t e r m of the isothermal work or the Nernst equation. This apparently wasteful use of energy is implicit in maintaining the improbable living system in being. One can visualize the thermodynamically irreversible, rapid turnover and utilization of metabolites (sugars) which build the structure of cells and also create, concomitantly, a “free energy component represented by concentrated soluble molecules” which is the counterpart of the energy component that ap17ears as “structured free energy” or negative entropy of the cytoplasm.

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Thus, two contrasted events (the formation of new structure in the cytoplasm of dividing cells and the increase of enclosed volume with minimal new structure in enlarging cells) are mutually complementary aspects of cell growth. Between these two events, there is a needed turnover of metabolites which results in end products that are excreted (CO,), in energy converted to heat (which is lost), and which, overall, represents the inefficiency with which the organism converts chemically stored energy via the sun into biological structure that can persist. The energy relations of solute accumulation may also require, however, the concept that as the system grows, replicates, and develops it communicates a driving force to the movements of water and solutes which is the consequence of entropy relations in the growing cells. This “driving force” is then a feature of the entire system, not a required property of an inherently partial process involving water and/or solutes as illustrated in the following analogy. It would not be, for example, thermodynamically reasonable or necessary to equate the work done to carry a letter in the mail bags of a moving train or aircraft directly to the energy needed to furnish the power, or to any specific part of the machinery of its power plant-but its destination and rate of arrival there is an intimate function of that system in being, of its sources of direction, and of the overall pace of its operations. This earlier analogy (Steward, 1937) appropriately updated, still seems apt. This reasoning gives to those visible (e.g., cell replication) or measurable (increased protein content by synthesis) criteria of complexity a special significance as indications of the working of an “entropy machine.” Such an analysis also points to the significance of “turning points” in cellular ontogeny when growth is first expressed largely by cell multiplications ( - S ) , i.e., replication with but little increase of cell size, and later when it is expressed largely in terms of cell volume and of a protoplast which perforce emphasizes surface rather than bulk and an enclosed cell volume, with its concentrated solutes ( + F ) , rather than replication. A necessary part of this approach is a modified view of the protoplasmic membranes and their role. Essentially they set off the boundaries of different compartments-relatively unstructured compartments such as the vacuole and the ambient medium, and highly structured compartments such as the ground cytoplasm. The essential vital metabolic functions (e.g., respiration, nitrogen metabolism, and so on) reside in the cytoplasm and its organelles, whereas the principal repository for the soluble substances in bulk is presumed to be in vacuoles. These soluble substances are those not immediately required for the anabolic functions of cells, for it is now clear that the intermediates of synthesis do not usually, or necessarily, mingle with their accumulated stored counterparts (Steward and Bidwell, 1966) ; vacuoles may also contain the by-products of metabolic “turnover” or the inorganic salts or ions absorbed from the ex-

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ternal medium. Finally, the cell wall permits an entire protoplast to exist in a dilute ambient solution in which, with its internal concentrations, it would otherwise burst since its wall pressure balances the diffusion pressure deficit between the vacuolar and external fluids. The structural properties of plasma membranes may facilitate or hinder movements of specific solutes, and reactions that occur there may be conducive to increased rates of passage of solutes. Thus, such concepts as those of perineases and/or other carriers, “statistical pores” in membranes, or the electrical potentials across membranes may all be useful in interpreting rates of movement in special cases. Nevertheless, the ultimate driving forces that determine whether a given vacuole or internal aqueous phase can be a “source” or a “sink” depend on events in the cytoplasm of cells and the relations between competing cells or communicating protoplasts. Obviously, there is a wide range of possible salt and solute concentrations here, which in the isolated cells or tissue explants (Section V ) is a demonstrable function of growth regulatory substances and systems. In the intact plant, however, it is also a function of the supply and demand between cells at “sources” and “sinks” which, in turn, are subject to external environmental control even though it may also be mediated by regulatory substances. Thus, cells can no longer be competely conceived of as heat engines doing work by the production of metabolic heat and its conversion into work; they can no longer be fully comprehended even as “molecular machines” which produce certain molecules (reduced coenzymes, phosphorylated carriers of metabolically released energy) that cause reactions (synthesis of high energy molecules) and, having done this, “keep the metabolic wheels turning, by breakdown and turnover.” In order to grow, cells seem to function as “entropy machines.” These nonequilibrium machines do work with minimal heat change. By the creation of complexity, i.e., of “structured reduced entropy,” they mediate movements of solutes and water into a cell system which, when it does not replicate, must perforce increase its outer surface to secure the solutes for its autotrophic nutrition and also develop its inner volume with an attraction for water, both to maintain turgor and to “spread out its limited cytoplasm in a very thin layer.” In this sense, essentially autotrophic totipotent plant cells behave as “potassium-mediated entropy machines.” The system is described as “potassiLinidependent” advisedly. In the first place, there is no element more intimately and generally (albeit obscurely) concerned with growth and protein synthesis than potassium (Steinbach, 1962; Evans and Sorger, 1966) and, furthermore, there is no ion more generally “accumulated” by plant cells from their environment than potassium. Such cells also convert randomness into reduced entropy, or complexity, and grow first by replication, or later by an increase of enclosed

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volume with maximum surface and, as they achieve the latter, the driving forces of growth must be mediated through the properties of water. In this context, the possible role of potassium as a key solute which niay regulate the properties of the water of the ground cytoplasm, i.e., its fluidity versus viscosity or the amount which is liquid versus that which is more structured, may be relevant as indicated by Tracey (1969). Modern measurements on the properties of cytoplasmic water (Cope, 1969) and on “poly water” (Lipincott et al., 1969) and ideas on the degree of order resulting from “ice-like” configurations that may exist in liquid water (Berendsen, 1966) are indicative of the ways in which even water may contribute to the “ordered array” of molecules in cytoplasm; as these balances are disturbed, energy and entropy changes are implicit. When cells increase their volume and acquire their solutes, they essentially activate, not specific solute or ion pumps, but “water pumps” in the sense that they utilize their endogenous or innate capacities mediated by their exogenous nutrients and stimuli, to create the internal demand for total aqueous volume, for total solute concentration, and finally for nonmetabolizable, soluble, inorganic salts. Thus, the de novo formation of vacuoles (cf. Fig. 2 ) and of their first complement of solutes is essentially an active secretion, and in the sense that water must occupy the vacuolar space, an active internal secretion of water. Hence, the inorganic salt concentrations, or the ionic imbalances, or the electric potentials across membrane, i.e., the EMF’S, between phases with different concentrations of salts, should be seen as the “aftermath,” that is, the less direct consequences and not the primary causes, of the original solute concentrations that occurred. The problem, therefore, begins with cells that can grow and which are in an environment that permits them to grow. It ends with quiescent mature cells which either maintain the solutes they have acquired or which merely exchange these in response to superimposed factors in their environment, to the demands of cells in other regions of the plant body, or to endogenous changes as they inevitably decline toward senescence and death when they will assuredly reach true diffusion equilibrium with the ambient medium. The concept now is that living plant cells have the genetic instructions to perform all these acts, but they nevertheless require that the nutrients that can nourish them in so doing need to be engaged by exogenous growth factors or stimuli. In all this, cells cannot be regarded as equilibrium systems. The cytoplasmic increase in bulk, which growth requires, converts the free energy (ultimately from sunlight) and the random, small molecules of their environment into structured complexity with its attendant reduction of entropy ( - 3 ) . The “built-in” capacity of cells to grow, which is their heritage from the zygote, is thus expressed in energetic terms by the reduction of entropy they achieve at the expense of the free energy of the complex (or substrate) molecules they

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consume, even as catabolism is the price paid for anabolism (see Fig. 1.1, Steward, 1966). As long as cells replicate and divide, their decrease of entropy (-S), acquired in terms of their complex substance, may be redirected and expressed in terms of a free energy moity (e.g., in unpaired DNA or unfilled protein templates) which permits them to repeat the process at the next division. After replication and separation of daughter cells is no longer conspicuous, a “turning point” is reached-this may be identified (Fig. 5 ) with the manufacture of protoplasmic surface instead of bulk as preformed cells now enlarge. Throughout the development of a given cell, however, the energy (A) represented by complexity within the cell should be seen as “shared” among its component parts and “balanced” among all phases within the boundary of that cell, i.e., any --S of increased complexity in the organized cytoplasmic phase is to be “balanced-off’ against the free energy of solute concentration (+F) in the newly formed, unorganized solution of the vacuole. In order to exist in a steady state of turgor, plant cells balance out the energy difference of their contents and the ambient medium, as measured by their attraction for water, by the energy content of their stretched cell walls. These events may be mediated by the so-called “cytokinins” which seem to produce protoplasmic volume by synthesis with minimum surface (externally or internally) or the so-called auxins which seem to produce maximum protoplasmic surface (internally and externally) with a minimum of new protoplasm. However the growth-regulating substances may act in detail, they seem to influence or activate by the very form they create the balance between cytoplasm and vacuole and between cell and medium with respect to water and to solutes. These complex events will embrace many different energy components or moieties in which free energy of internal solute concentrations is increased, in which entropy (represented by complex molecules, proteins, enzymes, and by structured compartments and organelles) is reduced. It is the function of the exogenous growth-regulating substances and the challenge of growth induction in quiescent cells that they engage the otherwise idling, uncoupled, suppressed metabolic machinery and direct it to the ongoing processes of growth which carry with them such far-reaching implications for the absorption and internal secretion of water, solutes, ions, and inorganic salts. Such theoretical problems are therefore faced anew, hopefully to outline concepts that potentially embrace the entire range of phenomena and lead to a new and more broadly based general schema. To do this in ways that embrace the controlled movements and interactions of such diverse solutes as nonelectrolytes, weak electrolytes, and the charged ions of inorganic salts there must be some common principle, some common denominator. This will align complexity in cytoplasm with content of solutes in

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vacuoles and replication at limited bulk with enlarged internal aqueous volume in a cytoplasm that no longer replicates but enlarges its surface, both internal and external. Such disparate events must be linked through their energy relations and their impact on the balance of solutes and water, which is more generally mediated than by electrical events, and must be expressed through the activity of water. The “built-in” capacity to grow, which harnesses nutrients to metabolism, requires “division of labor” between organelles in cells and between cells in organs. “Division of labor” may be translated into a concept of “energy dis-mutation” by which the complexity of organization (-5) , as in cytoplasm, is incompatible with small molecules being randomly distributed (+S) therein. In the economy of essentially autotrophic plants, however, their solutes are conserved; their surfaces of contact with their ambient media are enlarged and they develop an unstructured, internal volume in which the entropy (+S) of the random small molecules and ions that are incompatible with the organization of the cytoplasm may appear as the “free energy of concentration” (+F) of solutes in the vacuole. Thus, a positive outflow or export of “randomness” ( + S ) from the cytoplasm in the form of solutes which preserves the degree of order (-5) in the cytoplasm also builds up free energy ( + F ) in the vacuole and, in this way, work is performed (--TAX). In the outcome, a cell preserves order in the cytoplasm by exporting “randomness” to the vacuole (Fig. 1 5 ) , and the price is paid in terms of heat transfers under the isothermal conditions that prevail. The overall process is irreversible as there is an increase in entropy represented in part by the molecules that appear in free solution in the vacuole. A formal theoretical approach to these nonequilibrium problems is entirely beyond the scope of this article. In general terms, however, it visualizes that cells partition their available energy between two moieties that are seen in apposition, the “structured free energy” (-5) of their ordered cyptoplasm3 and the “unstructured free energy of concentration” in their vacuole. The synthesis, the metabolism, the heat release, the usable energy in ATP, the electrical events which influence or result from ion movements, all these are part of the detailed 3 Other areas of thought and theory seem to require an energy term or component which is an expression of the degree of organization in biological systems. Thus, genetic models and their relation to cell differentiation are now being conceived (Goodwin, 1963) in terms based on oscillators, in which the oscillator comprises the alternative production of a product and the feedback stimulus to its further control. A term “talandic temperature” has been devised to represent the degree of that coupled feedback control and denotes a thermodynamic energy term that covers the organizational complexity of such a system. Although limited to the periodic phenomena interpretable as oscillators, and which are applicable to biochemical genetic systems, there is here again a need for an energy term to represent the role of complexity and organization in the cell which, in the broad sense, we have referred to as negative entropy ( - S ) .

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machinery, but they do not explain the essential driving force without which the solutes would not move. Although these ideas are not expressed here in rigid theoretical terms, they can be visualized more generally in Fig. 15. Here the complementary effects in the behavior of nonelectrolytes and of electrolytes should be recognized, as well as the contrasting or complementary events as cells divide or expand, the implications of anabolism succeeded by catabolism as in metabolic “turnover” in cells, and the “source-sink” relationships that must obtain between the cells in different parts of the plant body. Progress along these lines should illuniinate many rapid turgor changes (whether attributable to stimulated movements of water or solutes) that permit “motor” cells to function as in certain responses to stimuli (e.g., of guard cells as in stomata1 movements, of motor cells in leaf rolling and “sleep” movements, as well as of the cells strategically placed in the regions of tropistic responses to stimuli). If this approach has merit, then it is unprofitable to equate ion movements alone with measurements of EMF-the EMF’S are as apt to be the coiz.iegzietzce of the conditions that make the ions move as their came. It is equally unprofitable to equate stoichiometrically the ions moved to any special moiety of respiration (i.e., the CO, produced) which is peculiar to the actual intake of ions. The initial driving force ultimately requires the nonequilibrium thrust that growing cells receive to put their D N A to work, to replicate, and to grow. As this creates orderly structured negative entropy a t one point, it carries with it, inevitably in plant cells which grow autotrophically and absorb simple solutes, the requirement to store solutes with an increase of “unstructured free energy of their concentration.” It is inherent in the nature of plant cells, however, that the solutes, whether of endogenous or exogenous origin, are secreted internally. This device provides simultaneously for maximum contact of the outer surface with the environment, which is a necessary corollary of autotrophic nutrition, and a maximal internal volume that both allows metabolic by-products to be stored against reuse and to maintain the stability of the protoplast by turgor. The contrast here between animal cells and plant cells is very pertinciit. Tile former discharge their metabolic waste products into their environment, whereas plant cells retain them (other than CO, or minor volatiles) internally, largely in vacuoles, in forms in which they can be reused. It is crucial to the understanding of the problem so conceived that it be investigated on systems capable of carrying out the internal secretion of solutes in cells that build LIP their concentration levels de n o w . One cannot expect to understand this process by endowing the experimental system with properties too restricted for this process to occur. This inevitably means the facing, dif-

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MEDIUM

CYTOPLASM Phase I of uptoke

VACUOLE Anabolism

/ /

/

Entry of small molecules and ions

/

Synthesis(-S) creates special “sites“ or /’ “places” for specific solutes

/ /

4

/L ’

,/

A

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releases solutes

Solutes may be removed from the vacuole provided they are replaced with other solutes

(+s)

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estricted fflux is

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f

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/

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//

J

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respiration

+$e Entrop selective

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Acts to increase free energy in ihe vacuole due to solute concentration, to reduce the internal activity of water and so develops an internal attraction for water that creates and maintains turgor

FIG. 15. The essential feature of this figure lies in the ability of long contorted molecules to provide ordered space within their conformation in which specific solutes may be held [there is precedent for such general binding of potassium to cytoplasmic proteins in E . coli, in the work of Damadian (1969)l; and, conversely, as they are broken down to release these solutes into free solution. As such events occur cyclically in the cytoplasm, solutes could be alternately held and released. There is precedent for the conformational changes in long, fibrous molecules being harnessed to the doing of work and, as in the “muscle machine” of Steinberg et ul. (1966) [cf. Lear (1966)1, to the facilitated movements of salt and of water. In the “muscle machine,” salt moves in one direction and water in the other and in this model, the reversible adjustment of the fibers to salts and to water creates the conformational changes that are capable of being transformed into work. The ultimate driving force in the model is a salt concentration and the resultant ion movements are with, rather than against, a free energy gradient. Nevertheless, if the driving force were to be the actual creation of complex molecules, which in their form could associate solutes, and the energy input were that required for synthesis, then one could visualize a machine working in the opposite direction from the “muscle machine.” As breakdown of the complex molecules occurred, the previously associated solutes would be released so that the cyclical events of synthesis and turnover could build up concentration differences. In this way, a machine could incorporate energy into its structure to create a situation in which entropy would be reduced and otherwise random solute molecules would be locally removed from free solution to reappear later as a free energy of concentration when the ordered, complex molecules so synthesized were broken down as visualized above.

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ficult or unpopular as this may seem to be, of the system that can grow and develop with all its complications. We are now witnessing a curious return to an earlier rigidity that strove to evade the fact that cells really grow. Prior to 1925-1930, the solute movements in cells were studied as though they were independent of all metabolism, and for awhile the Donnan equilibrium was the favored approach. Active transport, as currently defined and studied, has also become a highly restricted and artificial concept. By definition, or tacit consent among those who subscribe to this point of view, it is to be investigated only in cells or under conditions that preclude growth, in systems which as nearly as possible operate reversibly with respect to ion transfers and, hopefully, obey the essentially equilibrium equations which then apply. By convention, the system is not permitted the freedom of any total or irreversible change, even though any living metabolizing system is constantly changing, even in its complex molecules, by turnover. Again, by tacit consent, the organic solutes, sugars, weak acids, amino acids and amides, hexitols, and so on, do not enter into the overall interpretations of solute balance even though they are soluble and may be retained within the cell, often against zero concentrations outside, and they are as effectively accumulated or as actively transported into aqueous inclusions as are the ions. These conventions, if accepted, effectively ensure that the essential problem cannot be solved because, in this way, it i s ignored. Thus, active transport as currently investigated, is so remote from the nutritional reality of growing plants that it may have but little bearing upon it. Any satisfying explanation should explain how cells acquire and retain the total osmotic concentrations on which their turgor depends, whether the osmotic value is made up of organic or inorganic solutes. It should explain the paramount role of potassium as the essential inorganic ion and of carbohydrates, organic acids, and simple nitrogen compounds as the essential organic ones that comprise the osmotically active solution. Moreover, a satisfying concept should bridge the enormous gap between the developing vacuole system of active growing cells as in growing points or free cell cultures, on the one hand, and, on the other, such sluggish, virtually salt-saturated, special cases as the relatively mature almost nongrowing systems such as large Nztellu cells or Vdoniu vesicles and the equally nongrowing system of long-washed, nutrient-limited, growth factor-limited discs of carrot root xylem. In the latter systems, the preformed “sinks” are full; they can make little effective call on their external solution unless their preformed steady state is disturbed by a change in their environment. But relative maturity of living cells in the angiosperm plant body does not preclude all further participation in the internal nutrition of the plant body; this is true because the still-growing cells of the active growing regions can deplete older ones of their solutes (salts and nitrogen compounds). Thus, salts may be moved to active centers (or “sinks”) while the osmotic balance in the

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depleted cells may be restored either by renewed absorption from outside or, more often, by the return of other translocated organic solutes to the older cells so depleted. Admittedly this somewhat unconventional review does not neatly solve the problems of the accumulation of solutes or of their active transport in plant cells. Insmuch as it summarizes current attempts to do so and still finds them wanting it requires a fresh approach. It is suggested that this new approach should pay close attention to cells that create their internal solute concentrations de no00 as they grow. In fact, it is the behavior of such cells that has prompted the views here expressed. If the concepts here outlined help to bridge a long standing gap between experiments that are often performed because they seem interpretable and the events that so obviously occur in cells, even though they seemingly defy easy explanation, then the purposes of this review will have been served.

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Author Index Numbers in italics refer to the pages on which the complete references are listed.

A Abbott, B. C., 299, 368 Abbott, N. J., 59, 62, 63, 66, 85 Adams, P., 262, 272 Adrian, R. H., 82, 85 Aggarwal, S. K., 128, 129, 152, 165, 167 Aggarwal, U., 128, 129, 167 Albers, R. W., 303, 365 Alexander, hl., 180, 207 Alexander, M. L., 153, I65 Allfrey, V. G., 172, 175, 206 Allison, A. C., 102, 103, 122 Amalric, F., 174, 184, 188, 205, 209 Amano, M., 174, 205, 208 Ames, A,, 111, 67, 85 Amici, A,, 121, 123 Ammirato, P. W., 316, 370 Amsterdam, D., 201, 208 Anderson, B., 302, 365 Anderson, E., 51, 86 Anderson, E. G., 142, 163 Anderson, J. D., 299, 368 Anderson, R. M., 191, 207 Andres, K. H., 200, 205 Anraku, Y . , 302, 365 Ansfield, F. J., 194, 207 Antropova, E. N., 148, 165 Aoki, M., 201, 206 Aoki, S., 120, 124 Aoyama, Y . , 201, 206 Arendell, J. P., 174, 210 Arisz, W. H., 298, 365 Arnold, J. M., 39, 42 Arnold, N. J., 189, 205 Arvanitaki, A., 74, 85 Asai, J., 293, 366 Ashhurst, D. E., 47, 48, 49, 61, 67, 85 Ashworth, C. T., 189, 205 Asrijan, I. S., 175, 209 Atkins, L., 161, 165 Atkinson, M. R., 307, 327, 365

Baker, W. K., 149, 165 Balis, M. E., 397, 205 Balogh, K., Jr., 225, 236 Bang, F. B., 170, 205 Bannasch, P., 189, 210 Barigotzi, C., 149, 165 Barka, T., 177, 191, 205, 210 Barnes, D. W. H., 234, 236 Barr, R. A,, 337, 354, 365 Barton, R., 93, 122 Bartsocas, C. S., 161, 165 Bassett, C. A. L., 228, 237 Bauer, A,, 170, 205 Bauer, H., 112, 122 Batham, E. J., 56, 85 Bawden, F. C., 100, 115, 122 Beadle, G. W., 142, 150, 152, 153, 154, 158, 165, 168 Beard, J. W., 177, 207 Beaver, D. L., 203, 205 Beenakkers, A. M. T., 2, 43 Beetschen, J. C., 201, 206 Beevers, H., 327, 337, 365, 366, 367, 369 Belling, J., 140, 165 Bellini, O., 180, 197, 206 Benditt, E. P., 177, 210 Benedeczky, I., 193, 208 Bensch, K. G., 94, 96, 122 Bentley, R. M., 128, 167 Berendes, H. D., 141, 165 Berendsen, H. J. C., 359, 365 Bern, H. A., 51, 57, 88 Bernhard, W., 170, 172, 174, 177, 179, 181, 183, 185, 188, 189, 198, 200, 201, 205, 206, 207, 208, 209 Berry, W. E., 310, 311, 341, 343, 365, 3 70

Best, R. J., 100, 101, 122 Bevelander, G., 225, 236 Bezem, J. J., 2, 4, 13, 30, 35, 36, 41, 44 Bhuyan, B. K., 180, 197, 198,205 Bidwell, R. G. S., 311, 337, 354, 357, 365, 369, 370

B

Bierling, R., 175, 205 Binding, H., 92, 108, 122 Bingham, P. J., 217, 220, 226, 236, 237

Bacon, J. S. D., 327, 367 Baker, P. F., 79, 85, 303, 365 371

372

AUTHOR INDEX

Birt, L. M., 337, 365 Blaauw-Jansen, G., 3, 42 Blackman, S. S., 203, 205 Bloom, W., 227, 237 Blount, R. W., 299, 365 Bluemink, J. G., 4, 12, 42, 43 Boer, H. H., 48, 57, 85 Bogdanov, Y. F., 148, 165 Boistel, J., 79, 8 6 Bond, V. P., 217, 218, 236 Bonting, S. L., 303, 365 Borysko, E., 170, 205 Bosisio-Bestetti, M., 181, 210 Bouteille, M., 201, 206 Bowes, B. G., 111, 122 Bowling, D. J. F., 299, 365 Boycott, A. E., 36, 42 Bracken, C. E., 111, 122 Bradbury, S., 63, 8 6 Bradfute, 0. E., 92, 97, 122, 123 Bradley, M. V., 265, 271 Branton, D., 290, 365 Bratell, I. A,, 217, 220, 226, 236 Brenner, S., 197, 208 Bretschen, M. S., 137, 165 Bretschneider, L. H., 1, 13, 16, 17, 33, 37, 42 Bridges, C. B., 135, 142, 145, 151, 152, 160, 165 Bridges, J. B., 215, 229, 236 Bridges, P. N., 145, 165 Briggs, G. E., 297, 298, 300, 365 Brinton, C. C., 141, 165 Brown, E. H., 128, 165 Brown, F. A,, 64, 87 Brown, S. W., 161, 165 Browyer, F., 301, 365 Broyer, T. C., 295, 333, 366 Brunnekreeft, F., 2, 22, 43 Bubel, H. C., 197, 206 Bucciante, L., 182, 206 Buchanan, J. M., 179, 207 Bucher, N. L. R., 191, 206 Bucz, B., 104, 123 Buffe, D., 175, 209 Bullock, T. H., 47, 53, 8 6 Bungenberg, de Jong, H. G., 7, 42 Burdick, C. J., 135, 167 Burger, M., 301, 365

Buring, K., 230, 231, 236, 238 Burstrom, H., 287, 367 Burton, R. F., 64, 74, 75, 76, 86 Burwell, R. G., 229, 235, 236 Busch, H., 170, 172, 174, 175, 177, 179, 182, 189, 191, 200, 206, 208, 209, 210 Butcher, D. N., 111, 122 Butt, V. S., 337, 36s Byrde, R. J. W., 111, 122 Byvoet, P., 172, 189, 206

C Cabrini, R., 224, 236 Caffrey-Tyler, R. W., 214, 236 Caldwell, P. C., 303, 36s Calendi, E., 180, 197, 206 Callan, H. G., 140, 165 Calonge, F. D., 111, 122 Camus, G., 262, 263, 264, 271 Caplan, S. R., 308, 365 Caplin, S. M., 310, 311, 317, 36S, 369 Caputo, C., 70, 88 Caravaggio, L. L., 303, 365 Cardot, H., 74, 85 Carlson, A. D., 53, 65, 66, 69, 76, 77, 78, 79, 82, 86, 88 Carlson, H. L., 153, 165 Carnahan, J., 141, 165 Cameiro, J., 222, 236 Case, D. B., 259, 272 Caspersson, T., 170, 20G Cassingena, R., 172, 182, 209 Catchpool, J. F., 291, 365 Cattaneo, S. M., 217, 236 Cessac, G. L., 359, 367 Chamberlain, S. G., 75, 77, 8 6 Chance, B., 291, 368 Chandler, W. K., 73, 86 Chandley, A. C., 134, 151, 16>, 166 Chapman-Andresen, C., 92, 93, 122 Chase, M., 100, 122 Chase, S. W., 230, 237 Chauveau, J., 200, 208 Chevremont, M., 193, 206 Chevremont-Combaine, S., 193, 206 Chiang, K. S., 142, 165 Chiga, M., 181, 207 Chino, M., 160, I61

373

AUTHOR JNDEX

Chomyn, E., 51, 86 Chovnick, A,, 142, 165 Christensen, J. J., 289, 367 Clement, A. C., 39, 42 Clever, U., 152, 166 Clifford, J. I., 181, 200, 206 Clowes, F. A. L. 93, 122 Cockerell, R. S., 292, 305, 366 Cocking, E. C., 90, 91, 92, 93, 97, 99, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 119, 121, 122, 123, 286, 366 Coggeshall, R. E., 46, 48, 52, 55, 56, 59, 62, 63, 65, 8 6 Cohen, G. N., 297, 301, 366 Cohn, Z . A,, 99, 122 Cole, K. S., 79, 8 6 Collander, R., 328, 366 Collins, G. H., 59, 62, 65, 87 Comings, D. E., 142, 166 Confer, D . B., 191, 210 Cope, F. W., 359, 366 Coraboeuf, E., 79, 86 Cornwall, C. C., 203, 209 Counce, S. J., 38, 42 Court Brown, W. M., 161, 166 Cowden, R. R., 59, 62, 65, 8 7 Crane, J. C., 265, 271 Creighton, H. B., 142, 166 Crelin, E. S., 228, 236 Cronkite, E. P., 217, 218, 224, 228, 236, 23 7 Cummings, M. R., 128, 162, 166 Curran, P. F., 308, 367 Curtis, A. S. G., 37, 38, 41, 42 Curtis, H. J., 79, 86

D D’Agostino, A. N., 181, 207 Dainty, J., 298, 299, 308, 326, 366, 367 Dalcq, A., 37, 42 Dales, S., 100, 101, 102, 103, 122, 123 DAlessio, G., 92, 122 Dalton, J. C., 79, 8 6 Damadian, R., 363, 366 Danielli, J. F., 40, 43, 298, 301, 366 Danielson, J., 262, 272 Darlington, C. D., 134, 161, 166, 167

Darnell, J. E., 116, 117, 123, 174, 182, 209 Das, N. K., 141, 166 Davidson, E. H., 135, 167 Davies, R. E., 326, 366 Davis, A R., 283, 328, 366 Degani, N., 312, 313, 315, 317, 366, 369 de Groot, A. P., 2, 3, 6, 21, 22, 42 de Harven, E., 49, 8 6 Delarue, J., 201, 206 DeMaggio, A E., 266, 272, 273 Demel, R. A , 5, 42 de Recondo, A. M., 181, 200, 207 Dessau, F. I., 207 de The, G., 201, 206 de Voogd van der Straaten, W. A , 224, 236 de Vries, G. A,, 3, 43 de Vries, L. G., 8, 43 Digby, J., 265, 266, 272, 273 Dijkstra, J., 118, 122 Dilly, P. N., 55, 86 D i Marco, A., 180, 197, 206 Dingman, C. W., 181, 210 Diver, C., 36, 42 Dobzhansky, T., 155, 156, 166 Dolfini, S , 149, 165 Dorigotti, L., 180, 197, 206 Doty, S . B., 225, 235 Douma, E., 48, 57, 85 Drinkwaard, A. C., 6, 7, 8, 44 Dubuc, F., 231, 238 Duchlteau, G., 65, 8 6 Dudok de Wit, S., 2, 43 Dumont, N. J., 51, 8 6 Duprat, A M., 177, 183, 201, 206, 209 DuPraw, E. J., 135, 166 Durwald, H., 181, 208 Duval, M., 74, 8 6

E Eakin, R. M., 177, 206 Eames, A. J., 259, 272 Eber, L , 193, 197, 207 Eckermann, G., 307, 327, 3 6 ~ Edward, B. A., 289, 370 Edwards, C., 79, S6 Edwards, G. A., 49, 86 Eisenberg, H. W., 181, 206

374

AUTHOR INDEX

Elbers, P. F., 4, 11, 12, 43, 288, 366 Eldefrawi, M. E., 66, 8 6 Eldjarn, L., 189, 208 Ellem, K. 0. A., 193, 208 Elliott, W. H., 198, 206 Ellis, R. J., 327, 367 Elsdale, T. R., 177, 201, 207 Elzam, 0. E., 297, 366 Emerson, S., 142, 16s Endo, H., 181, 201, 206 Engleberg, J., 183, 209 Ennis, H. L., 302, 367 Epstein, E., 297, 298, 328, 343, 366 Erhan, S., 143, 166 Errera, M., 181, 208 Esau, K., 104, 122, 239, 240, 243, 266, 272 Eschrich, W., 241, 243, 244, 245, 263, 272 Estrada-0, S., 292, 366 Etherton, B., 299, 366 Evans, H. J., 358, 366 Everett, N. B., 214, 236

F Fihrmann, W., 51, 8 6 Fan, D. P., 147, 167 Fanning, T., 148, 167 Farber, E., 180, 191, 193, 194, 197, 206, 209, 210, 211 Fawcett, D. W., 46, 52, 53, 55, 59, 62, 63, 65, 8 6 Fedorko, M. E., 99, 122 Feng, T. P., 65, 86 Fielding, A. H., 122 Firket, H., 193, 206 Fischnich, O., 262, 272 Fish, J. C., 161, 168 Fischman, D. A., 226, 236 Fitzgerald, 193, 197, 207 Fitzhugh, D. G., 189, 206 Fiume, L., 181, 206 Fliedner, T. M., 217, 218, 236 Florkin, M., 65, 8 6 Ford, E. H. R., 140, 168 Fosket, D. E., 263, 272 Foster, R. J., 299, 366 Fraccaro, M., 149, 165 Franke, W., 120, 121, 122 Frankenhaeuser, B., 72, 73, 8 6

Franklin, R. M., 197, 206 Frayssinet, C., 170, 172, 177, 179, 181, 198, 199, 200, 205,206, 207, 208 Freese, E., 197, 206 Freireich, E. J., 170, 210 Frenster, J. H., 172, 175, 206 Frey-Wyssling, A., 288, 366 Friedenstein, A. J., 230, 231, 232, 235, 236 Friedman, B., 230, 236 Frolova, G. I., 231, 232, 236 Frommes, S. P., 201, 211 Frost, L. C., 151, 167 Fry, R. J. M., 217, 218, 237 Furth, J., 180, 207 Furuyama, J., 181, 207

G Gaetani, M., 180, 197, 206 Galavazi, G., 265, 272 Gall, J. G., 172, 206 Galle, P., 203, 206 Ganotte, C . E., 185, 206 Gardner, D. R., 75, 87 Gardos, G., 303, 366 Gassner, G., 163, 166 Gautheret, R., 265, 272 Geduldig, D., 74, 86 Geelen, J. F. M., 4, 44 Geilenkirchen, W. L. M., 6, 8, 9, 10, 43 Gemski, P., 141, 163 Georgiev, G. P., 172, 174, 175, 206, 209 Gerasimov, V. D., 74, 75, 76, 86, 87, 88 Geuskens, M., 174, 181, 188, 206 Gharpure, M., 188, 206 Gimenez, M., 73, 88 Ginszburg, B. Z., 308, 366 Glass, M. D., 189, 205 Glinka, Z., 308, 366 Gloor, H. J., 148, 167 Goldberg, I. H., 180, 198, 211 Goldblatt, P. J., 180, 184, 193, 194, 197. 200, 206, 209, 210 Goldhaber, P., 230, 237 Goldman, D. E., 79, 87, 298, 366 Goldstein, D. A,, 308, 366 Goldstein, M. N., 175, 206, 207 Gomatos, P. J., 101, 122 Goodman, R. N., 113, 123

AUTHOR INDEX

Goodwin, 361, 366 Gordon, G. B., 94, 96, I 2 2 Gorman, J., 301, 366 Gowen, J. W., 131, 160, 166 Grady, H., 181, 191, 198,207,210 Graham, A. F., 174, 209 Granboulan, N., 170, 172, 174, 189, 200, 205, 206 Granboulan, P., 170, 174, 206 Grant, B. R., 337, 366 Grant, M., 307, 327, 365 Grant, W. H., 359, 367 Grasveld, M. S., 3, 5, 6, 43 Graubard, M. A., 150, 166 Graven, S. N., 292, 366 Gray, E. G., 53, 55, 86 Green, D. E., 291, 293, 305, 366 Greenberg, H., 174, 208 Greenberg, J., 179, 210 Gregory, D. W., 91, 92, 106, 122, 286, 366 Grell, R. F., 134, 151, 152, 162, 163, 166 Gropp, A., 170, 205 Guillery, R. W., 53, 86 Gulline, H., 300, 369 Gupta, B. L., 46, 51, 53, 59, 63, 78, 8G, 88

Gupta, D. N., 189, 207 Gurdon, J. B., 185, 188, 207 Gutknecht, J., 299, 366

H Hackett, E. M., 150, 167 Haeck, J., 6, 7, 8, 44 Hagen, C. E., 297, 298, 328,366 Haguenau, F., 170, 191, 205, 207 Hajek, J. V., 225, 236 Halvorson, H. O., 301, 302, 366, 368 Ham, A. W., 214, 237 Hama, K., 62, 86 Hamori, J., 59, 63, 86 Hanaoka, H., 230, 236 Hanneforth, W., 57, 86 Hanney, C. E. A., 265, 266, 273 Hansen, A. J., 120, 122 Harley, J. L., 337, 367 Harris, C., 181, 198, 200, 207, 209 Harris, E. J., 292, 305, 366 Harris, H., 174, 207

37 5

Harris, R. A,, 293, 366 Harrison, G. A., 232, 237 Harrison, J. A., 341, 352, 370 Hart, P., 158, 159, 166 Hartman, S. C., 179, 207 Harvey, E. N., 40, 43 Hay, E. D., 170, 172, 185, 188, 207, 226, 236 Hay, P. H., 231, 238 Hayes, F. R., 74, 86 Hayman, D. L., 151, 166 Haymore, B. L., 289, 367 Heald, W. R., 328, 368 Hedley-Whyte, E. T., 201, 207 Heideberger, C., 194, 207 Heine, U., 177, 207 Heiple, K. G., 230, 236, 237 Hejtnova, L., 301, 365 Hell, A., 181, 208 Hemingway, R. J., 303, 369 Henderson, S. A., 139, 147, 150, 166 Herman, L., 193, 197, 207 Herndon, C . H., 230, 237 Hershey, A. D., 100, 122 Hewitt, G. M., 158, 166 Hibbard, P. I.., 283, 328, 366 Higa, A., 147, 167 Higginbotham, N., 299, 366 Higgins, G. M., 191, 207 Higginson, J., 181, 191, 198, 200, 210 Hildebrandt, A. C., 119, 120, 122 Hind, G., 305, 367 Hinton, C. W., 133, 158, 161, 166 Hirai, T., 121, 123 Hird, J. R., 337, 365 Hirsch, J. G., 99, 122 Hirshberg, G., 271, 272 Hoagland, D. R., 282, 283 Hodgkin, A. L., 53, 72, 73, 77, 78, 79, 85, 86, 87, 303, 36fj Hodnett, J. L., 174, 182, 208 Hoffman, J. F., 303, 366 Hokin, L. E., 303, 367, 368, 369 Hokin, M. R., 303, 367 Holleman, J. M., 337, 367 Holter, H., 93, 122 Holtrop, M. E., 228, 237 Holtzman, E., 174, 208

376

AUTHOR INDEX

Hope, A. B., 298, 300, 304, 365, 367, 368 Hopkins, J. W., 182, 208 Horridge, G. A., 59, 63, 86 Horstmann, E., 59, 87 Hotta, Y., 139, 142, 147, 148, 166, 168 Howard, A,, 217, 237 Howard, E. F., 148, 166 Hoyle, G., 66, 87 Huang, C., 289, 368 Hudig, O., 3, 4, 6, 43 Hudson, W., 117, 124 Huggins, C . B , 230, 237 Hughes, R. D., 160, 166 Hugues, B , 175, 179, 201, 209 Huppert, J., 103, 123 Hurst, V., 262, 272 Hurwith, J , 180, 207 Huxley, A. F , 73, 87

H Isings, J., 2, 44 Israel, H . W., 113, 122, 293, 312, 354, 367, 370 Ito, M., 129, 139, 142, 147, 166, 168 Iyer, U. N., 179, 181, 207 Izatt, R. M., 289, 367

J Jackson, B , 207 Jacob, F., /39, 166 Jacob, J., 172, 174, 177, 207 Jacobs, W. P., 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 264, 266, 267, 269, 271, 272, 273 Jagendorf, A. T., 305, 367 Janne, J , 194, 209 Jedlinski, H., 118, 122 Jeffs, R. A , 265, -372 Jensen, W. A , 92, 122 Jezequel, A. M., 177, 179, 181, 207 Johansen, D. A,, 248, 272 Johnes, E. A., 112, 124 Johnson, P. L., 225, 236 Joklik, W. K , 116, 117, 122, 123 Jones, J. W., 177, 185, 188, 201, 207 Journey, L. J , 175, 206, 107 Julian, F. J., 79, 87 Junge, D., 74, 86, 87

Juniper, B. E., 93, 122 Jura, C., 38, 43 J u u ~ J., , 182, 207

K Kabus, B., 246, 272 Kadem, O., 308, 367 Kalifat, S. R., 201, 206 Kamiya, N., 90, 123 Karasaki, S., 174, 185, 207 Karney, D. H., 175, 181, 201, 209 Kassanio, B., 104, 119, 123 Kasuga, T., 201, 206 Katchalsky, A,, 308, 363, 367, 369 Katz, B., 73, 77, 78, 87 Kavanau, J. L., 291, 367 Kember, N. F., 217, 218, 219, 220, 224, 23 7 Kemp, T., 182, 207 Kennedy, E. P., 303, 370 Kent, A. E., 316, 370 Kepes, A., 301, 367 Kephatt, J. E., 92, 124 Kerkut, G. A,, 48, 51, 75, 77, 86, 87 Key, J. L., 337, 367 Keynes, R. D., 303, 3GS Khrushchov, N. G., 234, 236 Kidd, F., 328, 370 Killy, S., 183, 210 King, R. C., 127, 128, 129, 130, 131, 132, 133, 136, 142, 152, 161, 162, 165, 166, 167, l G 8 Kinsky, S. C., 5, 42 Kirk, S . C., 266, 272 Kisieleski, W. E., 217, 237 Kitsato, H., 299, 367 Kleczkowski, A., 118, 123 Kleinfeld, R. G., 188, 189, 191, 207 Kleinzeller, A., 301, 36> Klercker, J. A. F., 89, 108, 123 Klomp, H., 4, 6, 43 Koch, E. A , 127, 128, 129, 162, 166, 167 Koch, W. E., 228, 236 Kohn, H. I., 218,237 Koksma, J. M. A., 48, 57, 85 Kolattukudy, P. E., 327, 368 Komamine, A., 327, 368 Korn, E. D., 290, 367 Kostyuk, P. G., 74, 75, 86

377

AUTHOR INDEX

Koulish, S., 191, 207 KovHcs, E., 104, 123 Krasts, I. V., 76, 88 Krause, G., 38, 43 Kress, Y . , 203, 209 Krichevskaya, A,, 175, 209 Krishan, A., 201, 207 Kroeger, H., 152, 167 Kroon, D. B., 227, 237 Kuboda, Y.,181, 207 Kiinning, H., 259, 272 Kuffler, S. W., 55, 59, 64, 6 5 , 66, 61, 68, 69, 70, 71, 8 7 Kume, F., 181, 207 Kundig, W., 302, 365 Kupchan, M., 303, 369 Kurolesova, A. Y . , 231, 232, 236

L Lafarge, C., 170, 172, 177, 179, 181, 198, 199, 200, 205, 206, 207, 208 Laird, A. K., 189, 191, 207 Lalykina, K. S., 230, 231, 236 Lamb, B. C., ,151, 167 LaMotte, C. E., 247, 248, 249, 250, 251, 252, 253, 254, 259, 272 Lamport, D. T. A,, 109, 110, 123 Lane, N. J., 66, 71, 81, 83, 84, 87, 112, 207 Langlois, A. J., 177, 207 Lapis, K., 181, 193, 208 Lardy, H. A,, 292, 366 Laschi, R., 181, 206 Latham, H., 174, 209 Laties, G. G., 297, 326, 327, 331, 342. 367, 358 Lazarus, S. S., 201, 208 Lear, J., 363, 367 Leblond, C. P., 174, 205, 208, 222, 236 Le Breton, E., 177, 179, 181, 198, 205 LeClerc, G., 134, 167 Leclercq, J., 65, 8 6 Ledoux, L., 103, 123 Leeson, T. S., 214, 237 Leggett, J. E., 303, 3G7 Lehmann, F. E., 37, 43 Lehninger, A. L., 292, 305, 367, 370 Lejeune, J., 161, 168 Lerman, L. S., 180, 197, 208

Lesher, S., 217, 218, 237 Lethatn, D . S., 313, 367 Levedahl, B. H., 299, 365 Levenberg, B., 179, 207 Levi, J. U., 65, 85 Levinthal, C., 147, 167 Levy, H. B., 201, 208 Lewin, P. K., 181, 208 Lewis, E. B., 131, 167 Lezzi, M., 152, 167 Liapunova, N. A., 148, 165 Lindsley, D. L., 159, 167, 168 Ling, G. N., 298, 367 Lippincott, E. R., 359, 367 Lips, S. H., 331, 367 Littan, V. C., 135, 167 Little, C., 74, 87 Liu, Y . M., 65, 86 Lloyd, L., 140, I65 Loos, G. M., 327, 370 Loutit, J. F., 215, 234, 236, 237 Love, R., 193, 208 Lubin, M., 302, 367 Lucchesi, J. C., 158, 159, 167 Lukanidin, E. M., 175, 209 Lundegirdh, H., 287, 305, 367 297, 367 Luttge, 180, 197, 208 Luzzati, P., Lwoff, A., 184, 208 Lwoff, M., 184, 208 Lyndon, R. F., 352, 353, 354, 367

u.,

M MacCarty, W. C., 189, 208 McClintock, B., 142, 145, 153, 154, 166, 167 McConkey, E. H., 182, 208 McCready, C. C., 255, 256, 259, 262, 272 MacDonald, I. R., 299, 326, 327, 367 Macklon, A. E. S., 299, 365, 367 McLaren, A. D., 97, 123 McLean, F. C., 215, 227, 229, 233, 235. 237, 238 McLendom, D. E., 181, 208 MacLennan, D. H., 337, 367 McNelly-Ingle, C., 151, 167 MacPherson, S., 218, 219, 237 MacRobbie, E. A. C., 298, 299, 303. 367 I

378

AUTHOR INDEX

Maddrell, S. H. P., 46, 47, 49, 51, 57, 61, 63, 65, 79, 80, 81, 83, 84, 87, 88 Maddy, A. H., 40, 43, 289, 368 Maeno, T., 76, 87 Maguire, M. P., 151, 167 Magura, I. S., 76, 87, 88 Maiskii, V. A., 74, 75, 86 Malamy, M., 180, 207 Malhotra, S. K., 64, 88 Malkin, M. F., 181, 208 Maloof, F., 189, 208 Malzone, W. F., 59, 62, 87 Mankin, H. J., 229, 237 Mapes, M. O., 312, 316, 3 5 5 , 370 Marcus, P. I., 103, 123 Marinozzi, V., 170, 208 Marshall, J. M., 95, 123 Maruyama, S., 181, 207 Masson, F., 180, 197, 208 Matile, P. H., 95, 97, 123 Mauchline, J., 62, 87 Mawdsley, R., 232, 237 Maxwell, F. G., 272 Maxwell, R. E., 179, 208 Mayo, M. A., 99, 1 0 2 , 103, 123 Mednis, B., 102, 123 Meech, R. W., 75, 87 Mellon, D., 46, 51, 5 3 , 59, 62, 63, 65, 66, 67, 69, 76, 86, 87 Menzel, M. Y . , 145, 167 Menzel, R. G., 328, 368 Mesquita, J. F., 293, 368 Meves, H., 59, 73, 75, 86, 87 Meyer, G. F., 134, 141, 16S, 167 Michel, W., 92, 123 Micklem, H. S., 215, 237 Mighorst, J. C. A,, 5, 6, 43 Millar, F. K., 326, 352, 353, 369 Millen, J. W., 140, 168 Miller, C . O., 313, 368 Miller, D. M., 299, 368 Miller, L. R., 94, 96, 122 Miller, S. L., 291, 368 Minganti, A., 26, 30, 43 Mirsky, A. E., 1 3 5 , 167, 172, 175, 206 Mitchell, P., 294, 305, 368 Mitra, J., 3 5 5 , 370 Mitra, S., 147, 167 Miyai, K., 191, 193, 200, 208

Mizusarsa, Y., 121, 123 Moens, P. B., 129, 145, 167 Mohan Ram, H. Y . , 284,369 Mollenhauer, H. H., 92, 124 Molliard, M., 264, 272 Moncel, C., 181, 209 Moner, J. G., 188, 208 Monneron, A., 172, 175, 179, 198, 199, 200, 208

Monod, J., 139, 166, 297, 301, 366 Montgomery, P. O’B., 175, 179, 181, 197, 201, 208, 209

Moor, H., 95, 97, 123 Moore, J. W., 79, 87 Morasca, C., 183, 210 Morel-Maroger, L., 203, 206 Moreton, R. B., 75, 87 Morgan, C., 102, 123 Morgan, H. E., 301, 368 Morgan, L. V., 142, 167 Morgan, T. H., 134, 142, 167 Morley, F. H. W., 158, 168 Morohashi, Y . , 327, 36R Morrow, I. B., 263, 267, 268, 269, 270. 271, 272

Moscarello, M. A., 181, 208 Moses, M. J., 129, 135, 136, 167 Motomura, I., 37, 43 Mode, Y . , 175, 200, 208 Moyle, J., 305, 368 Miihlethaler, K., 112, 113, 118, 123, 288. 291, 366, 368

Mueller, G. C., 174, 210 Mueller, P., 289, 368 Mulder, M. P., 148, 167 Muller, H. J., 145, 167 Mundry, K. W., 106, 117, 123 Muramatsu, M., 174, 175, 182, 208

N Nachimias, V. T., 95, 123 Nakajima, Y . , 51, 53, 87 Nakken, K. F., 189, 208 Naqvi, S. M., 260, 261, 272 Narahashi, T., 72, 79, 80, 83, 87, 88 Narayan, K. S., 174, 177, 208, 210 Nash, D., 148, 167 Nebel, B. R., 150, 167 Neras, O., 110, 114, 123

AUTHOR INDEX

Nelson, A. D., 189, 206 Nelson, D. P., 289, 367 Nesbett, F. B., 67, 85 Neumann, K. H., 313, 355, 368, 370 Nevis, A. H., 66, 67, 87 Newman, G., 48, 51, 87 Newton, W. C. T., 134, 167 Nicholls, J. G., 59, 62, 64, 65, 66, 67, 69, 70, 71, 87 Nickel, V. S., 179, 208 Nicoletti, B., 159, 167, 168 Nishioka, R. S., 51, 57, 88 Northcote, D. H., 113, 123, 265, 272 Novikoff, A. B., 103, 123 Nur, U., 153, 167 Nygaard, O., 189, 208

0 Oberling, C., 170, 2 o j OBrien, R. D., 66, 86 Oda, A., 177, 208 Oertli, J. J., 297, 368 Okada, H., 301, 302, 366, 368 Okamura, N., 191, 210 Olenov, J. M., 104, 123 Oomura, Y., 76, 87 Oparin, A. I., 276, 368 Oplatka, A., 363, 369 Orkand, R. K., 71, 87 Orgel, H., 197, 208 Osmond, C. B., 327, 337, 368 Otsuki, Y., 120, 124 Owen, M., 217, 218, 219, 220, 221, 222, 224, 225, 226, 236, 237 Oyashi, M., 201, 206 Ozaki, S., 76, 87

P Painter, R. H., 272 Pallaghy, C. K., 300, 369 Parchman, L. G., 148, 166 Pardee, A. B., 302, 368 Paris, A. J., 2, 43 Park, C. R., 301, 368 Parry, G., 49, 64, 87 Parsons, P. A,, 151, 166 Pasteels, J., 37, 42, 43 Paul, J. S., 181, 208

379

Pading, L., 291, 368 Pease, D. C., 37, 43 Pecora, P., 197, 205 Pelc, S. R., 217, 237 Pelluet, D., 74, 86 Peltrera, A., 37, 43 Penman, S., 174, 208 Penniston, J. T., 293, 366 Perdue, J. F., 291, 305, 366 Perley, J. E., 259, 273 Perry, R. P., 174, 181, 182, 208 Persons, D. F., 291, 368 Petrakova, K. V., 231, 232, 236 Pfuderer, P., 104, I23 Phelps, H. L., 181. 210 Piatetzky-Shapiro, I. I., 231, 236 Pichon, Y., 79, 82, 83, 87 Picken, L. E. R., 46, 87 Pickett-Heaps, J. D., 112, 123 Pilet, P. E., 259, 272 Pitman, M. G., 300, 327, 365, 368 Plaut, W., 148, 166, 167 Plough, H. H., 151, 152, I67 Plowe, J. Q., 89, 90, 123, 286, 368 Poduska, P. R., 161, 168 Poel, L. W., 327, 368 Pojnar, E., 93, 103, 106, 107, 109, 110, 112, 113, 114, 119, 121, 122, 123 Pollard, J. K., 293, 337, 355, 368, 369, 370 Poole, R. J., 299, 368 Porter, P. J., 161, 16s Post, R. H., 230, 237 Potter, D. D., 55, 59, 64, 67, 68, 70, 71, 87 Potts, W. T. W., 46, 47, 49, 64, 65, 87 Poux, N., 293, 368 Power, J. B., 92, 99, 106, 113, 114, 123 Pressman, B . C., 292, 305, 366, 368 Prestidge, L. S., 302, 368 Preston, C., 311, 340, 341, 349, 350, 369, 3 70 Preston, R. D., 113, 123 Prevot, P., 341, 352, 368, 370 Price, J. M., 145, lG7 Priestly, J. H , 310, 368 Pritchard, J. J., 221, 224, 225, 237 Pritchard, R. H., 134, 167 Prosser, C. L., 64, 8 7

AUTHOR INDEX

380

Q Quastler, H., 217, 218, 236, 237 Queva, C., 269, 272

R Racela, A., 191, 210 Racker, E., 291, 368 Raimondi, G. R , 149, I65 Raina, A., 194, 209 Rains, D. W., 297, 366 Rake, A. V., 174, 209 Ramamurti, T. K., 311, 341, 370 Ramsay, J. A., 65, 88 Rao, K. V. N., 313, 355, 370 Rao, P. N., 183, 209 Rather, L. J., 189, 209 Raven, C . P., 1, 2, 3, 4, 5, 6, 7, 8, 12, 13, 16, 17, 18, 19, 20, 22, 24, 26, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43, 44 Ray, R. D., 229, 237 Raychaudhuri, S. P., 119, 123 Recheigl, M., Jr., 191, 209 Recourt, A., 13, 14, 16, 44 Redfield, H., 152, 167 Reddy, J , 180, 198, 200, 209 Reed, D. J., 327, 368 Rees, K. R., 181, 189, 200, 206, 209 Regen, D. M., 301, 368 Reggiani, B., 180, 197, 206 Reich, E., 180, 182, 198, 209, 211 Reid, R. A., 305, 368 Reinhold, L., 308, 366 Remmers, A. C., Jr., 101, I68 Rendel, J. M., 158, 167 Revel, J. P., 172, 207 Reynolds, R. C., 175, 179, 181, 197, 201, 208, 209 Rhoades, M. M., 154, 156, 167 Richards, A. G., 48, 8 s Richter, G., 203, 209 Rier, J. P., 264, 265, 266, 273 Riley, P. A., 203, 209 Rimbaut, C . , 175, 209 Ris, H., 172, 209 Riviere, M., 201, 206 Roberts, L. W., 263, 272 Roberts, P. A., 149, 153, 154, 156, 157, 158, 167

Robertson, J. D., 64, 88 Robertson, R. N., 287, 298, 300, 304, 307, 327, 328, 365, 368 Robineaux, R., 175, 181, 209 Robinson, R. A,, 225, 236 Robison, R., 225, 237 Roborgh, J. R., 2, 3, 43 Rodin, A. E., 161, 168 Rodriguez, T. G., 177, 209 Roeder, K. D., 66, 80, 88 Rogers, S., 104, 123 Rondez, R., 189, 209 Rose, H. M., 102, 123 Roseman, S., 302, 365 Rosenberg, T., 301, 368, 369, 370 Rosenbluth, J., 48, 51, 56, 59, 62, 63, 88 Rosenkranz, H . S., 181, 206 Rosenthal, A. S., 185, 206 Rosselli, L., 181, 209 Rossen, J. M., 142, 168 Roth, T. F., 129, 136, 139, 146, 168 Rothstein, A., 301, 302, 369 Rott, R., 197, 209 Rouiller, C., 189, 209 Rowland, G. F., 189, 209 Rubinson, A. C., 128, 129, 166 Rudin, D. O., 289, 368 Rudkin, G. T., 145, 149, 168 Ruesink, A. W., 91, 109, 123 Ruoho, A. E., 303, 369 Ruska, H., 49, 86 Ruttner, J. R., 189, 209 Ryser, H. J. P., 103, 123 Rytting, J. H., 289, 3G7

S Sabet, T. Y., 229, 237 Sabnis, D. D., 271, 272 Sacher, G., 217, 237 SadIer, H. D. W., 300, 368 Salmon, W. D., 194,210 Salomon, J. C., 189, 209, 210 Salomon, M., 189, 209 Salpeter, M. M., 66, 86, 113, 122, 293, 354, 367 Salser, J. P., 197, 205 Samarina, 0. P., 172, 175, 209 Samuel, E. W., 327, 370 Sandelin, K., 102, 103, 122

AUTHOR INDEX

Sandeman, D. C., 49, 62, 88 Sander, E., 104, 123 Sander, K., 38, 43 Sandler, L., 158, 159, 160, 166, 167, 168 Sarles, H. E., 161, 168 Scarpinato, B., 180, 197, 206 Scarth, G. W., 369 Schaffer, F. L., 197, 209 Scherrer, K., 174, 182, 209 Schlote, F. W., 48, 57, 88 Schmekel, L., 57, 88 Schoefl, G., 175, 179, 181, 209 Schofield, B. H., 225, 2.?6 Scholtissek, C., 197, 209 Schumacher, W., 246, 272 Scott, B. L., 217, 226, 237 Scott, B. I. H., 300, 369 Scott, T. K., 260, 261, 262, 272 Seal, P., 203, 209 Shalla, T. A,, 121, 123 Shankar Narayan, K., 177, 189, 191, 209 Shantz, E. M., 312, 313, 317, 337, 355, 369, 370 Shapiro, S. H., 201, 208 Shaw, G., 313, 369 Shaw, J., 49, 80, 88 Shaw, J. G., 118, 123 Shaw, J. L., 228, 237 Shaw, T. I., 79, 85, 303, 365 Sherman, F. G., 217, 218, 236, 237 Shemin, D., 181, 206 Sherudilo, A. I., 148, 163 Sherwood, E. R., 125, 168 Shetlar, M. R., 221, 237 Shiga, M., 177, 208 Shigematsu, A,, 121, 123 Shimokoriyama, M., 327, 368 Shinozuka, H. P., 191, 193, 194, 197, 200, 209 Shreeve, M. M., 181, 207 Shull, K. H., 194, 210, 211 Sidransky, H. P., 191, 193, 200, 209 Siefriz, W., 286, 369 Siegel, A,, 117, 123 Silverstein, S. C., 101, 103, 123 Simard, R., 170, 172, 174, 177, 179, 181, 182, 183, 184, 185, 188, 194, 197, 198, 200, 201, 205, 206, 207, 209

38 1

Simmons, D. J., 219, 237 Simon, G., 189, 209 Simon, S., 241, 272 Simoni, R., 302, 36J Simpson, L., 51, 57, 88 Sirlin, J. L., 172, 177, 207 Sirtori, C., 181, 210 Sisken, J. E., 183, 210 Sjostrand, S., 290, 369 Skoog, F., 313, 368 Skou, J. C., 303, 369 Slayman, C. L., 299, 369 Slayman, C. W., 302, 369 Slimes, M., 194, 209 Slizynski, B. M., 145, I 6 8 Slotnick, I. J., 175, 206 Smetana, K., 170, 172, 177, 189, 200, 206, 210 Smith, C. G., 180, 205 Smith, D. S., 47, 49, 60, 63, 73, 81, 84, 88 Smith, G. H., 161, 168 Smith, I., 174, 208 Smith, P. A., 127, 128, 129, 130, 131, 132, 133, 136, 142, 161, 167, 168 Smith, R. C., 194, 210 Smith, R. F., 128, 129, 166 Smithers, A. G., 352, 369 Smuckler, E. A., 177, 210 Snoab, B., 183, 210 Soding, H., 263, 272 Soeiro, R., 174, 211 Soga, J., 198, 210 Soldatti, M., 180, 197, 206 Solomon, A. K., 291, 298, 308, 366, 369 Soodak, M., 189, 208 Sorger, G. J., 358, 366 Sorokin, S., 264, 273 Sorokina, 2. A., 62, 64, 74, 75, 76, 88 Sotelo, J. R., 129, 141, 168 Spanswick, R. M., 299, 365, 369 Splittstoesser, W. E.,327, 369 Sporn, M. D., 181, 210 Spyrides, G. J., 302, 369 Stachelin, T., 191, 209 Stadelmann, E., 90, 123 Staehlin, A,, 112, 123 Stalfoort, T. G. J., 6, 44 Steele, W. J., 172, 174, 177, 182, 189, 191, 200, 208, 210

382

AUTHOR INDEX

Stein, W. D., 297, 301, 369 Steinbach, H. B., 358, 369 Steinberg, I. Z., 363, 369 Steiner, J. W., 181, 191, 193, 200, 207, 208

Stenger, R. J., 191, 210 Stenram, U., 177, 181, 191, 193, 194, 197, 210 Stephens, P. R., 55, 56, 88 Stern, C., 125, 134, 142, 152, 168 Stern, H., 139, 142, 147, 148, 166, 168 Stevens, B. J., 172, 177, 179, 210 Stevens, J. G., 188, 210 Steward, F. C., 113, 122, 280, 282, 283, 284, 293, 296, 304, 310, 311, 312, 313, 315, 316, 317, 326, 327, 328, 337, 340, 341, 343, 349, 350, 352, 353, 354, 355, 357, 360, 365, 366, 367, 368, 369, 370 Stiles, W., 277, 328, 370 Stiller, M. L., 337, 365 Stirpe, F., 181, 206 Stobbart, R. H., 49, 80, 88 Stolarck, J., 299, 369 Stonehill, E. H., 103, 123 Stowell, R. E., 184, 200, 210 Streiblova, E., 109, 123 Stromberg, R. R., 359, 367 Strong, F. M., 313, 368 Studzinski, G. P., 193, 208 Sturtevant, A. H., 36, 44, 142, 152, 153, 154, 156, 158, 166. 768 Sueoka, N., 142, 16S Sugii, M., 312, 337, 369 Sullivan, R. J., 180, 206 Sutcliffe, J. F., 282, 283, 296, 297, 304, 321, 327, 328, 340, 352, 369, 370 Suter, E., 189, 210 Suzuki,D. T., 150, 158, 159, 167, 168 Sved, J. A,, 141, 168 Svoboda, A., 110, 114, 123 Svoboda, D., 180, 181, 191, 198, 200, 207, 209, 210 Swift, H., 170, 172, 200, 210 Szybalski, W., 179, 181, 207

T Takahama, M., 177, 191, 210 Takayama, S., 201, 206

Takeba, I., 120, 124 Tamaoki, T., 174, 210 Tarlow, A. R., 303, 370 Tatum, E. L., 302, 369 Taylor, J. H., 135, 142, 148, 168 Teorell, T., 298, 370 Terawaki, A., 179, 210 Terzuolo, C. A., 79, 86 Thimann, K. V., 91, 109, 123, 327, 370 Thoenes, W., 189, 191, 210 Thompson, N. P., 243, 251, 254, 255, 256, 257, 258, 259, 260, 263, 270, 272, 273 Thompson, T. E., 289, 368 Threadgold, L. T., 93, 94, 95, 124 Tien, H. T., 289, 368 Tiepolo, L., 149, 165 Tolmacheva, A. A,, 230, 231, 235, 236 Tomlinson, G., 112, 124 Tonna, E. A,, 218, 219, 224, 228, 237 Toppozada, A., 66, 86 Torrey, J. G., 264, 273 Townsend, C. O., 108, 109, 124 Tracey, M. W., 359, 370 Treherne, J. E., 46, 47, 49, 51, 53, 57, 59, 60, 61, 62, 63, 65, 66, 67, 69, 71, 1 2 , 76, 77, 78, 79, SO, 81, 82, 83, 84, 86, 87, 88 Trim, A. R., 92, 122 Trippa, G., 159, 167, 168 Tristram, G . R., 48, 88 Troshin, A. S., 298, 370 l’rueta, J., 229, 218 Trujillo-Cen6z, O., 59, 88 Trump, B. F., 784, 200, 210 Turpin, R., 161, 168 Twarog, B. M., 66, 80, 88

U Ubbels, G. A,, 4, 13, 16, 3 0 , 33, 35, 36, 41, 44 Unuma, T., 170, 174, 179, 210 Uribe, E., 305, 367 Urist, M. R., 215, 229, 230, 231, 233, 2 3 5 , 236, 237, 238 Usenik, E., 201, 211 Ussing, H. H., 298, 370 Uzman, B. G., 201, 207

AUTHOR INDEX

V Valdovinos, J. G., 259, 273 Valentini, L., 180, 197, 206 van Deenen, L. L. M., 5, 42 van den Broek, E., 24, 44 van der Wal, U. P., 22, 24, 44 van Duijn, P., 148, 167 van Erkel, G. A., 6, 44 Van Harreveld, A,, 64, 88 van Loo, R. P., 4, 44 Van Praag, D., 181, 206 VanSteveninck, J., 301, 302, 368 Van Steveninck, R. F. M., 300, 326, 327, 3 70 van Zeist, W . , 6, 24, 44 Varcoe, J. S.,189, 209 Vaugh, D., 327, 367 Veprintsev, B. H., 76, 88 Verdonk, N. H., 4, 6, 7, 8, 28, 30, 19, 44 Verhoeven, L. A., 6>7,8, 44 Verney, E., 191, 200, 209 Vessely, J. C., 230, 236 Vethamany, V. G., 201,208 Vignais, P. M., 292, 370 Vignais, P. v . , 292, 370 Villalobos, J. G., 191, 210 Villa-Trevino, S., 194, 210,211 Villegas, G. M.,5 5 , 63, 72, 73, 79, 88 Villegas, L., 70, 72, 73, 88 ViIIegas, R., 5 5 , 63, 70, 72, 73, 79, 88 Van Gaudecker, B., 174, 211 Von Haam, E., 189, 191, 207 von Kaan Albest, A., 241, 242, 243, 245, 247, 263, 273 von Saltza, M. H., 313, 368 Vreugdenhil, D., 90, 124

W Wachstein, M., 203, 211 Walker, D. G., 225, 238 Walker, N. A., 299, 367 Walker, R. J., 48, 51, 87 Ward, D. C . , 180, 198, 211 Wareing, P. F., 265, 266, 272, 273 Waring, M. J., 180, 198, 212 Warner, J. R., 174, 211 Washizu, Y., 79, 86 Wattleworth, A., 230, 237

383

Watson, D. H., 102, 124 Watson, M. L., 172, 211 Weber, A. F., 201, 211 Wechsler, W., 57, 88 Weinstein, A,, 144, 168 Werner, D . J., 189, 205 Wescott, W . C., 289, 368 Westergaard, M., 142, 168 Wetmore, R. H., 264, 265, 266, 273 Wettstein, R., 129, 141, 168 Whaley, W. G., 92, 124 Wheeler, K. P., 289, 370 Whipp, S., 201, 211 White, M. J., 158, 168 White, P. R., 310, 370 Whittam, R., 289, 370 Widdas, W. F., 301, 370 Wierzejski, A., 30, 44 Wigglesworth, V. B., 47, 49, 51, 5 3 , 57, 60, 65, 81, 88 Wilbrandt, W., 301, 368, 369, 370 Wildman, S . G., 117, 124 Wilkie, D. R., 308, 370 Wilkins, &I. J., 287, 304, 326, 328, 366, 3 68 Willen, R., 197, 210 Williams, E. J., 299, 369 Willison, J. H. M., 109, 112, 113, 119, 123 Wilson, D. V., 313, 369 Wilson, E. B., 39, 44 Woffenden, L. M.,310, 368 Wogan, G. M., 181, 210 Woledge, R. C., 308, 370 Wolf, D. A., 197, 206 Wolfe, D. E., 59, 62, 64, 66, 67, 87 Wollam, D. H. M., 140, 168 Wright, R., 310, 343, 370 W u , J. H., 117, 124 Wyatt, G. R., 49. 65, 88

Y Yamasaki, T., 72, 79, 80, 83, 87, 88 Yemm, E. M., 311, 354, 370 Yin, H . C., 267, 273 Yoshida, Y., 90, 108, 124 E’otsuyanagi, Y., 89, 90, 124, 170, 211 Young, J. Z., 5 5 , 56, 86, 88

AUTHOR INDEX

384

Young, R. W., 216, 217, 218, 219, 220, 221, 223, 224, 227, 228, 238

z Zahalsky, A. C., 181, 208 Zaitlin, M., 117, 123

Zalta, J. P., 174, 184, 188, 201, 205, 206, 209 Zelenskaya, V. S., 64, 74, 75, 76, 88 Zimmerinann, R. A,, 147,167 Zohary, D., 161, 165

Subject Index A

Cytoplasm, classic concepts, membranes and their roles, 285-287 cortical, composition, nature and behavior of,

Actinomycin D. nucleolus and, 175-182 Active transport, concepts, origin and development, 282283 Aflatoxin, nucleus and, 198-200

11-12

B Bone, fracture repair, bone-forming cells and, 233-2111 Bone cells, see Osteoprogenitor cells

C Carrot, clones, responses to different stimuli, 317-322 explants, simple salt solutions and, 326-330 time course of metabolism and absorption in, 322-326 Cells, see alro Invertebrate nerve cells, Osteoprogeiiitor cells, Plant cells growth requirements, solute uptake and, 3 10-316 with potential for bone formation, 229230 bone fracture repair, 233-234 direct transplants, 231-233 millipore filters and, 230-23 1 solutes in, concept of active transport, 282-283 division of labor, 283-285 genesis of vacuoles, 280-282 plant contrasted with animal, 276-279 vacuolar contents, 279-280 Cell wall, regeneration by plant protoplast, 108-115 Chromatin, margination, proflavin and, 195-198 Chromosomes, attachment site on nuclear membrane, 140-142 Crossing-over, factors influencing, 148-160 meiotic mutants and, 160-164 mitotic, without synaptonemal complexes, 134-135 recombinases and, 142-145

formation of, 12-18 physiological properties of, 3-11 fine structure and biochemistry, 287-289 subcortical, accumulation of, 24-32 animal pole plasm, 21-24 vegetative pole plasm, 18-21 Cytoplasmic organelles, current concepts. 289-294

D Differentiation, osteoprogenitor cells, kinetics of, 218-224 D~osophila, cytology of oocyte nucleus, 120-134 meiotic prophase of oocyte, light microscopy of, 129 ultrastructure of, 129-131 ovary, morphology of, 127-129

E Electrical activity, invertebrate nerve cells, ionic requirements for, 70-84 Ethionine, nucleolus and, 193-195

385

G Glial cells, invertebrate nerve cells and, 5359 Growth, solute accumulation and, 337-311

I Indoleacetate, movement, sieve tubes and, 262-263 sieve tube regeneration and, 258-262 Infection, viral, initiation of, 100-107 Inorganic ions, distribution and exchanges in invertebrate nerve cells, 64-70 Interchromatin granules, agents affecting the nucleus and, 200 lntersynaptomeric distances, travel times and, 145-147

386

SUBJECT INDEX

Invertebrate nerve cells, distribution and exchanges of inorganic ions and molecules, 64-70 electrical activity, ionic requirements for. 70-84 structural considerations, extracellular system, 59-61 extraneural structures, 46-47 glial cells and, 53-59 nerve sheath and, 47-53 Ions, uptake by cultured tissue, 352-355

L Lasiocarpine, nucleus and, 198-200 Lymnaea egg, cortical cytoplasm, composition, nature and behavior of 11-12

formation of, 12-18 physiological properties of, 3-11 cortical field of, 32-37 subcortical cytoplasm, accumulations of, 24-32 animal pole plasm, 21-24 vegetative pole plasm, 18-21

M Macromolecules, entry into plant cells, 92100 Meiotic mutants, crossing-over and, 160-164 Meiotic prophase, biochemistry of, 147-148 Drosophila oocyte, light microscopy of, 129 ultrastructure of, 129-31 Membranes, current concepts, 289-294 Millipore filters, bone-forming cells and. 230-231

N Nerve cells, invertebrate, extracellular system, 59-64 extraneural structures, 46-47 glial cells and, 53-59 nerve sheath and, 47-53 Nondisjunction, meiotic mutants affecting crossing-over and, 160-164

Nuclear inclusions, agents affecting the nucleus and, 200-203 Nuclear membrane, chromosomal attathment sites on, 140-142 Nucleolus, agents primarily affecting, actinomycin D, 175-182 ethionine, 193-195 supranormal temperature, 182-188 thioacetamide, 188-192 normal fine structure, 170 Nucleus, agents primarily affecting, aflatoxin and lasiocarpine, 198-200 interchromatin granules, 200 nuclear inclusions, 200-203 proflavin, 195-198 normal fine structure, 170-175

0 Oocyte nucleus, Dmrophila, r ( . ? ) Gmutation and, 131-133 cytology of, 129-131 .rbd105 mutation and, 133.134 Osteoprogenitor cells, 216-21 7 composition of, 228-229 electron microscopy, 226 histochemistry, 224-225 kinetics of differentiation, 218-224 parathyroid hormone and, 226-227 proliferative activity, 218 transformation of, 227-228 tritiated thymidine uptake, 217-218 Ovary, Diosophila, morphology of, 127-129

P Parathyroid hormone, osteoprogenitor cells. 226-227 Perichromatin granules, aflatoxin or lnsiocarpine and, 198-200 Pinocytosis, isolated plant protoplasts and. 92-100 Plant(s), cultured tissue explants, absorption studies with, 3 17.355 endogenous capacities and exogellous requirements, 312-316

SUBJECT INDEX

cut discs, endogenous capacities and exogenous requirements, 310-311 indicators of salt uptake in, 348-352 isolated protoplast system, 89-92 cell wall regeneration, 108-115 entry of macromolecules, 92-100 virus multiplication in, 115-122 virus uptake, 100-107 Plant cells, contrasted with animal cells, 276-279 physiological studies of salt accumulation, period 1925-1945, 234-296 period 1945-1960, 296-297 recent trends, 297-309 Potassium halides, behavior in tissue, 330337 Proflavin, nucleus and, 195-198 Protoplasmic surfaces, current concepts, 289-294 Protoplasts, plant, cell wall regeneration, 108-115 isolated system, 89-92 macromolecule entry, 92-100 virus multiplication in, 115-122 virus uptake, 100-107

R Recombinases, crossingover and, 1 4 - 1 / 1 5 Regeneration, sieve tubes, 2/11-263

s Salt accumulation, physiological studies in plant cells, period 1925-1945, 294-296 period 1945-1960, 296-297 recent trends, 297-309 Salt uptake, in cells and plant body, 3183 52

387

Sieve tubes, differentiation. in cultures, 264-266 normal, 266-269 regeneration, 241-246 chemicals and, 252-256 control by indoleacetate, 258-262 excision of organs and, 251-252 factors controlling, 246-25 1 generality of results, 256-257 indoleacetate movement and ,262-263 Sodium halides, behavior in tissue, 330-337 Solute, accumulation, growth and, 310-316, 337341 membranes and fine structure, 285-294 perspectives and prospects, 355-365 salt relations, cellular basis, 342-348 Synaptonernal mmplexes, mitotic crossing-over and, 134.135 origin and functioning of, 135-160 synaptome-zygosome hypothesis, 136-140

T Temperature, supranormal, nucleolus and, 182-188 Thioacetamide, nucleolus and, 188-192 Thymidine, uptake by osteoptogenitor cells, 217-218 Transplants, bone-forming cells and, 2 3 1 233

V Vacuoles, contents in autotrophic system. 279-280 genesis, creation of solute content, 2 8 0 282 Virus, multiplication in plant protoplList. 1 15I22 uptake by plant protoplasts, 100-107

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