International Review of Cytology: A Survey of Cell Biology, Volume 144

VOLUME 144

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1988 1949-1 984 19671984-1992 1993-

ADVISORY EDITORS Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay Mark Hogarth Keith E. Mostov

Audrey Muggleton-Harris Andreas Oksche Muriel J. Ord Vladimir R. Pantic M. V. Parthasarathy Lionel I. Rebhun Jean-Paul Revel L. Evans Roth Jozef St. Schell Hiroh Shibaoka Wilfred Stein Ralph M. Steinman M. Tazawa Alexander L. Yudin

Edited by Kwang W. Jeon Department of Zoology The University of Tennessee Knoxville, Tennessee

Jonathan Jarvik

Department of Biological Sciences Carnegie Mellon University Pittsburgh, Pennsylvania

VOLUME 144

Academic Press, Inc. Harcouti Brace Jovanovich. Publishers

This book is printed on acid-free paper. @ Copyright 0 1993 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. 1250 Sixth Avenue, San Diego, California 92101-431 1 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX Library of Congress Catalog Number: 52-5203 International Standard Book Number: 0-12-364547-6

PRINTED IN THE UNITED STATES OF AMERICA 93 94 95 96 97 98

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9 8 7 6 5 4 3 2

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CONTENTS

Contributors ....................................................................................

ix

Karyosphere in Oogenesis and lntranuclear Morphogenesis Mira N. Gruzova and Vladimir N. Parfenov I. (I. Ill. IV. V. VI.

Introduction ..................... ............. ............................. Occurrence of Karyosphere d in Animals ................ Karyosphere in Oocytes of Some Insects ......................................... Formation of Karyosphere in the Oogenesis of Vertebrates .................... The Karyosphere Capsule and the Nuclear Matrix ...... ................. General Discussion and Concluding Remarks.. ............ .............. References ..................................................... ..........

1

8 8 25 40 41 47

Applications of Arabidopsis thaliana to Outstanding Issues in Plant-Pathogen Interactions Jeffery L. Dangl I. II. 111. Iv. V. VI.

.................................. s: A Brief Overview ........... Arabidopsis thaliana: Weed No More ................................ Arabidopsis Pathogens: Interaction Phenotypes and ..... ................................... Biochemical Responses.. . ................................... Current Impact of Aradopsis as a Mode References .............................................................................

V

72 76 79

vi

CONTENTS

Toward a New Concept of Cell Motility: Cytoskeletal Dynamics in Amoeboid Movement and Cell Division Yoshio Fukui I.

II. 111. IV. V. VI. VII.

Introduction ................................ Conventional Theories.. . . . . . . . . . .. .. .. . . . Current Concepts , , , ........ . . . . . . . . . . .. .. Cytoskeletal Components.. . . . . .... . . . . . . Dynamic Changes of Dictyostelium C Critical Evaluation of the Mechanism Summary and Perspective ..... ..... . . . . . References . . . . ... . . , , , , , . ..... . . . . . . . . .. ...

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

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

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

85 86 94 103 110 114 120 121

Fine Structure, Innervation, and Functional Control of Avian Salt Glands Riidiger Gerstberger and David A. Gray Introduction.. , ... ... , , , , . . .... , . , , , , , , , ..... . . . . . . .. .... . ... . . Secretory Tissue of the Avian Salt Gland ................. Blood Supply to the Salt Gland .............................................. Salt Gland Innervation., ..... ,., , , , .. .... ,.. . . . ........ , , . , , , . . . ..... . . . . . . ... . ............................................... Secretory Mechanism . . ... . . . . . Receptive Systems for the Control of Salt Gland Secretion ............. .. ...... ....... ....... ..................... . VII. Hormonal Control of Salt Glands VIII. Stimulus-Secretion Coupling , , ., .. .. , . , . . , . . .... , . , , . , . , , ....... . , . , ....... , IX. Concluding Remarks , , . . .... , . ........ ............. .... References ............................................................... I. II. Ill. Iv. V. VI.

. . . . . . . . . . . . . . . . . . I

I . . . . .

129 130 148 155 168 177 189 200 205 206

Mitosis: Dissociability of Its Events Sibdas Ghosh and Neidhard Paweletz I. II. 111. IV.

Introduction.. . . . . . .... . . . . . . .. ...... . . . . . . ...... . . . . . . .. ...... . . ., .. . Mitotic Events , , , .... , , , . . . ...... , , , , , , , , ...... . . . . ........ ... ....... Dissociation of Mitotic Events , .... ..... . . . . . ........ ... . . . .. . . . . . Conclusions. . , , , , , .... , , , . .. ., .... , . , , , , ., ...... . . . . . . .. ...... . . . . . .. References .............................................................................

217 218 226 252 252

vi i

CONTENTS

The Endosymbiotic Origin of Chloroplasts Jean M. Whatley I. II. 111. Iv. V. VI . VII . VIII .

Introduction., ........................... Cyanobacteria, Red Algae, and Cyanelles Prochlorophytes, Green Algae, and Land Cryptomonads and Chlorarachnion ....... Heterokont Algae or Chromista ...... Euglenoids ................................ Dinoflagellates ............................ Conclusions., ................................. References .................................

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

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

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

Index ............................................................................................

259 261 265 272 277 282 285 293 296 301

This Page Intentionally Left Blank

Numbers in parentheses indicate the pages on which the authors' contributions begin

Jeffery L. Dangl (53), Max-Delbruck Laboratory in the Max-Planck Society, 0-5000 Koln 30, Germany Yoshio Fukui (85), Department of Cell, Moleculal; and Structural Biology, Northwestern University Medical School, Chicago, Illinois 6061 1 Riidiger Gerstberger (129), Max-Planck-lnstitut fur Physiologische und Klinische Forschung, W G. Kerchkoff-lnstitut, 0-6350 Bad Nauheim, Germany Sibdas Ghosh (217), Centre for Advanced Study in Botany, University of Calcutta, Calcutta 700019, lndia and Research Program /I!German Cancer Research Centel; 0-6900 Heidelberg, Germany David A. Gray (129), Max-Planck-lnstitut fur Physiologische und Klinische Forschung, W G. Kerchkoff-lnstitut, D-6350 Bad Nauheim, Germany Mira N. Gruzova (l),Laboratory of Cell Morphology, lnstitute of Cytology, Russian Academy of Sciences, St. Petersburg 196064, Russia Vladimir N. Parfenov (l), Laboratory of Cell Morphology, lnstitute of Cytology, Russian Academy of Sciences, St. Petersburg 196064, Russia Neidhard Paweletz (217), Research Program /I!German Cancer Research Centel; 0-6900 Heidelburg, Germany Jean M. Whatley (259), Department of Plant Sciences, Oxford University, Oxford OX1 3RB, England

ix

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Karyosphere in Oogenesis and lntranuclear Morphogenesis Mira N. Gruzova and Vladirnir N. Parfenov Laboratory of Cell Morphology, Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064. Russia

1. Introduction The karyosphere was named and first described by Blackman (1901, 1903, 1905, 1907), who observed that the chromosomes in spermatocytes of millipedes (Chilopoda) join and form a knot. Our long-standing interest in this nuclear formation and the lack of investigation on the subject compelled one of us to analyze the available data (Gruzova, 1975). This study revealed that the karyosphere is a form of chromosomal apparatus that sometimes exists for long periods in the oocytes of many animals, from hydra to higher vertebrates. However, although lampbrush chromosomes (often preceding karyosphere formation) have been discussed in numerous studies, karyosphere formation has received less attention, being mentioned only briefly in studies on oogenesis and meiosis in animals. Thus, evidence on the morphology and genesis of the karyosphere was extremely scarce, and there was no certainty of its functional role in sex cells. The clarification of these questions was complicated by the lack of a distinct definition of karyosphere and confusion over terms (Table I). We currently regard the karyosphere as the result of all chromosomes of the gametocyte joining in a limited nuclear volume with final formation of a single complex chromatin structure-a type of nucleus inside the germinal vesicle. It is thought that karyosphere formation is the result of relative inactivity of chromosomes during RNA synthesis, since it is formed either in nutrimental oogenesis (for example, in insect oocytes with meroistic ovarioles) or after a long period of lampbrush chromosome activity (as in amphibian oocytes). Formation of the karyosphere is accompanied by intensive nuclear cytoplasmic exchange that results in the appearance of numerous protein granules and bodies in the karyosphere and their subsequent transfer toward the nuclear periphery. The similarity between the karyosphere and a nucleus becomes stronger in cases where a peculiar species-specific zone or “capsule” develops around the lnrernurronrrl Review, of Cytology?..lid 144

1

English translation capynght 0 1993 by Academlc Resa. Inc. All nghh 01 reproduction in any fomi reserved.

2

M. N. GRUZOVA AND V. N. PARFENOV

TABLE I Species of Animals in which the Karyosphere Is Formed in the Gametocytes Phylogenetic position of the animals studied Coelenterata Hydrozoa Hydra fusca Nemathelminthes Nematoda Ascaris niegalocephala Strnngyhs frlaria Sclerostoniunr sp. Cordiacea Cordiirs tolosanus Annelides Hirudinea Nephelis vulgaris Glossiphonia coniplanata Arthropoda Arachnida Acarina Pediculoides ventricnsids Pediculopsis graminum Myriopoda Scutigeromorpha Scutigera forceps Scolopendromorpha Scolopetidra heros Sc. subspitripes Lithobiomorpha Lithobius niorda.r L . muitidentatus L. sp.

Sex

Reference

Other terms used to designate karyosphere

P

Brien, 1950

Reseau chromatique

0

Pasteels, 1948 Kroning. 1923 Kuntz. 1913

Karyosome

P P P

Vejdovsky, I9 I 1-19 12

Innekern

P 0

Jorgensen, 1909 Gruzova and Zaichikova, 1967; Aisenshtadt et al., 1967

P P

Patau, 1936 Cooper, 1939

d

Medes, 1905

6 3

Blackman, 1901

3

3

Blackman, 1907 Blackman, 1907 Blackman, 1907

P

Bauer. 1933

3 3

P 0

Pantel and Sinety, 1906 Brown, 1913 Brown, 1913 Brown, 1913 Vejdovsky. 191 1-1912 Kozhanova, 1974

d

Arnold, 1908

s

Karyospheroid

Insecta

Dermaptera Forfrculu auridaria Hemiptera Notonecru glauca N . iindriluta N . irrorata N . insuluta Aphrophora alni Euryxaster intepiceps Coleoptera Hydrophilidae Hydrophilus piceus

6

Chromosomenknauel

(continues)

3

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS TABLE I Continued Phylogenetic position of the animals studied Tenebrionidae Phanaeus carnife.1P. igneus Tenebrin moliior Slaps Iethifera Bruchidae Acanthoscelides ohtectus Scarabaeidae Geotrupes silvaticus G . vernalis Dytiscidae Dytiscus marginalis

Acilius sulkatus Carabidae Curahus nemuralis C . granulatus Pterostiehus vulgaris PI. niger Ahax alter Neuroptera Chrysopa perla

Sex

c7 c7 P 0

Hayden, 1925 Hayden, 1925 Schlotman and Bonhag, 1956 Gruzova, 1962b, 1979, 1982

0 8

Mulnard, 1950, 1954 Mulnard. I95 1

0

B. Matuszewski, unpublished

9

Saint-Hilaire, 1895 (cited in Nath, 1924); Debaisieux. 1909; Bauer, 1933; Urbani and Russo-Caia, 1969: Bier et 01.. 1967 Bier et al., 1967

0

Bier el a/.. 1967 Bier et a/.. 1967 Bier et a/.. 1967 Bier et al., 1967 Bier et a/.. 1961

P

P 0

P P P

Ch. vulgaris

P

Ch. vitfafu

9

Ch. sp.

P

Mecoptera Panorpa communis P. hyhrida Trichoptera Stenophy1a.v stellatus Hymenoptera Nemeritis canescens Rhogogaster picta Lepidoptera Deilephila euphorhia D. sp.

Reference

P

Other terms used to designate karyosphere

Karyonucleolus Chromosome knot

Gruzova, 1960: Gruzova er al., 1912 Gruzova, 1960; Gruzova et al.. 1972 Gruzova, 1960 Gruzova et al., 1912 Gruzova, 1960; Gruzova et al.. 1972

9

Bauer, 1933; Gruzova. 1962a Gruzova, 1962a

P

Gresson, 1933

P

Speicher, 1937 Bobrova and Gruzova. 1967

Amphinucleolus

P

P P

Kedrovsky, 1959 Kedrovskv. 1959

Chromosome knot

(conti

4

M. N. GRUZOVA AND V. N. PARFENOV

TABLE I Continued Phylogenetic position of the animals studied Laspere-yresia pomonella Galleria melonella Diptera Tachinidae Calliphora erythrocephala

Sex

Reference

0 0

Gruzova, 1974 M. N. Gruzova (unpublished)

0

Gruzova, 1967; Bier et al.. 1967 Naville, 1932 (cited in Bauer, 1933); Bauer, 1933

Lucilia caesar

0

Muscidae Musca domestica

0

Mahover, 1959; Bier et al.. 1967

Drosophilidae Drnsophila melanogaster

0

Metz. 1927; King, 1970; Rasmussen, 1975 Metz, 1927 Metz, 1927 Metz. 1927 Metz, 1927

Dr: virils Dr. pseudoohscura Dr. gihherosa Dr. funehris Cecidomyiidae Mikiola fagi Rhahdophaga rosaria Mayetiola poae Oligotrophus schmidti Wuchtiella persicaria Aphidoletes aphidimyza Tipulidae Tipula paludosa T. lateralis T. oleraceae T. marginata Culicidae Stegomia fasciata (Aedes) Aedes sp. Anopheles maculipennis Aedes aegipti

Culex.pipiens C.fatigans Orthoptera Diestramena marmoratu Chordata Cyclostomata Petromezones Lumpetra fluviatilis

P

P 0

P 0 0 0

Other terms used to designate karyosphere

Chromatin clump

0

Matuszewski, 1960, 1982 Iazdowska-Zagrodzinska and Matuszewski, 1978 Matuszewski, 1960, 1966 Kunz et al., 1970 Gruzova et (11.. 1987

0 P 0 P

Bauer, 1933 Bayreuter, 1952, 1956 Bayreuter, 1952, 1956 Bayreuter, 1952, 1956

0

0 0

Bauer, 1933 Gruzova, 1967 Nicholson, 1921; Bauer, 1933 Roth. 1966; Fiil and Moens, 1973 Fiil and Moens, 1973 Nath, 1924

Definitive nucleus

0

Vejdovsky, 1911-1912

Innenkern

0

Chubareva, 1957

P P

P

9 P

Inner part of the nucleus

(continues)

5

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS TABLE I Continued Phylogenetic position of the animals studied

Pisces Elasmobranchii Pristiurus Sciflum canicula Torpedo marmorata Actinopterygii Teleostei Zoarces viviparus Trigla sp. Perm Jluviateles Acerinu cernua Esox lucius Coregonus albula C. lavaretus Gasterosteus aculeatus Amphibia Urodela Siredo sp. Triton cristatus T. iaeniatus T. alpestris Amblystoma sp. Triton sp. T. cristatus carnife.r Proteus anguineus Anura Rana fusca R . temporaria

R . ridibunda Hynobius retardatus Reptilia Chelonia Testudo gracea T. europea Squamata Lacertidae Lacerta saxicola Ophiops elegans Eremias velox

Sex

P

0

P ?

0

P

0

P

0 0 0 ?

0 0

0 0 9

0 ?

9 9

Reference

Riickert, 1892 Markchal, 1907 Riickert, 1892; Sterba, 1961

Schultze, 1887 Schultze, 1887 Born, 1894; Camoy and Lebrun, 1898 Camoy and Lebrun, 1898 Camoy and Lebrun, 1898 Lubosch. 1903 Callan, 1952 Jorgensen, 1910

9 9

9 0

Loyez, 1906 Loyez. 1906

P

Darewski and Kulikova, 1961 Arronet, 1969 Kulikova. 1963

?

Kary osome

Wallace, 1903 Marechal, 1907 Meien, 1927 Latif, 1966 Sakun, I96 I Sakun, 1961 Sakun, 1961 Sakun, 1961

Schultze. 1887 Camoy and Lebrun, 1898; Wagner, 1923; Duryee, 1950: Parfenov, 1974; Parfenov and Gruzova, 1975a.b Gruzova and Parfenov. 1973 Makino. 1934

9

Other terms used to designate karyosphere

Centraler knauel Centralkorper Massif central Massif central Centralkorper Central mass of chromoson Centralkorper Centraler knauel Chromosome frame, capsul

Chromosome knot

(conti

6

M. N. GRUZOVA AND V. N. PARFENOV

TABLE I Continued Phylogenetic position of the animals studied Agamidae Phrynocephalus reticulatus P. helioscopus Agama caucasica Anguidae Anguis fragilis Chalcides ocellatus Lacertidae Lacrera viridis L. mubalis L. stirpium L. vivipara Uromasti.r achantinurus Geconidae Platydactylis muralis Ophidia Viperidae Vipera aspis Colubridae Tropidonotus viperinus

T. nutrix Crocodilia Crocodilus niloticus

Aves Galiifomes Gallus domesticus

Columbifomes Columba livia Passeriformes Passer domestica Embriza citrinella Fringilla coelebs

F. montifringilla Carpodaca erythrinus Spinus spinus An thus triviales Turdus iliacus Alcedo ipsida

Sex

Reference

P

Arronet, 1975 Arronet, 1975

0 0

Arronet, 1975 Arronet, 1969, 1975

P 0

Loyez, 1906 Loyez, 1906

0 P 0 P

Loyez, Loyez, Loyez, Loyez, Loyez,

0

Loyez. 1906

0

Loyez, 1906

P

Loyez, 1906

0

Loyez, 1906

9

Loyez, 1906; Sonnenbrodt, 1908; Brambell, 1924; Gaginskaya, 1972

0

Loyez, 1906; Gaginskaya, 1972

9 P 0

Loyez. 1906; Gaginskaya, 1972 Loyez, 1906; Gaginskaya, 1972 Loyez, 1906; Gaginskaya and Gruzova, 1969; Gaginskaya, 1972 Gaginskaya, 1972 Gaginskaya, 1972 Gaginskaya, I972 Gaginskaya, 1972 Gaginskaya, 1972 Loyez, 1906

P

P 0

0 0 P P

Other terms used to designate karyosphere

1906 1906 1906 1906 1906

Corpuscule pyrenichromatique

(continues)

7

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS TABLE I Continued Phylogenetic position of the animals studied Apodifones Apus upus Rapacifones Polvboroides madugascurensis Fulco chrvsuetos Echassiers Himantopus autumnalis Palmipodes Anus domesticus

Sex

Reference

P

Gaginskaya, 1972

0

P

Loyez, 1906 Loyez. 1906

P

Loyez. 1906

P

Loyez. 1906

P

Kiknadze. 1966

0

P

Austin and Braden, 1953; Odor, 1955: Dalmane, 1967 Zybina, 1967, 1968 Zybina, 1967 Zybina et al.. 1990

P

Zybina, 1967, 1968

P

Kuhlmann, 1970; Sanyal et ul., 1976: Kurilo, 1982: Parfenov erul., 1984, 1989

Other terms used to designate karyosphere

Mammalia Carnivora Lutereolu luteriola Rodentia Rattus norvegicus

Mus musculus Spalac leucodon Microtus anzalis Leporidae Oryctolugus cunirulus Hominidae Homo sapiens

P

0

karyosphere, analogous to the nuclear envelope. Some authors termed this karyosphere innenkem (Table I). The origin, fine structure, and function of these capsules have not been determined, but recently these questions have been answered in part with results from ultrastructural and cytochemical investigations. One of the aims of this review is to demonstrate that the karyosphere is quite common in gametogenesis, in particular, oogenesis. However, a more important task is the description at the ultrastructural level of the events taking place in the nucleus during the formation of the karyosphere capsule or its analogs. In our studies aimed at determining the structures involved in the capsule formation, nucleoli and their derivatives appeared to be more accessible for examination at the outset. Electron-microscope studies on the self-assembling of synaptonemal complex (SC) elements and its derivatives in various meiotic cells proved to be most helpful in this work (Moses, 1968; Rasmussen, 1975). Morphogenesis of complex species-specific capsules is probably the result of genetic programming in the nucleus, but it remains obscure how this programming works. On the basis of morphological and cytochemical data on oocyte

8

M. N. GRUZOVA AND V. N. PARFENOV

nuclei of some invertebrates and vertebrates, we put forward the hypothesis that DNA may be directly involved in the organization of the capsules. We hope that further studies will give insight into the molecular biology of intranuclear morphogenesis. It is important to elucidate the contribution of the karyosphere and its associated capsule to the organization of a mature egg, and the segregation of ooplasm, in particular. Interest in the study of the karyosphere also comes from the fact that it represents a transformationmof meiotic chromosomes often occurring just prior to the completion of meiotic divisions.

II. Occurrence of Karyosphere during Gametogenesis in Animals Table I gives data available in the literature since the end of the last century concerned with karyosphere formation during gametogenesis in both invertebrates and vertebrates. Listed are the different terms used by different authors for the same phenomenon; these different terms undoubtedly caused much confusion and complicated the attempts at comparative analysis of this phenomenon in gametes. Naturally, some old data should be reconsidered, especially those dating back to the years prior to 1924, when the Feulgen reaction came into use. The data show that karyosphere formation has been observed in gametocytes of more than 120 species of animals representing more than 50 orders of 12 classes belonging to 4 phyla of the animal kingdom. In most cases the karyosphere occurs in oocytes and, much more seldom, in spermatocytes. The stage of gametogenesis in which the karyosphere is formed is always a longterm diplotene of meiosis.

111. Karyosphere in Oocytes of Some Insects The karyosphere and the structures around it are so small that they can only be detected by electron microscopy. In this study the oocytes of insects, namely Diptera, Neuroptera, and Coleoptera, were examined.

A. Nucleus and Karyosphere in Oocytes of Diptera Of the Diptera, noteworthy are the oocyte nuclei of fruit flies, mosquitoes, and gall midges. During oogenesis of Drosophila melanogaster the karyosphere is formed immediately after the pachytene in the S,-S, stage (Smith and King, 1968). In wild-type flies the karyosphere represents a dense chromatin mass, initially

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS

9

associated with the nucleolus, that is devoid of any elements of a capsule, The nucleus is relatively small and reaches about 20 pm in diameter. The appearance of polycomplexes close to the karyosphere and nucleolus has been recorded in a few single oocytes in the germarium of heterozygous mutant flies of this species (Rasmussen, 1975), but these polycomplexes and the ultrastructure of the oocyte nuclei at later stages of oogenesis have not been studied further. A complex capsule around the karyosphere has been described in oocytes of mosquitoes. The first to describe this was Roth (1966). Studying SC during the meiotic prophase in oocytes of Aedes aegipti, Roth showed that typical SC, which first appear in paired homologs in pachytene, with a start of diplothene separate from chromosomes and are released into the peripheral zone of the karyosphere, not as single complexes any more, but as aggregates of SC; hence, they were named polycomplexes. Roth assumes that polycomplexes arise as a result of agglomeration or “polymerization” of single fragments of SC. Roth’s observations were supported and further developed by Fiil arrd Moens (1973) (Fiil, 1976a,b, 1978). These authors studied the giant branched nucleus in oocytes of mosquitoes Aedes aegipti, Anopheles gamhiae, and Culex pipieps. Using ultrathin serial sections, they showed that in oocytes of Aedes after the accumulation of chromosomes into a karyosphere, around the latter there first appears single polycomplexes, similar to the structures described by Roth. Then their number significantly increased, which, according to these authors, implies that these polycomplexes are formed de now. Elaborating on Roth’s observations, Fiil and Moens discovered among the polycomplexes a large number of annuli. These annuli looked like the pore complexes of the nuclear envelope and were either autonomus or associated with polycomplexes or with each other, and arranged in long rows by fibrillar material, forming pseudomembranes. Fiil and Moens also discovered intranuclear annulata lamellae associated with both polycomplexes and the nuclear envelope. These complex interrelations are schematically represented in Fig. 1. Having considered all the sequences of patterns at different stages of oogenesis, Fiil and Moens concluded that the fibrillar material connecting these structures is the same as that in the modified central elements of SC. Finally, around the chromosomes is a wide network of structures, which possibly holds the chromosomes together in this enormous nucleus (40 X 140 X 3pm). The organization of the karyosphere in oocytes of C. pipiens is somewhat different (Fiil and Moens, 1973). In the nucleus there appears rather early an electron-transparent round body to which bivalents are attached. Then the chromosomes organize around this body and are surrounded by pseudomembranes on the outside. The latter also join single bivalents; pseudomembrane regions occur in the fibrillar round body as well. They represent lamellae that are made up of filaments (derivatives of the central elements of SC). Regarding A . gambiae, the material of the capsule seems to have no visible similarity to the elements of SC. The nucleus outside the capsule is filled with

10

M. N. GRUZOVA AND V. N. PARFENOV

FIG. 1 Diagrams of modified synaptonemal complexes in Aedex aegypti and Culex pipians (Fiil and Moens, 1973). (a) A . aegypti. There is a normal complex at A with lateral element (le) and central element (ce). The lateral elements are connected by sheets of transverse filaments. The polycomplex (B) has lateral element equivalents (lee) and central element equivalents (cee). The lateral element equivalents break up into short bars at F. The bars are arranged in hexagonal arrays ( G ) maintaining the normal spacing between lateral elements. The transverse filaments between the bars form sheets (H). Some bars give rise to annuli (I). Annuli (an) also form on the lateral element equivalents of the polycomplexes (D). The annuli dissociate from the polycomplex and then reassociate with wider spacing into annulated pseudomembranes (apm) at C and E. Some of these become intranuclear annulate lamellae (ial). (b) C. pipiens. While synaptonemal complexes are still associated with the chromosomes (ch) the sheets of transverse filaments, which normally lie between lateral elements of a given complex, become much extended and make contact with lateral elements (le) of other synaptonemal complexes. The bivalents thus become interconnected by pseudomembranes (pm). (From Fiil and Moens, 1973, with permission.)

fragments of the nucleolus, although the evolution of the latter has not been determined in detail (Fiil and Moens, 1973; Fiil, 1976b, 1978). In A. garnhiae granular aggregates are often seen in association with the capsule material (Fiil, 1978). A comparison among three species of mosquitoes clearly demonstrates the species specificity of the organization of the capsule material.

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS

11

The capsule material, which to some extent is similar to that of the polycomplexes in Drosophilu and mosquitoes, appears in abundance in oocyte nuclei of the representatives of lower Diptera of the family Cecidomyiidae. It was first described at the light-microscopic level by Matuszewski (1960), and later some data on its ultrastructure appeared (Kunz et al., 1970; Matuszewski, 1982; Gruzova et al., 1987). I n gall midges this lamella or fibrillar strand material surrounds the central group of S bivalents and separates it from the E chromosomes; this particular case will be discussed below. The origin of nuclear lamellae remains obscure thus far (Kunz et al., 1970; Matuszewski, 1982), although there are some data available indicating that these structures represent modified elements of SC (Gruzova and Batalova, 1990). Such an interpretation cannot contradict the fact that nuclear lamellae of gall midges appear as early as the polar cell stage in early embryogenesis (Junquera, 1983) and apparently are also present in oogonia, i.e., long before the meiosis begins. In oocytes the system of lamellae becomes more developed. In this regard, it is noteworthy that these structures, similar to SC, have also been found in the cytoplasm of sex cells of ascarids prior to conjugation of homologous chromosomes (Bogdanov, 1977; Fiil el al., 1977).

8. Nucleus and Karyosphere in Oocytes of Some Neuroptera

1. Some Generalities on Oocyte Development The first object of our light-microscopical and ultrastructural analysis of karyosphere formation were oocytes of the golden-eyed fly Chrysopa perla. We also studied oocytes of Ch. wittata and Ch. carnea (Gruzova, 1960, 1966; Gruzova et al., 1972). The oocytes of these insects develop in polytrophic ovarioles. In trophocytes supplying the oocytes with RNA there are several complex nucleoli (Zaichikova and Gruzova, 1975). However, ploidy of the trophocytes is relatively low, reaching about 300 n (Zaichikova, 1976). The organization of an oocyte nucleus is unusual. During earlier stages of oogenesis through the onset of diplotene it contains one to four extrachromosomal bodies (Figs. 2a and 2f) (Gruzova et al., 1972; Rousett, 1977) containing rDNA (extra-DNA) (Gaginskaya and Gruzova, 1975) (Fig. 2f) and one to three pronucleoli (Figs. 2b and 2c). The “dispersal” of the DNA bodies in the nucleus and the transformation of pronucleoli (Figs. 2c and 2d) lead to the appearance of numerous small nucleoli (Fig. 2e) synthesizing RNA (Fig. 2m) (Gruzova et al., 1972). Early on, transcriptionally inactive chromosomes join into a karyosphere around which a complex capsule forms. Small DNA bodies are constantly present in the protein material of the capsule (Figs. 4D-4F). The capsule does not disintegrate before the metaphase of meiosis I.

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13

Thus, the development of oocytes of golden-eyed flies could be classified as nutrimental oogenesis, but actually is intermediate between solitary and nutrimental oogenesis, since the oocyte nucleus synthesizes RNA as well as trophocytes.

2. Electron-Microscope Data The processes of transformation of the nucleolar apparatus and karyosphere formation are closely interrelated in the nucleus. For convenience, the material dealing with transformation of nucleoli and capsule formation is considered separately. The transformation of nucleoli is shown schematically in Fig. 5 (bottom). Pronucleoli in early diplotene comprise purely fibrillar structures. They are directly associated with bodies of the extraDNA (Figs. 2k and 5a) and carry nonuniform dense material on the periphery; similar material can be seen inside the pronucleoli (Fig. 2k). This peripheral material is apparently fragmented from the surface of pronucleoli, and around them clusters of clumps, each 70-80 nm in diameter, appear. Similar clumps can be seen in cavities and on the surface of DNA bodies, as well as around the DNA bodies (Fig. 2k). As the oocyte grows, the nucleus becomes filled with such clumps. Electron-micrograph analysis shows that these clumps, as well as the surface area of pronucleoli, contain DNA and apparently represent chromatin associated with nucleoli. This is also evidenced by autoradiography, in which the periphery of pronucleoli is labeled with [3H]actinomycin D, [3H]~ridine, and [14C]adenine(Fig. 2i) and, in the case of nucleic acid hybridization in situ, with [3H]rRNA (Fig. 2j) (Gruzova, 1966; Gaginskaya and Gruzova, 1975); however, this zone is too narrow to be detected as a Feulgen-positive ring.

~

FIG. 2 Oocyte nucleus in Chrysopu perla. Transformation of nucleolus apparatus. (a) Young oocyte nucleus in pachytene. Large body of extrachromosomal DNA is seen (arrow). Squash preparation, Feulgen stained. (be) Young oocyte nuclei. Subsequent stages of pronucleolar fragmentation as observed on living ovarioles (phase contrast). K, karyosphere. Bars = 10 pm. (f) Young trophocyte (top) and oocyte (bottom) nuclei. Hibridization in siru with ['HJrRNA of Drosophilu. Labeled are DNA bodies in the oocyte. Bar = 5 pm, (8. h) [3H]Thymidine incorporation in oocyte nuclei at the stage of nucleolar fragmentation. Small nucleoli are labeled (h) (8, X 1360; h, x 15,640). (i) [14C]Adenine incorporation after a 15-min incubation of ovarioles with precursor. The peripheral zone of nucleolus is labeled. Bar = 5 pm. (j)Hybridization in siru with [3H]rRNA of Drosophilu: tracks are located at the periphery of a large pronucleolus. Bar = 5 pm. (k) Part of young oocyte nucleus. Close to DNA bodies (arrows) fibrillar pronucleoli (PN) are located. Dense clumps are accumulated around DNA bodies and pronucleoli. Some of the clumps are directly on the surface of pronucleoli ( X 6800). (I) Fibrillar pronucleolus of young oocyte with dense strands coming off its surface. Shown by arrows is the material of the forming karyosphere capsule ( X 17,000). (m) [3H]Uridine incorporation in diplotene oocyte nucleus, 1 hr after precursor injection. Nucleolar material is heavily labeled; rare tracks over karyosphere (K). Bar = 10 prn. (n) Two ring nucleoli. Coming off their surface are granular strands (arrows) ( X 13,600).

14

M. N. GRUZOVA AND V. N. PARFENOV

FIG. 3 Oocyte nucleus of Chrysopa. Formation of karyosphere capsule. (A) Part of the nucleus with the forming karyosphere capsule. Electron-dense strands and clumps among and around the chromosomes (Ch). PN, fibrillar pronucleoli ( X 6700). Inset: general view of a nucleus at the same stage; thick section; K, karyosphere with thin capsule ( X 670). (B) Part of the nucleus with fibrillar pronucleoli (PN).Oblique and cross-sections of strands (arrows) are seen ( X 11,390). Inset: crossand oblique sections of tubular strand at higher magnification. (C,, C,) Tubular assemblies characteristic of nucleolar complexes. ce, central element; le, lateral element. (From Moses, 1968, with permission.) (D,, D,) Schematic interpretations of electron micrographs of the possible inner structure of tubular strands of Chrvsopa.

In addition to these clumps, dense fibrillar strands appear near the pronucleoli (Figs. 21, 3A, and 3B). Sometimes they leave the nucleolar surface in concentric layers (Figs. 21 and 5c). Meanwhile, large DNA bodies disappear, and in the nucleus smaller DNA clumps of different sizes can be observed. Simultaneously, large pronucleoli (Figs. 2b-2d) are substituted for abundant small nucleoli, often ring-shaped (Figs. 2e, 2h, 2n, and 5d). Narrow internal and external

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS

15

layers of ring nucleoli carry proribosomal particles (Fig. 2n). Starting with this stage, the nucleus will incorporate ['Hluridine (Fig. 2m) and [3H]thymidine (Fig. 2g), the latter being localized in the small nucleoli (Fig. 2h). Part of the ring nucleoli unravels into granular nucleolar strands, which leave the surface of the nucleoli (Fig. 2n). As a result, in later previtellogenesis the entire nucleus (100 X 40 pm in diameter) is filled with granular strands (Fig. 5e). By the completion of vitellogenesis the nucleonema disappears; among rare RNP clumps, clumps and ring structures containing DNA occur. The major stages of the karyosphere and capsule formation in Chrysopa are shown in Fig. 5 (top). In young diplotene oocytes the bivalents are found in the center of the nucleus, with large pronucleoli on the periphery (Fig. 5b). The number of fibrillar strands, derivatives of the pronucleolar surface, increases dramatically (Figs. 3A and 5c). The strands are represented either by sheets 40 to 60 nm wide with cross-striation or by cylinders 70-80 nm in diameter, which on cross-section look like ringlets with double walls connected with transverse fibrils (Figs. 3B, 3D,, and 3D,). The sheets resemble the central element of SC, whereas the cylinders are similar to modified SC arising in gametocytes of some insects in association with nucleoli (Fig. 3C) (Moses, 1968). The fibrillar strands surround the knot of chromosomes (Fig. 3A), and simultaneously, on the outside in association with the strands, there appear aggregates of fibrillar material of average density (Fig. 5c). Next the closed external zone of the karyosphere capsule is formed (Figs. 4A and 5D). The inside of this zone is lined with the dense strands described above, to which chromosomes are attached (Fig. 4A). In the loose chromatin of the karyosphere electron-dense clumps (Fig. 4A) and ring structures appear here and there. Contrast in the latter decreases after EDTA treatment, which is indicative of the DNA content in them. Similar clumps and ring structures occur in abundance on the outside of the capsule, in close contact with it (Figs. 4A and 4B). Coiled filaments (40 nm thick) made up of granules (50 nm) are often attached to the clumps and ring structures (Fig. 4A). The clumps and ring structures often consist of the same filaments, but the packing densities are different (Fig. 4A). The filaments of these coils enter the capsule material (Fig. 4A). Similar, but larger, complexes are scattered in the nucleus (Fig. 5e, bottom). These complexes resemble the secondary nucleoli that have been described in cricket (Jaworska and Lima-deFaria, 1973) and dragonfly oocytes (Halkka and Halkka, 1968). By the start of vitellogenesis the formation of the karyosphese capsule is complete. The outer zone of the capsule is uneven, but significantly thicker, reaching 0.5 to 1 pm in width, and acquires lobes (Fig. 4G and 5e). The lobes assume irregular shapes and sometimes have inner partitions; they are filled with karyoplasm with different inclusions described above. By the end of oogenesis the chromosomes are spiralized inside the capsule and the walls of the capsule are noticeably thicker (Fig. 5f).

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M. N. GRUZOVA AND V. N. PARFENOV

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS

17

Thus, the process of karyosphere capsule formation involves fragmentation of the extrachromosomal DNA body and the associated transformation of nucleoli. The formed complex capsule consists of a closed outer fibrillar zone with lobes; from the inside the capsule is lined'with dense strands or cylindrical structures (Fig. 5e, top). Both are derivatives of the surface layer of pronucleoli and extra-DNA bodies and are regarded as anomalous SC. The presence of DNA in them is assumed. During capsule formation there appear on its surface some complex structures (a type of secondary nucleoli), consisting of small clumps and ring bodies containing DNA.

3. Discussion A peculiarity of oocyte nuclei of golden-eyes is that the rDNA amplification is nor confined to earlier stages of oogenesis, when the extra-DNA body is formed. A new cycle of extra-DNA synthesis has been found in mid diplotene (Gruzova, 1977). Apparently, the supply of rDNA, amplified at earlier stages of oogenesis, is sufficient only for the formation of several tens of, or maybe a hundred, ring nucleoli, whereas for subsequent transformation of these nucleoli into a large net of nucleolar strands additional rDNA synthesis is necessary. Another peculiarity of these nucleoli is the presence of a fragmented narrow peripheral zone containing DNA. This zone in early oogenesis is capable of forming fibrillar strands required for capsule formation. Subsequently, proribosoma1 particles appear both in the peripheral zone of nucleoli and in the strands. The evolution of extra-DNA bodies and the appearance of multiple nucleoli

FIG. 4 Karyosphere with capsule in Chrvsopa perla at pre- and vitellogenesis. (A) A region of the karyosphere with a young closed capsule. Closely attached to the fibrillar material of the capsule (C) are dense clumps and ring structures (arrows) of extra-DNA as well as granular coils (GC) of various density. Some of the GC and dense strands fuse with the material of the capsule (arrowheads). Ch, loose and dense chromatin inside the capsule ( X 17,500). ( B ) Fragment of the karyosphere capsule in C. carnea treated with EDTA. The double body, ring structure (arrows), and chromatin (Ch) show a decrease in contrast (RNA-containing structures tend to retain it) ( x 14,000). (C,C2) Double bodies as in (B) from the nucleus. (C,) Control section; (C,) after EDTA treatment ( X 14,000). (D) Autoradiograph of karyosphere; hybridization in siru with [3H]rRNA of Drosuphila. The label is localized in the capsule. Chromosomes (Ch) are not labeled. Bar = 10 pm. (E, F) Regions of nucleus with karyosphere. Clumps and ring structures are seen in the capsule. (E) Ag-NOR reaction. (F) Azur+osine staining. Bar = 10 pm. ( G ) Karyosphere in oocytes at mid vitellogenesis. The lobes of the capsule are enlarged significantly and filled with nucleolar strands (NS) and dense clumps (CL) of extra-DNA ( X 7,000). Inset: thick section of karyosphere at a similar stage after aurarnine 00 staining; chromosomes are heavily fluorescent; karyosphere capsule ( C ) is also fluorescent ( X2100).

18

M. N. GRUZOVA AND V. N. PARFENOV

a

b

C

d

e

f

FIG. 5 Changes in the nuclear structures in the oogenesis of Chrysopa. Top row: formation of the karyosphere capsule; bottom row: transformation of the nucleoli. ( a d ) Previtellogenesis. (e, f ) Vitellogenesis. B, body of extra-DNA; PN, pronucleoli; FS, fibrillar strands (polycomplexes); NS. nucleolar strands; RN, small ring nucleoli; C, capsule; Ch, chromosomes; GC, granular coil. See text for further details.

have been studied in oocytes of crickets and some beetles (Cave and Allen, 1974; Lima-de-Faria, 1974; Matuszewski et al., 1977; Trendelenburg et al., 1977). In these insects the DNA body represents an aggregate of mostly ring molecules of nucleolar DNA. As a result of fragmentation and dispersal of the DNA bodies, thousands of small ring nucleoli appear in the nucleus. In these nucleoli short chains of actively transcribing ribosomal cystrones (one to five) have been detected (Trendelenburg et al., 1977). In Chrysopa the process of transformation of nucleoli and extra-DNA is in principle similar to that in Achetu and Dytiscus, although it is multistaged, and finishes with the formation of nucleolar strands, rather than ring nucleoli. It should be noted that in beetles, as in Chrysopa, the karyosphere is formed in the oocyte nucleus (Bier et al., 1967; Matuszewski et al., 1977). Similar morphology and description of the capsule material have been reported for the karyosphere of the mosquito A. gambiae (Fiil, 1978). It should be pointed out that in golden-eyed flies as well as in mosquitoes (Fiil and Moens, 1973; Fiil, 1976b) the formation of a karyosphere with a capsule is paralleled by the development of complex nucleoli. At present the question of the amplification of nucleolar DNA in mosquitoes remains unclear. It might be that this same process occurs in them, which is evidenced by the presence in oocytes of A. fasciata (Bauer, 1933) and other species of Aedes (M. N. Gruzova, unpublished) of a Feulgen-positive nucleolus in addition to a karyosphere. The body of “metabolic” DNA has also been described in oocytes of the mosquito Tipula oleracea (Lima-de-Faria and Moses, 1966). Karyosphere formation in Tipula has been shown by light-microscopical observations only (Bayreuter, 1952, 1956).

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS

19

C. Nucleus and Karyosphere in the Oogenesis of Tenebrionid Beetles 1. Some Generalities on the Development of Oocyte and Its Nucleus The beetle oocytes of Tenebrionidae family develop in meroistic telotrophic ovarioles. Despite some species-specific differences, the general pattern of changes in nuclear structures during oogenesis in Blups lethiferu, B. mortisagu, Gnuptor spinimanus, and Tentyriu nomus tuuricu is the same (Gruzova and Batalova. 1979; Gruzova, 1982; Alexandrova, 1992). The organization pattern of the oocyte nucleus is typical of nutrimental oogenesis: nucleoli are absent and karyosphere formation is accompanied by the cessation of chromosome transcription. Let us consider this pattern on B. lethiferu. As oocyte growth begins, chromosomes become loose and they will incorporate [3H]uridine. The nucleus contains one large (3-5 pm) and several small nucleolus-like bodies (NLBs) (1 pm) (Fig. 6a); the NLBs do not synthesize RNA to any noticeable extent. As early as mid previtellogenesis, karyosphere formation begins; the chromosomes shorten and, while fine threadlike structures appear among them, increase in number (Fig. 6a). By the end of previtellogenesis around a compact knot of chromosomes there appears a large fibrous region (Figs. 6c, 6f, 6g, and 6j), which occupies up to one-third of the nuclear volume. The nucleus in that stage reaches 300-400 pm in diameter. The karyosphere persists until the disintegration of the germinal vesicle. Besides fibers, the capsule contains numerous small granules or microbodies (0.2-1 pm) (Figs. 6c, 6e, and 6g) and large protein NLBs (3-6 pm) (Figs. 6b, 6d, and 6g). Nucleolus-like bodies are formed on chromosomes and then move toward the periphery of the nucleus. Cytochemical tests reveal low pyroninophilia in NLBs. They mostly consist of acid proteins (Gruzova, 1962b; Gruzova and Batalova, 1979). In the AgNOR reaction they, as well as capsule micro-bodies, are impregnated with silver (Figs. 6d and 6e) (Gruzova and Mamaeva, 1986). The fibrous material of the capsule also consists mostly of protein formations. Nevertheless, gallocyanine staining of sections and DAPI reaction on isolated karyosphere reveal the presence of DNA in bodies (Figs. 6h and 6i). 2. Electron-Microscope Data

Studies have been done on the ultrastructure of oocyte nuclei of four species of tenebrionid beetles: B. lethiferu, B. mortisagu, T. nomus tauricu, and G. spinimanus. The stages of karyosphere formation have been studied in different species and to a different extent. Therefore, the data obtained are arranged according to the stages of karyosphere formation, taking into account the evidence

20

M. N. GRUZOVA AND V. N. PARFENOV

FIG. 6 Karyosphere in oocytes of tenebrionid beetles. (a-f) Karyosphere in B h p s lethiferu. Note that the capsule microbodies in (c) are absent in the later karyosphere capsule (f). (b) Section, phase contrast: (c) section, stained with hallocyanine; (d, e) Ag-NOR reaction after Howell and Black (1980). Note that NLBs and microbodies are silver stained. (9, i) Karyosphere in Tenfyriu nomas tuurica; (i) isolated nucleus, DAPI stained. Note the fluorescence of karyosphere and microbodies. (h, j) Karyosphere in Gnuptor spinimunus; (h) fragment of karyosphere isolated from the nucleus, DAPI stained after RNase treatment. Note the fluorescence of capsule and microbodies. (a, f, g, j ) Sections, stained with iron hematoxilin after Heidenhain. N, nucleus: K, karyosphere; KC, karyosphere capsule: NLB, nucleolus-like body; MB, microbody. For full explanation see text. Bar = 10 pm.

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS

21

on different species. In nuclei of young diplotene oocytes of B . lethifera one can see dense and rather loose chromatin regions (sections of chromosomes) bleached after EDTA treatment, one large NLB (3-5 pm in diameter), and single fibrillar masses of moderate electron density. The latter are often in contact with chromatin clumps. Somewhat later fibrillar strand-like structures appear. Such strands consist of bundles of longitudinally oriented filaments 10 nm thick-anlagen of capsule strands. During late previtellogenesis in the nuclei of all the beetles studied, the karyosphere with a capsule is already present. The chromatin of the karyosphere of B . mortisaga is represented by loose fibrillar material. In it, here and there one can see numerous electron-dense fibrogranular clusters (granules 15 to 50 nm) with halos around them (Fig. 7a). Also visible in the chromatin is one ring structure (Fig. 7a), which is possibly related to the complex of sex chromosomes (White, 1973; Gruzova and Mamaeva, 1986). Located along the periphery of the chromosome knot are large (3-8 pm in diameter) NLBs (Fig. 7a). They consist of densely packed fibrils 8-10 nm thick (Fig. 7b). The peripheral, more dense, narrow zone of NLBs a1-e bleached after EDTA treatment. Nucleolus-like bodies are more often surrounded by fibrillar strands (Fig. 7a). On the surface of NLBs are granular-fibrillar aggregates. As a rule, one or several fibrillar strands branch from them (Fig. 7b); these strands pierce the entire chromosome knot. Numerous thin strands are abundant in the vicinity of the karyosphere. The width of capsule strands varies from 10 to 300 nm. Depending on the section, they either consist of granules or are cross-striated, which makes them similar to the central element of SC. As previously mentioned, in all four species of beetles there are microbodies (0.1-1 pm in diameter) in the karyosphere capsule and in the karyolymph (Fig. 8). They are fibrogranular structures with organization similar to that of aggregates on the surface of NLBs and in the chromosomal part of the karyosphere. It should be pointed out that many microbodies are in close contact with the capsule strands (Figs. 8b, 8d, 8f. and 8i). The fibrillar components of the bodies after EDTA treatment of sections are bleached to a greater extent than their granular counterparts (Figs. 8c, 8e, and 8g). This indicates that in the oocyte nuclei of beetles there are structures containing extra-DNA. Analysis of electron micrographs shows that the granular component of the microbodies consists of coiled filaments 10-20 nm thick made of granules 30 nm in size. This organization is clearly seen in T. nomas taurica (Fig. 8h). It should be noted that these bodies closely resemble the secondary nucleoli of Acheru (Jaworska and Lima-de-Faria, 1969) and are similar to some extent to the “granular coils” of Chrysopa. In mid and late vitellogenesis the capsule strands become wider due to an in-

crease in the number of filaments they contain. On average, the thickness of strands reaches 0.3-0.5 pm (Fig. 8a). The strands not only interweave with the knot of chromosomes from outside but also penetrate across the chromosomes.

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KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS

23

often forming large aggregates in the chromatin (Figs. 7c, 7e, and 7f). At this stage bundles of fibers not only are linear but also sometimes acquire a circular shape, forming a type of vacuolated NLB (Figs. 7c and 7d).

3. Discussion Oocyte nuclei of tenebrionid beetles, different from those of golden-eyed flies, are characterized by the absence of extra-DNA bodies and nucleoli; the latter are evidently substituted by NLBs. As in Chrysopa, the development of the karyosphere capsule is accompanied by a decrease in transcriptional activity of chromosomes. The karyosphere capsule of the beetles studied is not a closed cavity. The capsule strands pierce the entire chromosome knot and go far outside it, forming a wide (up to 200 pm) fibrous zone. The fibers of the capsule are considered to be derivatives of lateral elements of SC. Multistranded structure of lateral elements of SC become evident under certain conditions in some animal and plant gametocytes (Mazo and Gil-Alberti, 1986; Pujol et al., 1988; Grebennikova and Golubovskaya, 1991). In the capsule, in close apposition with filaments, there are numerous fibrogranular microbodies often showing a ring structure. The microbodies contain DNA and sometimes resemble secondary nucleoli of other insects. Their origin remains obscure thus far. By the end of oogenesis the bodies disappear from the capsule. During the period of capsule formation several generations of NLBs are formed on chromosomes. They evidently move through the capsule toward the periphery of the nucleus. A comparative analysis of nuclear structures of the four species of tenebrionid beetles clearly reveals the species specificity of these structures. This is shown by the structural organization of microbodies, the NLBs, and the “architecture” of the capsules.

FIG. 7 Ultrastructure of karyosphere in tenebrionid beetles. (a) General view of karyosphere and part of the capsule in Slops morrisugu (previtellogenesis). NLBs are surrounded by strands (mows). Ch, chromatin ( X4830). Inset: (top right) granular aggregate among chromosomes at higher magnification, X 28,980; (bottom left) another section of chromatin ring structure (arrowhead) at higher magnification, X 27,600. (b) Fragment of a NLB as seen in (a) at higher magnification; a strand of the capsule comes off the chromatin clump on the surface of NLB (arrows) ( X27.600). (c) General view of karyosphere and part of the capsule in B. lethiferu at late vitellogenesis. Ch, chromatin; KC, material of karyosphere capsule. For further explanation, see text ( x 4830). (d) NLB of B. lerhiferu from late karyosphere. The fibrous structure of NLB is similar to that of the capsule ( X 17,250). (e, f ) Parts of the same karyosphere as seen in (c) at higher magnification, X 20,700. (e) Treatment of the section with EDTA. Chromatin (Ch) is bleached; the material of karyosphere capsule (KC) retains contrast. (f) Control. Chromatin clump and capsule material are in close contact (arrows).

24

M. N. GRUZOVA AND V. N. PARFENOV

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS

25

The formation of a karyosphere with a capsule has been described in oocytes of other families of Polyphaga beetles by Matuszewski and co-authors (see references in Tables I and 11). In all these beetles the nucleus of the oocytes is extremely large (see Table 11). In some species of the Silphidae family, the oocyte nuclei are very similar to those of Tenebrionidae. The authors report the appearance of numerous small basophilic NLBs that do not incorporate [3H]~ridine and their movement from chromosomes toward the periphery of a large capsule. In the capsule formed in Geotrupes, as in Blups, the numerous microbodies disappear (B. Matuszewski, personal communication). Nevertheless, there are species of beetles (Creophilus maxillosus, Dytiscus marginalis) with oocyte nuclei organization similar to that in Chrysopu: the karyosphere with a capsule is formed and there are bodies of extra-DNA and numerous small nucleoli (Matuszewski and Kloc, 1976). Unfortunately, no electron-microscopical analysis of these nuclear structures has been performed.

IV. Formation of Karyosphere in the Oogenesis of Vertebrates The phenomenon of the accumulation of chromosomes in the center of the nucleus is quite common in the oogenesis of vertebrates (Table I). As early as 1925, Wilson noted that the concentration of chromosomes in the nucleus in some reptiles and birds can be so high that it much resembles the karyosphere in some insects. However, this phenomenon in vertebrates is even more poorly studied than that in insects. In autumn oocytes of minnows, in the second half of their development, Chubareva (1957) reports the presence of a karyosome, which includes all the nuclear chromosomes. In contact with the karyosome are several nucleolus-like protein bodies. Some time later a fibrillar capsule is formed around the karyosome. As can be seen from the earliest studies published about a century ago (Ruckert, 1892, 1893; MarCchal, 1907), the formation of a chromosome coil in the oocyte nucleus of cartilaginous fishes is not infrequent. This phenomenon has also been reported for teleosts. However, these data are not abundant (Table I).

FIG. 8 Microbodies from karyosphere capsule of tenebrionid beetles. Note the fibrillar-granular structure of some of the bodies and their attachment to strands of the capsule. For further explanations, see text. (a, c) B . lethifera; (b, e ) B . mortisaga; (d, f, g, i ) G. spinimanus; (h) complex microbody of T. nomas tourica; ( c , e, g) ultrathin sections after EDTA treatment; fibrillar components of bodies are bleached. Bar = 0.5 pm.

26

M. N. GRUZOVA AND V. N. PARFENOV

TABLE II Dependence of Karyosphere Capsule Formation on Dimension of Oocyte Nucleus and Presence of Extra-DNA

Species Chrysopa perla Gnaptor spinimanus BIaps lethifera B . mortisaga Culex pipiens Aedes aegypti Rana temporaria R. ridihunda

Diameter of nucleus (in pm)

Extra-DNA in nucleus

+

Karyosphere capsule

+

220 X 140 450 300 350 400 X I40

+ + +

400-500 500-700

+ +

+ +

+

+

?

+

+ + +

Reference GNZOVa, 1960 Gruzova el a / . , 1972 Gruzova and Batalova, 1979 Gruzova, 1962b, 1979 Gruzova, 1979 Fiil and Moens.1973

?

300X 16 Gyrinus natator Necrophorus humator 950 X 150 500 Silpha sinuata S . atrata 500 S. thoracica 500 580 Necrodes littorales Creophilus maxillosus 700 X 160 Mikiola fagi 20 X 30 20 X 30 Oligotrophus schmidti 30 Aphidoletes aphidimysa 40 Calliphora erythrocephala 40 Musca domestica 35 Rhogogaster picta 40-50 Eurygaster integriceps 40-50 Galleria melonella 50 Laspeyresia pomonella 50-80 Panorpa communis Glososiphonia complanata 40-50 Drosophilia melanogaster 20-25

+ + + + + +

+ + + -

+

+ +

+ + + + +

+

-

-

-

-

-

-

-

-

-

-

-

-

Wagner, 1923 Gruzova and Parfenov, 1973; Parfenov, 1979 Matuszewski and Hoser, 1975 Matuszewski et al., 1977 Matuszewski et al., 1977 Matuszewski et al., 1977 Matuszewski et al., 1977 Matuszewski et al., 1977 Matuszewski and Kloc, 1976 Matuszewski, 1962 Matuszewski, 1966, 1982 Gruzova et al., 1987 Gruzova, 1967 Bier et al., 1967 Bobrova and Gruzova, 1967 Kozhanova, 1974 M. N. Gruzova, unpublished Gruzova, 1974 Gruzova, I962a Gruzova and Zaichikova, 1967 King, 1970

Regarding amphibians, many authors have reported the formation of a chromosomal knot in the oocyte nuclei (Table I). However, these studies focused on lampbrush chromosomes (Callan, 1986). In fact, in oogenesis of many species of different vertebrates, the development of lampbrushes is observed. Together with nucleoli, they function during the first half of oogenesis. This active state of the nucleus is accompanied by a drop in the transcriptional activity of chromosomes and nucleoli and accumulation of chromosomes into a karyosphere. Often karyosphere formation is paralleled by the appearance around the chromosomes of newly formed capsule-shaped structures. In general, changes in the functional state of the chromosome apparatus

27

KARYOSPHERE IN OOGENESIS AND MORPHOGENESIS Pisces, Arnp;bia

ReDtilia

h

aK@ 1

1

.. .:

W

FIG. 9 Changes in the functional state of nuclear structures in the oocytes of vertebrates. N, nucleolus: PB, protein bodies: Ch, chromosomes; K, karyospheres; KC, karyosphere capsule.

during oogenesis of many vertebrates can be schematically presented as shown in Fig. 9' (Gruzova, 1975).

A. Karyosphere in Oocytes of Anurans 1. Formation of Karyosphere in Oocytes of Rana temporaria At present the oocyte nucleus of amphibians has become a classical object for studying a wide range of general cytological problems, such as structure and function of lampbrush chromosomes and nucleoli, transcription and amplification of genes, and karyoskeleton (see reviews: Franke et al., 1979; Callan, 1986; Macgregor, 1972, 1986; Scheer and Zentgraf, 1982; Scheer and Dabauvalle, 1985; Higashinakagawa, 1982; Hadjiolov, 1985; Gruzova, 1988; Zbarsky, 1988). However, the organization of the nucleus throughout oogenesis in amphibians is far from being completely understood. As pointed out above, the period that is more obscure is late oogenesis (stages 5-6 according to Duryee, 1950), i.e., the period just prior to maturation. The nuclear transformations in late oogenesis of R . temporaria were described in detail by Wagner (1923). He followed seasonal changes occurring in oocyte nuclei for a period of about 4 years. According to present classifications 'References on lampbrushes in oocytes of vertebrates can be found in the review by Callan ( I 986) and those on the karyosphere. in Table I.

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(Duryee, 1950) these changes correspond to stages 3-6 of the meiotic prophase I. According to Wagner, around lampbrush chromosomes (stage 3) a fibrillar capsule is formed, whose walls thicken as chromosomes shorten and their lateral loops reduce. Numerous nucleoli, migrating into central regions of the nucleus, appear to be gradually incorporated into the expanded network of the capsule strands. The capsule attains its maximum development in March, not long before ovulation, when chromosomes join into a tight knot. Wagner’s data have been reconsidered for almost 50 years. Material collected in nature at different seasons as well as that obtained in laboratories by hormonal stimulation of oocytes has been used. The bulk of Wagner’s data have been confirmed and practically all nuclear structures have been shown to be involved in the formation of a karyosphere capsule (Gruzova and Parfenov, 1973, 1976, 1977; Parfenov, 1974; Parfenov and Gruzova, 1975a,b). Karyosphere formation in R. temporuria starts in late summer-early autumn with a transference of nucleoli in the central region of the nucleus. Around the lampbrushes there appears a barrier of fibrous material 1-3 pm wide that separates them from nucleoli (Fig. 10a). Nucleoli accumulate around chromosomes in an asynchronous manner and the fibrous capsule around chromosomes develops first on the side of the nucleoli. Inside the capsule, among chromosomes, numerous DNA-containing ring structures are seen (3 pm in diameter) (Figs. 10a and lob). On the side of the nucleoli, on the surface of the fibrous component of the capsule, microbodies 1-3 pm in diameter are located in a row (Fig. 10a). In autumn the concentration of chromosomes increases, the lateral loops of lampbrushes are reduced, and a fibrous barrier closes around the chromosomes (Fig. 1Oc). This barrier shows a distinct reaction to proteins and lipids. According to Duryee, the formation of the karyosphere capsule is completed in stage 5-6 oocytes (late winter-early spring). A network of fibrous material

FIG. 10 Nuclear structures and the karyosphere formation in diplotene oocytes of Rana temporaria (light-microscopical data). (a) General view of the oocyte nucleus (an early stage of karyosphere formation). The fibrous zone (arrows) is developed between the nucleoli (Nu) and the chromosomes (Ch). The region of ring structures (RS) is seen. (b) Central part of the developing karyosphere at higher magnification. Ring structures (RS), lampbrush chromosomes (Ch), and fibrous zone (arrows) are seen (August). (c) The formed karyosphere with capsule from autumn oocytes (late October). Lampbrushes with reduced lateral loops are completely surrounded by fibrous material, which is clearly seen in the nucleolar capsule as well (arrows). (d) Peripheral zone of the karyosphere capsule from winter oocyte; nucleolar threads (tails) are visible (arrows). (e) A nucleolus of irregular shape from a winter oocyte. (f) Central part of the karyosphere with capsule from winter oocytes stimulated to maturation in virro (8 hr of stimulation with progesterone). The picture is the same in spring oocytes. Note the well-developed fibrous component of the capsule (arrows) surrounding highly contracted bivalents (Ch). (g) Detection of DNA-containing material (arrows) between segregated and fragmentated nucleoli from the capsule of winter oocytes (autoradiography, [3H]actinomycin D).All bars = 10 pm. (a-f) Iron hemotoxylin stain after Heidenhain; (g) gallocyanin.

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appears, which interweaves the chromosomes and goes into the midst of nucleolar zone (Fig. 10f). The formation of the karyosphere and its capsule, as shown autoradiographically, is paralleled by the extinction of synthetic processes in the oocyte nucleus. With the start of karyosphere formation FWA synthesis in the nuclei decreases almost three times compared with that during the summer period. The completion of karyosphere formation (winter) is associated with an almost complete lack of [3H]uridine labeling over the chromosomes and its significant decrease over the nucleoli (Parfenov and Gruzova, 1975b). The large size of the karyosphere capsule (up to 200 pm) allowed its isolation from the nuclei (500 pm in diameter) using Callan’s method (Callan, 1966). These experiments having convincingly shown that chromosomes are actually situated inside a sac of fibrous elastic material (Parfenov, 1974). By the end of April the nuclear envelope is disintegrated and the nucleoli accumulate and finally fuse, forming a mass around the inner fibrous zone of the capsule, still present around the chromosome. Analysis of ultrathin sections of the core of the formed karyosphere in winter oocytes (stage 5 according to Duryee) has shown that among dense material of condensed bivalents, sometimes in contact with them, there occur spheroidal fibrillar bodies or ring structures, 0.1 to 1.5 pm in diameter. The wall of the rings is of different thickness (0.1-0.5 pm) and consists of densely packed strands 25-30 nm thick (Figs. 1 lc and 1Id). The surface of the rings appears coated; the coating consists of granular fibrils 30 nm thick, resembling the granular coils in oocyte nuclei of insects (see above). A fibrous barrier separating bivalents from nucleoli consists of a network of anastomosing strands 40-50 nm wide (Fig. 1la) (Gruzova and Parfenov, 1976, 1977). The strands look like a dashed line because of the alternation of light and dark regions. The stretch of dark regions is 65-75 nm, and that of the light ones, 50 nm. At high magnification and different areas of sections these structures appear to be rows of pore complexes (Fig. 1lb). However, the pores in the strands are linked not by membranes, but by fibrillar material and resemble the pseudomembranes described in oocytes of mosquitoes (Fiil and Moens, 1973). It should be pointed out that there is similarity, at least on the exterior, between the pseudomembranes of oocyte nuclei of R. temporaria and the organization of the complex “lamina pores” the oocytes of Xenopus laevis, in which the pores are joined by the fibrillar material of the lamina (Krohne et al., 1982a,b).

FIG. 11 Structures of the karyosphere capsule in Rano temporaria (electronmicroscopical data). (a) Ultrastructure of the wall of a capsule’s fibrous component surrounding chromosomes. Note the specific accumulation of pore-like complexes (PIC) and ring structures (RS) connected with pore-like complexes of the fibrous zone of the capsule by fibrillar material (arrows). (b) Annuli of pore-like complexes from fibrous component of the capsule at higher magnification. (c, d) Ring structures (RS) on sections made at different levels. All bars = 0.5 nm.

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FIG. 12 Nuclear structures and karyosphere formation in oocytes of Rana ridibunda. (a) Karyosphere in the early period of formation. Nucleoli (Nu) are assembled in the center of a nucleus; numerous ring (RS)and polymorphous structures (PS) (November). (b, d) Fluorescence micrographs of ring (b) and polymorphous (d) structures after staining with DNA-specific aurarnin SO, (for method see Rosanov and Kudryavtsev, 1967). (c) Central part of young karyosphere (November)

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In this stage the nucleoli in the karyosphere capsule represent structures of irregular shape (Fig. 10e). They consist of fibrillar material and undergo segregation and fragmentation accompanied by a decrease in [3H]uridine incorporation to a minimal level (Parfenov and Gruzova, 1975b). On the periphery of the nucleoli one can see the fibrillar centers (Gruzova and Parfenov, 1977). Nucleolar fragmentation results in the appearance of numerous micronucleoli. During nucleolar segregation and fragmentation some of the nucleolar DNA is evidently eliminated: [3H]actinomycine D binds to the nucleoli and the tracks between them are localized as well (Fig. log). 2. Karyosphere in Oocytes of R. ridibunda The nucleus in late oogenesis has been studies in another species of frogs, R. ridibunda (Gruzova and Parfenov, 1973; Parfenov, 1979, 1983; Parfenov et al., 1983; Parfenov and Gruzova, 1984). In spring (March-April) oocytes (stage 6) of mature R. ridibunda the situation in the nucleus was as follows. The bivalents occupied a central area in the nucleus and were surrounded by numerous nucleoli. However, fibrous material both around the chromosomes and between the nucleoli was not visible. Instead, in the center of the karyosphere there was a large (20 pm) round protein body, with bivalents attached to its surface (Fig. 12e). Study of ultrathin sections revealed that the central body consisted of an accumulation of pore-like structures (Fig. 13a). The latter were joined to each other by fine fibrillar material, although, in contrast to pseudomembranes of the grass frog, not regularly arranged. Thus, in R. ridibunda the karyosphere had an inverted appearance: an accumulation of pore-like structures appeared to be inside the knot of bivalents rather than outside. It is possible to distinguish the stages of transformation of nuclear structures leading to karyosphere formation during the year prior to spawning in R. ridibunda. In autumn (October-November) in large oocytes with yolk, the nucleoli shift to the center of the nucleus and surround the reduced lampbrushes (Fig. 12a). In a wide chromosome-nucleolar region (up to 200 pm in diameter) numerous polymorphous and ring structures are located, mostly in its center (Figs. 12a and 12c). They are similar to those observed in R. temporaria (Figs. 10a

with ring and polymorphous structures. ( e ) The formed karyosphere (April); central body of a karyosphere (CB) and associated chromosomes (Ch) are present. (f) Ring structures (arrows) in contact with nucleoli of winter oocytes. (g) Nucleolus of irregular shape from a winter oocyte. (h) Nucleoli of young oocyte after silver staining (Howell and Black, 1980); some intensively stained rings of rDNA (arrows) in nucleoli are seen. (i) Oocyte nucleoli at late vitellogenesis after silver staining (Howell and Black, 1980).Rings of rDNA in nucleoli and free rings in karyolymph are present (arrows). (j, k) Nucleoli with nucleolar threads (tails) (arrows) from winter oocyte (February); numerous beads on nucleolar thread are seen. All bars = 10 pm. (k) Phase contrast; (a-j) Iron hemotoxylin stain after Heidenhain.

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and lob), although larger (2-5 pm) and more numerous. Staining with fluorochromes reveals the presence of the DNA (Figs. 12b and 12d). With spring coming, the number and size of these structures decrease and then they disappear. In winter oocytes stimulated for maturation in vitro, in the nucleus among lampbrushes and nucleoli several spheroidal bodies could be observed (3-5 pm in diameter). In ultrastructure and reaction to overall proteins they are similar to the central body. A characteristic feature of these anlagen of the central body (ACB) is the presence on their surface, in close contact with them, of polymorphous and ring structures (Fig. 13b). On ultrathin sections it can be seen that the fibrillar material of these structures borders on, and sometimes directly passes into, pore-like rings (Fig. 13a). The open cavity of these ring structures usually faces the ACB (Fig. 13c). Apparently, the ACB eventually fuse into a single body; however, we failed to observe this. It remains unclear how in late karyosphere the chromosomes appear to be attached to the central body. It is practically impossible to determine this on the sections of oocytes of previous stages. It can only be postulated that the ACB are associated with lampbrush chromosomes. The mediators in this association, and in ACB formation as well, are apparently ring structures. The nucleoli in winter oocytes, as in R . temporaria, unfold for a second time, acquiring complex irregular configurations (Fig. 12g). Their material undergoes segregation and fragmentation. During this stage ring structures appear on the surface of nucleoli (Fig. 12f); these structures are similar to those described above (Fig. 12c). The nucleolar origin of at least some of the ring structures is evidenced by their contact with nucleoli and by the Ag-NOR reaction as well. Argentophilic rings are detected in nucleoli of both young (Fig. 12h) and late oocytes (Fig. 12i). However, in the latter the number of rings in nucleoli is much reduced and some of them are located outside the nucleoli (Fig. 12i). Infrequently, long threads depart from the nucleoli (Figs. 12j and 12k); these threads are similar to those observed on the periphery of the R . temporaria capsule (Fig. 10d). Apparently, both are analogous to nucleolar tails described in

FIG. 13 Ultrastructure of the karyosphere in Rana ridibunda ( a x ) and human oocytes (d, e). (a) Part of the central body (CB) and ring structure (RS) of the karyosphere (arrows indicate the pore-like complexes). Bar = 0.5 pm. (b) Anlage of the central body (ACB) (February) and ring structures (arrows) connected with it (semithin section). Bar = 10 pm. (c) Pan of ACB in close contact with ring structures. Transfer of RS fibrils into the material of the central body is seen (arrows). Pore-like complexes of CB are detected near RS (triangles). Bar = 0.5 pm. (d) Part of the formed karyosphere in the oocytes from human antral follicle. The complex of closely associated structures (nucleolus, chromosomes, and NLB) is clearly seen. Note the two zones of the nucleolus. Bar = 0.5 pm. (e) Part of the cumulus oophorus with the same oocyte as in (d) (semithin section). Bar = 10 pm. Nu. nucleolus; N, nucleus, K. karyosphere; NLB, nucleolus-like body; Ch. chromatin; Zp, zona pellucida; CC. follicle cells of corona.

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oocytes of some Plethodons (Kezer et ul., 1971; Leon et ul., 1991). According to these authors, they are of membraneous origin and possibly contain DNA. Thus, it is thought that ring and polymorphous DNA-containing structures in the oocyte nucleus of R. ridibundu are of nucleolar origin.

3. Discussion The observation of karyosphere formation in oocyte sections of two species of frogs shows that in the nucleus, along with chromosomes and nucleoli, there are polymorphous and ring structures. The latter are more clearly seen in the nucleus of R. ridibundu. Also more evident in this species is the participation of these structures in the formation of the central body of the karyosphere consisting of pore complexes. Also evident is the formation de now and gradual disappearance of ring structures. The origin of these structures remains unclear at present. Some data points to the assumption of the nucleolar origin of ring and polymorphous structures. Also of importance is the presence of the DNA in the structures participating in the formation of the central body of the karyosphere. In this regard it is extremely important to find out the character of this DNA. If one compares the observations on karyosphere formation in frogs with those in insects (mosquitoes, in particular), similarities in the elements of the karyosphere capsule can be seen, primarily, the appearance of pore-like, pseudo-membrane structures in both of them, despite the enormous philogenetic distance between Amphibia and Insecta. In Aedes the pore complexes are joined to modified elements of SC that look like polycomplexes (Fiil and Moens, I973), which suggests the possibility of mutual transformation of these structures, one into the other. It is interesting that an accumulation of pore complexes, such as the central body in the karyosphere of R. ridihunda, has also been observed in the meiotic micronucleus of the ciliate Loxodes striutus (Bobileva, 1984) and Trucheloruphis totevi (Kovaleva and Raikov, 1990). Thus, a comparative morphological analysis allows us to follow in meiotic cells a series of structural states, transitory from a typical SC and its modifications, similar to it in appearance, to pore-like structures and lamellar elements in which this similarity is lost. All these structures can constitute the material of the karyosphere capsule. This suggests that underlying all these nuclear transformations are some common properties of meiotic cells.

B. Karyosphere in the Oogenesis of Reptiles and Birds As has been already mentioned, the phenomenon of chromosome concentration in the nucleus is very common in the oogenesis of reptiles (Table I), as follows from a review by Loyez (1906) and studies by Kulikova (1963; ArronetKulikova, 1969, 1975). Loyez reports the appearance of the central accumula-

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tion of chromosomes immediately after the lampbrush stage (“plumose chromosomes”) in oocytes of 14 species of reptiles, including lizards, snakes, turtles, and crocodiles. In Lacerfa murulis she describes the formation of a common envelope consisting of thin fibrils and lamellae around the chromosome knot. Arronet-Kulikova, who has studied the oogenesis in six subspecies of rock lizard, Eremia velox, and two species of Phrynocephalus, has confirmed the data of Loyez on the general pattern of changes in the nucleus in the oogenesis of reptiles and supplemented them with cytochemical data and new observations. She has shown that a large nucleolus appears in young oocytes; it is fragmented rather quickly and disappears. Further generations of multiple small nucleolus-like bodies are of protein origin. In addition to reptiles, Loyez studied the oogenesis of birds. In 10 species of birds, she found the situation in the nucleus to be on the whole similar to that in reptiles (Table I). The data by Loyez on nuclear structures in bird oocytes have been supported and partly reconsidered (Gaginskaya and Gruzova, 1969; Gaginskaya, 1972; Gaginskaya and Rodionov, 1993). In finches (Gaginskaya and Gruzova, 1969) and other passerine representatives (Gaginskaya, 1972) (Table I), it has been shown that spheroidal structures of plumose chromosomes thought by Loyez (1906) to be nucleoli are actually not; they do not incorporate [”Hluridine, do not contain amplified rDNA in any notable amounts (Gaginskaya and Gruzova, 1975; Gaginskaya and Rodionov, 1993), and show only fibrillar organization. These structures are formed on all the lampbrushes of the chromosome set and have been called protein bodies, being different from the single spheres described on some lampbrushes of amphibians (Callan, 1966, 1986). The evidence obtained in these studies has significantly changed the idea that in oogenesis of birds and amphibians, in addition to lampbrushes there are multiple nucleoli. The absence of nucleoli in oocytes rich in ribosomes can be explained by the fact that follicular cells of bird oocytes produce specific bodies, transosomes, which provide oocytes with ready ribosomes (Press, 1964; Bellairs, 1965; Schjeide et al., 1974; Gorbik and Gabaeva, 1975). Protein bodies are localized in paracentromere regions of lampbrushes. As indicated by olivomycine fluorescence, these chromosome regions contain GC-rich DNA, which is detected in protein bodies as well. As shown by electron microscopy, the formation of protein bodies starts in early diplotene as a result of the accumulation of fibrillar protein material around chromocenters formed by chromosomes. As typical lampbrushes develop, the protein bodies grow to 10-12 pm in diameter. In finches and other passerines it has been shown that during oogenesis the protein bodies move toward each other and finally fuse into one large protein body to which chromosomes are attached. This is how the karyosphere is formed in birds. A study of nuclei (nuclear matrixes) extracted from finch oocytes by the method of Berezny and Coffey (1976) has shown that in protein bodies karyoskeleton material is present (Tsvetkov and Gaginskaya, 1983). Inside the

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protein bodies, electron-dense structures have been detected: after treatment of sections with EDTA the structures lose contrast, indicating the possibility that they contain DNA. On ultrathin sections they appear to be “canaliculi” or ‘‘rings’’ formed by fibrils (30 nm diameter). The origin of these formations is unknown at present. According to Gaginskaya and Rodionov (1993), the chromatin located inside the protein bodies at the karyosphere stage is drawn out onto the surface of the protein skeleton where it is packed into spheroidal structures with the participation of nuclear matrix proteins, whereas large masses of chromatin apposed to the protein skeleton of the karyosphere apparently represent condensing bivalents. The topography of the karyosphere of birds whose morphogenesis has been

followed on diplotene oocytes of finches is similar to that of the lake frog, and such karyosphere is of the inverted type. In both cases the central body is apparently built of the proteins of the nuclear matrix.

C. Karyosphere in the Oogenesis of Mammals The oogenesis of higher mammals differs markedly from that in other animals and is characterized by a number of specific features when representatives of other classes of vertebrates are compared. The egg cells are relatively small in size (100-200 pm in diameter), are practically devoid of yolk inclusions, and can be classified as alecithal type. As early as the gastrulation stage, close contact between the embryo and the maternal organism is established due to the formation of the placenta, through which the embryo gets all the substances needed for development. A characteristic feature of mammalian oocytes is a stop in growth at the multilayered follicle stage; for a long period during the development of a follicle and its transformation into a Graaf vesicle the size of an oocyte remains unchanged (Moore et al., 1974). Oocytes enter the diplotene of meiosis in the prenatal period of development or immediately after birth and remain in such condition for a long time, for instance, in humans for several decades. Meiosis is resumed just prior to ovulation. According to some authors, diplotene chromosomes in mammalian oocytes look like lampbrushes (Baker and Franchi, 1967: Zybina, 1968, 1969: Zybina and Zybina, 1992). although the question whether they truly are lampbrushes is open to discussion (Callan, 1986). In oocytes of antral follicles of many mammals in late oogenesis a concentration of chromosomes around the nucleolus is observed (Table I). We believe that this complex formation can be regarded as an inverted karyosphere. The utmost expression of the concentration of chromosomes is the formation around the nucleolus of a Feulgen-positive ring (Austin and Braden, 1953; Mandl, 1963; Kiknadze, 1966; Zybina, 1969; Zybina et al., 1980). The formation of a karyosphere in human oocytes is accompanied by extinction of transcriptional activity of chromosomes and the nucleolus (Parfenov et al., 1989).

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This is apparently common for the oogenesis of all mammals. This phenomenon was first demonstrated on mice (Zybina, 1968, 1971). The ultrastructure of karyosphere has been studied only in human and vole mouse oocytes thus far (Parfenov et al., 1984, 1989; Zybina el al., 1990). In human oocytes of antral follicles ready for ovulation, a compact karyosphere (up to 13 pm in diameter) is round or, more often, lobe-shaped and occupies an eccentric position in the nucleus, often quite close to the nuclear envelope (Fig. 13e). The nucleolus, located in the center of karyosphere, consists of the densely packed, fine fibrillar material (each fibril 3 pm thick) (Fig. 13d). The lack of granules, purely fibrillar structure of the nucleolus, and lack of [3H]~ridine incorporations point to complete inactivation of the nucleolus. Two zones can be distinguished in the nuleolus. On the periphery a layer -0.5 nm in width of more contrasting material is seen (Fig. 13d). The nucleolus is closely surrounded by chromatin (fibrils 6-10 nm thick) and outside, in contact with the chromatin, are NLBs (5-10 pm in diameter); they consist of granules 18-20 pm in diameter and fine fibrillar material (Fig. 13d). Similar NLBs have been described in the oogenesis of other mammals (Chouinard, 1975; Zybina and Grischenko, 1977). In general, the organization of the karyosphere, as studied at a light-microscopic level, in all mammalian species considered is similar and differences are only in the time of its formation and duration of existence. Thus, in rodents the karyosphere appears just prior to ovulation and exists for a short time (e.g., in rat, for about 1 hr; Odor, 1955). Apparently, for this reason, it is very difficult to detect the karyosphere in this order of mammals, which is so well studied with respect to oogenesis. In contrast, in antral follicles of mink, the karyosphere is observed for almost the whole winter period (Kiknadze, 1966). In preovulatory follicles of humans the karyospheres exist for only a few days (Parfenov et al., 1984). The ultrastructure of the nucleolus in the karyosphere of humans is similar to that of the inactivated nucleolus from the antral follicles of mouse (Chouinard, 1975; Takeuchi, 1984, 1986; Zybina et al., 1990), rat (Antoine et al., 1987), pig (Crozet et al., 1981), and cow (Crozet et al., 1986). A component common to such fibrillar nucleoli is acid protein which does not show the argentophilic reaction (Antoine et al.. 1988). The protein material of these nucleoli possibly helps in alleviation of the meiosis block during maturation of oocytes. An attempt to follow the fate of this material in oogenesis and early embryogenesis has been made (Antoine ef al., 1988, 1989). In their intensive studies of the nucleolus these authors did not pay special attention to the state of chromatin in the nucleus, only pointing out the different extent of its aggregation around the nucleolus. However, this specific concentration of chromosomes, sometimes looking like a dense ring, as is observed in oocytes of humans and other mammals directly prior to the reductional division and maturation of the oocyte, should be very carefully studied.

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V. The Karyosphere Capsule and the Nuclear Matrix It is known that the nuclear matrix of a cell is represented by residual structures after treatment of nuclei with nucleases, nonionic detergents, and salt buffers of high molarity. After such extraction, there remain in the nucleus the chromosome scaffold, residual nucleoli, and fibrillar-granular network. The morphological integrity is more preserved in the peripheral lamina-pore complex (Comings, 1978; Berezney, 1979; Agutter and Richardson, 1980; Zbarsky, 1988), as well as SC in cases where gametocytes have been studied (Comings and Okada, 1976; Jerardi et al., 1983; Li et al., 1983; Raven and Ben-Ze’ev, 1984). As has been shown above, in the karyosphere capsule of insects and amphibians the same morphological components are present as in the nuclear matrix: nucleoli, microbodies, pore- and SC-like structures constituting the pseudomembranes. Therefore, it was no wonder that the electrophoretic pattern of proteins of the isolated karyosphere of the grass frog had many features in common with that of the nuclear matrix of normal and malignant cells (Zbarsky and Filatova, 1979; Engelhardt et al., 1982). The isolated karyosphere capsule treated with nucleases maintained its integrity. Clearly seen in nucleoli were the zones of polycomplexes and their association with pore-like pseudomembranes was detected. Subsequent dehistonization with 1.5 M NaCI, without breaking the integrity of the capsule, caused loosening of the nucleolar material, microbodies, and pseudomembranes. Electrophoresis of such preparations (protein matrix) of the capsule reveals about 30 bands of polypeptides: proteins of molecular masses of 220-180, 145-135, 70-60, and 45 kDa. These proteins have not been identified. Although, with respect to some of them the following assumptions can be made. The 145-kDa bands apparently correspond to the protein of numerous residual nucleoli of the capsule. A protein of similar molecular mass has been described as a major skeletal protein of oocyte nucleoli of X . laevis (Franke et al., 1981; Krohne et al., 1982b). The protein fraction of 60-70 kDa in all probability is associated with the presence in the karyosphere capsule of pore complexes within pseudomembranes. Pseudomembranes consisting mainly of pore-like structures joined by fibrillar material can be regarded as counterparts of the peripheral lamina-pore complex. Study of the latter from oocytes of X. laevis revealed the presence of the only karyoskeletal protein lamin I11 (L 111) of 68 kDa (Krohne et al., 1982a,b; Stick, 1987). Preliminary analysis (the data obtained in our laboratory) of the matrix of the nuclear envelope and nuclear gel, including the karyosphere capsule, of the grass frog also showed the presence of L I11 in these two fractions (E. Bugayeva, personal communication).* 2Electrophoresiswith subsequent immunoblotting has been carried out with the use of monoclonal antibodies to L 111 (L,167-1) protein, a kind gift of Dr. G. Krohne (Institute of Cell and Tumor Biology German Cancer Research Center, Heidelberg).

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The 45-kDa bands should apparently be attributed to the intranuclear actin found in the nuclear matrix of somatic cells (Nakayasu and Ueda, 1983, 1985a,b) as well as actin microfilaments among the nucleoli in the karyosphere capsule of R . temporaria (Drosdov and Parfenov, 1983; Parfenov and Galaktionov, 1987). The karyosphere capsule also shows the presence of glycoproteins. Their electrophoretic profile has some features in common with that of glycoproteids of both the nuclear matrix of rat liver (bands 200 and 96 kDa) and that of rat hepatoma-27 (bands 36-30 kDa) (Zbarsky, 1988). Besides proteins, the karyosphere capsule of R . temporaria also contains such components of the nuclear matrix as lipids. Using thin-layer chromatography the following substances have been detected in the karyosphere capsule: neutral lipids (triglycerides, diglycerides, cholesterol esters) and phospholipids (lecithin) (Nikolaenko et al., 1985). A significant concentration of [3H]glycerol label has been found in the sites of localization of the fibrous component of the capsule. The role of lipids within the karyosphere capsule is not quite clear at present, although it has been shown that in somatic cells the lipids are involved in the attachment of the DNA-containing structures to the nuclear matrix (Manzoli et al., 1982; Alesenko et a f . , 1983). The treatment of the matrix of the capsule of R . temporaria with hyaluronidase resulted in the obtainment of identical granules 25-30 nm in diameter. Similar granules represent a major component of the nuclear matrix of rat liver cells, nuclear envelope, and chromosome scaffold in Chironomus (Engelhardt et al., 1982). These authors assume that it is at the expense of these subunits that mutual transformations of the structures are possible. On the basis of all the above data and considerations it can be assumed that the karyosphere capsule represents a particular case of the nuclear matrix. In large germinal vesicles, where the association of chromosomes to the nuclear envelope is lost during oocyte growth, the karyoskeleton is organized in a limited space of the nucleus close to (or inside) the karyosphere, and there appears to be “a nucleus in a nucleus,” according to the terminology of past authors (see Table I).

VI. General Discussion and Concluding Remarks A. Protein Metabolism and RNA Synthesis in Karyosphere From the all data cited above we conclude that karyosphere formation is a phenomenon that occurs specifically during gametogenesis and quite commonly during oogenesis in various groups of animals, even those very distant phylogenetically. In different organisms the karyosphere appears at different stages of a

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long diplotene stage of meiotic prophase. Thus, for instance, in meroistic ovarioles of some insects, the karyosphere can be formed as early as the beginning diplotene. The karyospheres of animal oocytes have several common traits, which allows the karyosphere to be defined as a complex formation that includes the whole chromosome set of sex cells; relative inactivity of chromosomes in the karyosphere is accompanied by active protein metabolism. Morphologically, this is expressed in the formation on chromosomes of protein bodies containing small quantities of RNA and DNA and granules, their subsequent detachment from the karyosphere, their movement toward nuclear envelope, and their release into the cytoplasm. However, it still remains unclear what concrete morphobiochemical processes in oocytes are involved in the formation and transport of these granules and bodies. The studies aimed at elucidation of the role of some proteins in nuclear processes (Krohne and Franke, 1980a; Scheer and Dabauvalle, 1985; Krohne, 1985; Moreau et al., 1986; Roth and Gall, 1987) mainly deal with the stage of maximum activity of lampbrushes. Of special interest now are nuclear granules of RNP, the products of synthesis of lateral loops of lampbrushes (Sommerville et al., 1978; Krohne and Franke, 1980b; Pinol-Roma et al., 1989). Some studies have appeared giving evidence that at least part of such nuclear granules in an oocyte is associated with storage and packaging of snRNA, which is probably used in splicing of the newly synthesized mRNA in embryogenesis (Deon and Schultz, 1990). Immunocytochemical methods are now being intensively developed that will allow the identification of a series of proteins in these RNP complexes (Lacroix et al., 1985; Gall and Callan, 1989; Pinol-Roma et al., 1989; Roth et al., 1990). Characterization of RNP granules during karyosphere formation still remains obscure. As has been mentioned, karyosphere formation can be regarded as a state of relative inactivation of chromosomes. However, even if we confine ourselves to insects, there are wide variations, such as the range from complete absence of ['Hluridine incorporation in the karyosphere of Chrysopa (Gruzova, 1966) to rather heavy labeling in Calliphora (Bier et al., 1967). In fact, the question of RNA synthesis in the karyosphere remains unanswered, with respect both to the criteria of its intensity and to the types of synthesized RNAs.

B. Patterns of Chromosome Assembly into the Karyosphere Not much is known about the possible means of chromosome accumulation into the karyosphere. Apparently, it differs among different animals. For example, in scorpion fly three or four groups of chromosomes are first formed, joined by a common chromocenter; then these groups accumulate into a single knot, the

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karyosphere (Bauer, 1933; Gruzova, 1962a). In Drosophila oocytes, the karyosome is formed by the joining of the centromere chromosome regions into a common chromocenter in the mid pachytene (Nokkala and Puro, 1976; Puro and Nokkala, 1977). In other insects, for instance, some flies (Bier et al., 1967), mosquitoes (Fiil, 1978). and butterflies (Gruzova, 1974; M. N. Gruzova, unpublished data), chromosomes are joined around a large round body, or Binnen Korper, whose origin and fine structure are still obscure. Only quite recently it was reported in the literature (Gall and Callan, 1989) that the Binnen Korper of arthropod oocytes is possibly analogous to spheres of amphibian oocytes, and participates in the assembling of snRNP complexes. In some vertebrates the karyosphere is also formed by fusion of protein chromosome structures. For example, in birds it is formed by the fusion of protein bodies associated with chromosomes. As a result, a single giant sphere is formed, an analog of the round body in insects, and shortened chromosomes are located on its surface (Gaginskaya and Gruzova, 1969; Gaginskaya, 1972; Gaginskaya and Rodionov, 1993). In oocytes of lake frog the chromosomes also appear to be attached to a large protein body (Parfenov, 1979, 1983). Is the SC involved in karyosphere formation? According to some authors, the SC does not take part in the assembling of chromosomes into the karyosphere; in the absence of SC, karyosphere formation in oocytes of some mutants of Drosophila was observed to proceed quite normally (King, 1970). According to other authors, modified SC or its elements serve as binding material in chromosome assembly (Fiil and Moens, 1973; Gruzova, 1979) (see above). Whatever the manner of chromosome assembly in the karyosphere, they are detached from the nuclear envelope, and this is never the case in somatic cells.

C. Characteristic Features of Oocyte Nuclei of Animals in which the Karyosphere Capsule Is Formed A characteristic trait of karyosphere in some species is that it is surrounded by a multilayered capsule. However, the karyosphere capsule does not develop in all animal species. An attempt has been made to determine the characteristic features of oocyte nuclei of animals in which the capsule is formed. Listed in Table I1 are species in which the karyosphere, with or without capsule, has been observed (Gruzova, 1975). The first part of the table contains the species with maximum oocyte nucleus diameter over 100 pm, and the second part, those below 100 pm. In parallel, the presence of extra-DNA in nuclei is indicated. Analysis of these data shows that the capsule usually appears in large germinal vesicles (more than 100 pm in diameter) rather than in small nuclei. A second condition necessary for capsule formation is the presence in the nucleus of extrachromosomal DNA in one form or another. An exception might be gall midges, in which the nucleus is rather small (up to 25 pm). Their karyosphere,

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if it can be called so, is quite specific. A lamellar barrier separates the whole group of S-chromosomes from that of E-chromosomes. The latter are constantly despiralized and transcriptionally active in contrast to S-chromosomes (Matuszewski, 1982; Gruzova et al., 1987).

D. Function of the Capsule As previously mentioned in some animals the perikaryosphere material is arranged around the chromosomes as an envelope or capsule; in others it is found inside the chromosome knot in the shape of a central body. We have termed these “external” and “inverted” capsules, respectively. Apparently, one of the functions of both types of capsules is that they act as a sort of shell, fastening and fixing the chromosomes in a limited volume in a rather large germinal vesicle (GV). Another function of the capsule is maintaining the chromosomes in a certain region of the large and irregularly shaped GV. This seems to be of importance for the location of the future pronucleus, meaning its proximity to the egg surface, for instance, to the micropyle in insect eggs. Undoubtedly, these are not the only roles of the capsule in oocytes. As can be seen clearly in gall midges, the capsule plays a role in the compartmentalization of the nucleus, which is essential for packing and storage of synthesized products and for exchange processes between the nucleus and the cytoplasm (Jazdowska-Zagrodzinska and Matuszewski, 1978). It is possible that the material of the capsule itself represents a package of protein-nucleic acid complexes and a kind of “storehouse” of information, necessary for future development. The capsule plays also a protective role; it usually persists until the disintegration of the nucleus and the early metaphase of meiosis I. For example, in mosquitoes, when the conditions for oviposition are not suitable, the capsule, which has already started to disintegrate, is substituted by another one, and meiotic division does not begin (Fiil, 1976a). It cannot be excluded that the capsule or its material takes part in blocking meiosis.

E. Substructural Elements of the Capsule Closer scrutinization of the substructural elements constituting the capsule in oocytes of insects and anurans shows that these elements are rather limited, even though these species are extremely distant phylogenetically. The fibrous material of the capsule in both of these groups represents diverse combinations of SC derivatives and autonomous pore complexes. Apparently, after chromosome conjugation the SC are reutilized and new polycomplexes are formed (Fiil and Moens, 1973). The sites of assembly of the SC elements and poly-

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complexes appear to be either the nucleoli (Moses, 1968; Gruzova et al., 1972; Rasmussen, 1975) or the extra-DNA bodies (Jaworska and Lima-de-Faria, 1973). The question of how the pore complexes appear in central regions of the nucleus can be elucidated by experiments on the reconstruction of nuclei in vitro. A series of experiments showed rapid self-assembly of the nuclear envelope around phage DNA that were injected to oocytes of Xenopus. A prerequisite of such reconstruction is the transformation of phage DNA into chromatin and its compactation into a dense sphere with a loss of transcriptional activity (Newport and Forbes, 1987). It might be that the karyosphere to some extent meets these requirements, possibly by inducing the accumulation around it of pseudomembranes involving autonomous pore complexes. Moreover, electronmicroscopic data presented above show that in the formation of capsule fibers or strands the protein microbodies (sizes 0.1 to 1-2 pm) are very important. The bodies are of fibrogranular composition and sometimes of complex structure. (Variants of the latter can be seen in Figs. 9i and 11.) The size of granules within one body can vary from 10 to 50 nm. Ring structures are often present in these bodies. Despite the morphological diversity of these bodies, they have some features in common, such as the presence of RNP and DNP, and their close contact with the capsule strands. The presence of both nucleic acids in them is revealed by the comparison of the results of light-microscopical cytochemical and autoradiographic investigations with those of the treatment of ultrathin sections with EDTA according to Bernhard (1969) (Gruzova et al., 1972; Gruzova and Parfenov, 1976; Gruzova, 1979, 1988). As capsule formation progresses, these bodies begin to disappear, and in a fully formed capsule they are absent (Fig. 6j). Thus, various nuclear structures participate in capsule formation: derivatives of SC, elements of the nuclear envelope, nucleoli, extra-DNA bodies, and numerous protein microbodies; in the latter the presence of DNA cannot be excluded. Although these structures are involved in this process in different combinations and to different extents, the final result appears to be the same: in one way or another (with the aid of the “envelope” or “central” body) the chromosomes are localized and fixed in a certain region of the germinal vesicle and, in some cases, the nucleus is compartmentalized. The assumption about the direct involvement of DNA in the morphogenesis of capsules is in accordance with the viewpoint that the morphogenetic information in the cell is contained in nontranscribed DNA sequences, which can fulfill their morphogenetic functions because of their unique ability to form, maintain, and reproduce certain spatial conformation (Maximovsky, 1988). Maximovsky points out that in order to understand the mechanisms of storage and realization of chromosomal morphogenetic information it is necessary to consider the morphogenetic processes occumng in the nucleus. These theoretical postulations can be successfully used for the analysis of the morphogenesis

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of the karyosphere capsule. With such an approach another possible function of the capsule can be revealed, that of storing the morphogenetic information for future development; the possibility that the material of the capsule makes a contribution to ooplasmic segregation cannot be excluded.

F. Spatial Organization of Oocyte Nucleus Questions about capsule formation lie within the general context of the complex problem of spatial organization of the nucleus, questions that are currently the focus of attention of many specialists (Comings, 1980; Spector, 1990). This problem is outside the scope of our review. However, in this connection we briefly touch upon the question of localization and movement of some nuclear structures inside large GV. According to our observations, the movement of nucleoli and NLBs accompanies capsule formation and is visible on histological preparations when the events in the nucleus are followed from one stage

I

11

111

a

b

FIG. 14 The movement of intranuclear structures and karyosphere capsule formation. (I) Formation of the capsule is accompanied by the movement of NLBs from karyosphere toward the nuclear periphery (beetles). (11) In the period of capsule formation the nucleoli and the karyosphere are in close proximity (golden-eye flies, mosquitos). (111) Formation of the capsule is accompanied by the movement of nucleoli from the periphery toward the karyosphere (frogs). (a, b) Beginning (a) and end (b) of capsule formation. Arrows indicate direction of movement.

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to another. For instance, the primary capsule in R . temporaria appears when nucleoli and chromosomes are separated by a relatively small distance. In different types of GVs the movement of nucleoli and NLBs can be either centrifugal or centripetal, as is shown schematically in Fig. 14. It is thought that with movement of nucleoli and NLBs there appears around the karyosphere a kind of a morphogenetic field in which numerous granules begin to operate. This morphogenetic field obviously determines the size and shape of the capsule. Outside the field numerous microbodies or granules are free in the karyolymph. The determination of the area of this field and discussion on the development of the bodies operating within it are beyond the scope of this review, although these questions are, no doubt, directly related to the problem of morphogenesis. Questions of the type might be successfully answered in terms of the theory of positional information (Sonneborn, 1974; Wolpert, 1973; Frenkel, 1984). However, with respect to the cell nucleus, this theory has not yet been developed.

Acknowledgments The authors thank Dr. A. L. Yudin for useful suggestions, A. G. Tsvetkov for stimulating discussions, Yu. I. Gukina, G. N. Pochukalina for technical assistance, and K. A. Gindina for translating the text into English.

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Applications of Arabidopsis thaliana t o Outstanding Issues in Plant-Pathogen Interactions Jeffery L. Dangl Max-Delbriick Laboratory in the Max-Planck Society, D-5000 Koln 30, Germany

I. Introduction Plants rely on a diverse set of molecular and physiological mechanisms to monitor and exploit their environment. Sessile existence places demands on plants to find and transport soil-borne minerals, fine tune their ability to harvest and utilize energy from light, and regulate the uptake and dissipation of water. Each of these processes mandates specialized tissues and cell types displaying highly evolved molecular modes of perception and response to external and internal signals. Plants must also share their environment, indeed compete for resources, with a multitude of other life forms. Many of these have evolved life strategies to take advantage of the nutritional haven afforded them by the plant’s energy generation and storage systems. These organisms have become pathogens, commensals, or symbionts. A deeper understanding of how plants interact with the biota that surrounds them spurs research in botany, ecology, physiology, microbiology, and a host of other disciplines. From an essentially anthropomorphic view, mankind has a particularly keen interest in understanding how plants protect themselves from infection. This bias has led to voluminous research analyses of plant-pathogen interactions. Research in the field loosely termed “plant pathology” has become extremely multidisciplinary. Over the last decades, this research has certainly led to important understanding of basic processes, and application of that knowledge to problems of agronomic interest. Nonetheless, our understanding of the molecular and cellular events that are responsible for plant disease resistance remains rudimentary. This is especially true of the events controlling the earliest steps of “active” plant defense, recognition of a potential pathogen and transfer of that cognitive signal throughout the cell and surrounding tissue. A detailed understanding of lnrrrnarionol Review of Cvrology, Vol I44

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these processes is of extreme interest from both a basic and an applied perspective, since they ultimately trigger the resistant phenotype. Current and future research will unravel the molecular mysteries of how plants recognize and discriminate between friend and foe in the biota surrounding them. Based on our knowledge to date, these will be fascinating systems, from which we can expect also to learn more about basic plant and microbe physiology and ecology. The purpose of this review is, by comparison, narrow. I hope to highlight some specific research problems of critical interest through a short, and by no means exhaustive, review of some aspects of the genetics and biochemistry of plant-pathogen interactions. A few discrete examples will be introduced as a way of highlighting some key, unresolved issues. Then, I will introduce a species of plant that has become an extremely important model in all aspects of plant biology, Arahidopsis thaliana. This species has not been ignored by workers in the plant-pathogen interaction field. Much of the review will dwell on the impact its adoption has made, and should continue to make, in our understanding of the molecular mechanisms that dictate plant defense responses. Finally, it should become clear that not only academic paradigms but also agronomically relevant genes can be gleaned via a concentrated effort using A. thaliana to this end.

II. Of Plants and Pathogens: A Brief Overview It surprises no one that microbes have evolved to parasitize plants. It is also self-evident that on an evolutionary scale, plants must have developed ways to discriminate pathogen ingress from epiphytic microbial existence. Principal among these are physical barriers such as wax layers and lignified cell walls. This first-line defense renders nearly all plant species refractory to parasitization by nearly all pathogen species (termed “basic” or “non-host” resistance). In contrast, pathogens that are principally able colonize plants of a given species create a situation termed “basic compatibility.” Within the confines of a “compatible interaction” between an isolate from a “virulent” pathogen group (termed a “race”) and a given plant species, selection forces the appearance of new plant genotypes capable of specifically recognizing one or more pathogen races. This type of resistance is termed “race-specific,” and leads to an “incompatible interaction,” where the previously virulent pathogen race is rendered “avirulent.” This often-confusing nomenclature is schematically depicted in Fig. 1. Subsequent to recognition of a potentially pathogenic microbe, plants can activate an array of biochemical responses. The most common, but by no means universal, resistance response, observed in both nonhost and race-specific interactions, is termed the “hypersensitive response” (HR) (Klement, 1982). In the HR, cells contacted by pathogen, and often neighboring cells, rapidly collapse and dry in a necrotic fleck. This response is thought to deny the pathogen access

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Basic Compatibility

Non-Host

F i piLq piq Resistant

Susceptlble

Resistant

Avirulent

lncompatlble

Compatible

Incompatible

Virulent

Compatible

Compatlble

Incompatible

FIG. 1 Terminology used to describe the genetics of plant-pathogen interactions. Three interdependent sets of terminology are necessary to describe plant-pathogen interactions. Pathogen isolates are either virulent or avirulent; plant genotypes are either resistant or susceptible; and the interactions between these partners are either compatible or incompatible. Note that utilization of any single term from one perspective (i.e., “a virulent pathogen”) requires simultaneous definition of the other terms (i.e., “a susceptible plant” and “a compatible interaction”).

to nutrients. Many plant defense responses are related to the local reinforcement of primary defense mechanisms meant to thwart the pathogen’s continued ingress, and generally to protect the plant from a spreading, or secondary, infection (Crute et al., 1985; Hahlbrock and Scheel, 1989; Lamb et al., 1989; Dixon and Hanison, 1990; Dangl, 1992b). Successful recognition and repulsion of a particular pathogen race by a resistant plant genotype force selection of new pathogen variants that can evade, suppress, or overcome the plant’s cognitive capabilities; new resistant plant genotypes are selected, and the game is on. On natural ecological scales, as briefly discussed below, populations of plants and pathogens are involved in a constant tug of war. One logical outcome of this constant selection is commensalism, where a plant is tolerant to a biotrophic parasite at no catastrophic loss to its reproductive capacity.

A. Genetics of Plant-Pathogen Interactions The intervention of agriculture has intensified the selective pressure on pathogens to “overcome” the resistance mechanisms of selected plant genotypes introduced, on large scales, by humans. It was recognized by breeders, fast on the heels of the rediscovery of Mendel, that disease resistance in plants was

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heritable. The manipulation of “major resistance” (R) genes by breeders in nearly all crop species, and against nearly all pathogens, has done two critical things in terms of what will be discussed below. It has generated an immense amount of genetic material that basic researchers have used to advantage. And, perhaps shortsightedly, it has focused the research debate intensively on coming to an understanding of the structure and function of R genes. Although it will not be discussed at length here, breeders have also long been aware of non-Rgene components influencing the outcome of plant-pathogen interactions (Crute, 1985). We concentrate on R genes because their action is determinative for a successful race-specific resistance response, they are relatively .easy to assay, they are ubiquitous throughout the plant kingdom, and breeders have provided a wealth of genetic resources relating to R-gene specificities. Other genes whose products influence, modify, or enhance R-gene function surely exist, as do loci involved in signal transduction and loci required for realization of the resistant state. In fact, as discussed below, the adoption of A. thaliana as a genetically simple model will also hasten identification of these classes of loci. As alluded to above, heritability of disease resistance in plants was discovered near the turn of the century. Later, Flor showed that single genes in the plant (R genes) determined resistance, but only in the presence of complementary, corresponding genes in the pathogen (Flor, 1955, 1971). These pathogen genes were given the somewhat unfortunate name “avirulence” (avr) genes; this does not imply that alternative alleles at avr loci are necessary components of virulence. Flor’s gene-for-gene hypothesis states that complementary products of pathogen avirulence genes interact, directly or indirectly, with products of plant resistance genes to trigger a race-specific resistance reaction, also known as an incompatible interaction (again, see Fig. 1 for a diagrammatic representation of the nomenclature). In most cases, both avirulence and resistance are dominant functions, but exceptions do exist. Despite their obvious importance, and 50 years of genetic description, no R gene or R-gene product has been isolated. Moreover, very little convincing evidence exists regarding how these products trigger the resistant phenotype (Crute et al., 1985; DeWit, 1992a,b; Ellingboe, 1981, 1982, 1984; Flor, 1971; Gabriel, 1989; Gabriel and Rolfe, 1990; Keen, 1982, 1990; Keen and Staskawicz, 1988; Knogge, 1991; Pryor, 1987). As described in detail below (also Debener et al., 1991), A . thaliana also contains race-specific resistance genes, and their isolation should be hastened through concentration on this model species. In contrast to R genes, a large number of avr genes have been isolated, nearly all from bacterial pathogens, as well as a few fungal pathogens (Keen, 1990; DeWit, 1992a,b). Bacterial avr genes are identified via a functional cloning strategy, first utilized by Staskawicz and colleagues, which takes advantage of the dominant or epistatic nature of avr- gene function (Staskawicz, et a / . , 1984). A mobilizable cosmid library is constructed from high-molecular-weight DNA

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from an avirulent pathogen isolate. The presence of an avr gene renders a previously virulent pathogen isolate avirulent on the appropriate plant genotype. A critical control is that the conferral of the avirulent phenotype must be plantgenotype-dependent. The presence of a cloned avr gene in an otherwise virulent, merodiploid, bacterial background also simplifies genetic analyses of bacterial functions influencing both virulence and avirulence. Thus, there is a very large body of evidence, from Pseudomonas, Xanthomonas, and Eiwinia species, showing the involvement of a cluster of genes, termed hrp for hypersensitivity and pathogenicity, in processes leading to both virulence and avirulence (Willis et al., 1991). Mutations at any hrp locus cause loss of pathogenicity and loss of the ability to generate an HR on both nonhost species and resistant genotypes of the nominal host species. The function of proteins encoded in the hrp loci is hotly debated, but it is clear that sensory functions, signal transduction functions and, presumably, delivery of virulence functions are present. Several groups have also shown that avr gene function is regulated by hrp loci (Willis er al., 1991). One outstanding issue is how hrp control of the vast array of avr genes, and necessary pathogenicity factors, is manifested. It would be instructive to compare and contrast the modes of bacterial pathogenicity in plant systems with the much more thoroughly understood interactions of mammalian bacterial pathogens and their hosts (Gough et al., 1992; Fenselan et al., 1992; see also Cornelius et al., 1989; Mekalanos, 1992, for reviews). Not only are the functions of avr gene products unknown, but it is also unclear whether they render any selective advantage to microbes expressing them. avr function, per se, is an obvious detriment for microbes colonizing a resistant plant genotype. Surprisingly, however, conservation of avr genes has been observed, but in only one case did deletion of an avr gene result in a loss of bacterial fitness following inoculation onto susceptible plant genotypes (Keamey and Staskawicz, 1990). We remain very naive in our understanding of the normal role of avr genes in either epiphytic or pathogenic growth modes of bacteria and, for that matter, fungi. A greater emphasis on studies of the general ecology of microbial pathogens would help in understanding how these genes contribute to bacterial growth and how plants have come to recognize them (Hirano and Upper, 1990). Important in consideration of the evolution of race-specific pathogen recognition, it has recently been shown that gene-for-gene interactions can dictate recognition outside the nominal host species of a particular pathovar (Whalen et al., 1988; Kobayashi et al., 1989; Keen and Buzzell, 1990). These two groups demonstrated that plant species not normally thought of as hosts for either a particular Xanthomonas campestris or Pseudomonas syringae isolate could nonetheless recognize avr functions for them. Subsequent analysis in other bacterial pathovar-plant species combinations supports these findings (Taylor et al., 1989; Vivian et a/., 1989; Minisavage et al., 1980; Fillingham et a/., 1992). In fact, some plant species could recognize a heterologous avr function in a geno-

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type specific way; e.g., some cultivars were resistant and some susceptible. Thus, genetic elements determining cultivar specific resistance within a species can also play a role in cultivar specificity across species, and in host range determination. These fascinating results suggest that what has been called nonhost resistance may be simply the additive effects of many simultaneously acting gene-for-gene interactions. These observations were, in fact, the rationale for searching for bacterial pathogens of A . thaliana among phytopathogenic bacterial isolates from closely related plant species (see below and Debener et al., 1991; Dong et al., 1991; Whalen et al., 1991). It is important to note that this finding is not limited to bacterial pathogens, as has been clearly demonstrated by recent analyses in two fungal pathosystems (Tosa, 1989; Valent et al., 1990). An interesting scheme for the evolution of race-specific resistance within a species, and how those systems might result in race-specific recognition by traditional nonhosts, was recently expounded by Heath (1991). Genetic studies in many systems suggest that plants inherently carry a great variety of loci that can function as R genes. Taking one agronomically relevant example, over 150 R-gene specificities have been described in barley (albeit from five nomenclature systems), which are active against various powdery mildew (Eiysiphe graminis f. sp. hordei) isolates (Sogaard and Jorgensen, 1988). As well, several studies in the barley-barley leaf scald (Rhynchsporium secalis) pathosystem established at least 75 R-gene specificities that account for reactivities of pathogen isolates on various series of differential cultivars (Shipton et al., 1974; Ali et al., 1976; Jackson and Webster, 1976). Because these data were generated by different groups, using various barley cultivars and fungal isolates, it is virtually impossible to know how many different Rgene loci these resistance specificities represent, or how many are alleles at known loci (13 characterized encoding powdery mildew resistance, 11 encoding scald resistance; Jorgenson, 1991). It is often difficult, in fact, to distinguish between the presence of a true allelic series at a given R-gene locus, and the occurrence of multiple, very tightly linked but discrete, genes at that locus (Bennetzen et al., 1988b; Islam et al., 1989; Hulbert and Bennetzen, 1991; Dangi, 1992a). It could be suggested that this cognitive diversity is an artifact of breeding for race-specific resistance and the subsequent evolutionary pressure placed on the respective pathogens. Extremely interesting examples using wild plant species and their natural pathogens show, however, that the diversity in wild populations is even greater. One particularly illustrative example is the interaction of Senecio vulgaris and the powdery mildew fungus Erysiphe fischeri (Clarke et al., 1987, 1990). The plant has a short life cycle and is highly inbred, hermaphroditic, and prolific; the fungus does not infect any known crop species. Thus, this interaction has evolved outside the limits of agriculture. Among 51 individual plants gathered from 25 widely separated locales in Great Britain, 33 displayed race-specific resistance when tested with 8 fungal isolates, all derived from another locale. The 33 resistant plant isolates fell into 28 groups, each

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defined by the presence of one or more resistance specificities. A similarly astounding diversity of resistance phenotypes, 10, were observed within a population of 75 plants, isolated from only 1 m2 when tested with just 5 fungal isolates! Much of the diversity appears to be determined by variation within the fungal population, all the more amazing since the sexual cycle of this fungal species has not been observed in Great Britain (where all isolates are homothallic and express the same mating type). Other examples of this type exist (Segal et al., 1980; Jahoor and Fischbeck, 1987; Heckelbacher et al., 1991), and continued analysis of these systems will surely shed light on the ecology and evolution, as well as the genetic control, of plant-pathogen interactions. Unfortunately, the tools necessary for isolation and analysis of resistance genes from a species like S. vulgaris are essentially nonextant. As discussed below, however, it may be the case that the diversity of interactions observed in the S. vulgaris-E. fischeri system also operates in the interactions of A. thaliana and its fungal pathogens.

6. Biochemical Reactions of Plants to Pathogen Ingress The recognition of a pathogen is only the beginning of a series of events leading to the resistant phenotype. The plant’s overall defense strategy, briefly introduced above, includes induction of biochemical pathways leading to synthesis of antimicrobial “phytoalexins,” reinforcement of cells surrounding the infection site, release of membrane-associated oxidants (and antioxidants), and, in many cases, the systemic induction of putative defense molecules. An immense body of literature exists regarding the biochemical basis of plant disease resistance (Crute et al., 1985; Collinge and Slusarenko, 1987; Hahlbrock and Scheel, 1989; Lamb et al., 1989; Dixon and Harrison, 1990; Dangl, 1992b), and the topic will only be touched upon here in terms of how outstanding issues can be succinctly and advantageously addressed using A. thaliana. It is clear from many systems that perception of a potential pathogen triggers massive new transcriptional activity in surrounding plant tissue and may also take place in the forming hypersensitive lesion itself. Many genes whose transcription is induced by pathogen ingress have been cloned and analyzed. The proteins encoded by these genes can be involved in phytoalexin biosynthesis; these events, and the derived phytoalexins, are usually species specific. Often, induced proteins belong to the so-called pathogenesis-related (PR) protein family, whose members include degradative chitinases and glucanases. Accumulation of PR proteins is also very often observed during induction of an “immunized” state termed systemic acquired resistance (SAR). The precise role of PR proteins is as yet undefined. As well, many proteins involved in generation and metabolism of oxidants and oxygen radicals are locally induced around sites of hypersensitive cell death, suggesting that oxidative bursts may play a

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role in resistance (Siedow, 1991). Cell wall strengthening proteins, rich in hydroxyproline or hydroxyglycine, also accumulate and are cross-linked at and around the infection site. Finally, proteins whose functions remain unknown are also induced at and around infection sites (Somssich et al., 1989; Schmelzer et al., 1989). The complexity of the response is, in many ways, meant not only to retard the growth of the pathogen, but also to protect the potentially weakened tissue from secondary infection. Induction of part, or all, of this defense array occurs in both nonhost responses and race-specific interactions. It has, however, proven very difficult to find a gene whose activation is truly specific for incompatible interactions. Most inductive events show only slower kinetics, and often varied magnitude, during compatible interactions. This observation has led to the idea that the difference between resistance and susceptibility may only lie in the timing of critical events following pathogen recognition. Alternatively, many of the induced activities analyzed may not be directly related to triggering of resistance. Moreover, most of the genes induced by attempted pathogen ingress also have other roles during normal plant development (Crute et al., 1985; Hahlbrock and Scheel, 1989; Lamb et af., 1989; Dixon and Hamson, 1990; Dangl, 1992b). Normal developmental expression of “plant defense genes” is often both spatially and temporally complex. These modes of expression are usually consistent with the gene product’s function, if known, during the defense response (for example, phenylpropanoid pathway products involved in lignin and flavanoid biosynthesis). Just as often, however, we are forced to speculate about the developmentally regulated function, as in the recently reported cases of chitinase and glucanase expression in developing flowers (Lotan et al., 1989; Memelink er al., 1990; Neale et al., 1990). The problem has been to separate which of the induced weapons in the plant’s defense arsenal is truly needed for a successful resistance response, and which are induced only as nonspecific, “added protection.” As described below, most of these defense mechanisms have now been identified in A. thaliana. The use of genetics in this model can now be exploited to determine which of the plant’s many responses are truly necessary for the establishment of the resistant phenotype.

111. Arabidopsis thaliane: Weed N o More A. Development of a Genetic Model for Plant-Pathogen Interactions The discussion above was meant to illustrate that many loci in a given plant species are capable of satisfying the genetic criteria for definition as an R gene and that a very large number of resistance specificities exist. It was also meant

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to briefly introduce the complexity of subsequent biochemical events that are thought to play a role in the overall plant defense strategy, if not a direct one in generation of resistance. Both genetic and biochemical complexities have been shown for a great number of systems including cereals and their fungal pathogens and wild plant species and fungal parasites, as well as for crop species and bacterial pathogens (Clarke et al., 1987, 1990; Brinkerhoff, 1970; Ilot et al., 1989; Hulbert and Michelmore, 1985; Farrara et a[., 1987; Innes, 1983; Mew, 1987). The genetic diversity of recognition capabilities present in a given plant species has potential functional implications. How many loci encode plant resistance genes; how do these products mediate specific recognition in gene-for-gene interactions? Most models of R-gene action predict that R-gene products are surface receptors that recognize either the direct or the indirect product of avr genes. Yet, returning to the barley cases mentioned above, is it realistic to consider that at least 24 loci, encoding classes of potentially related surface receptors, have evolved to specifically recognize a spectrum of signal molecules from only two fungal species? As well, R-gene products are presumed to be constitutively expressed on all relevant cells, as expected since recognition events are probably cell-autonomous functions (Bennetzen et al., 1988a). Could multiple loci conditioning resistance to more than one pathogen represent not only cognitive functions, but also downstream steps in processing of the avr signal, where polymorphism evolved to amplify pathogen signals more effectively (Dangl, 1992a)? Is pathogen recognition, in fact, even the primary function of resistance genes? Or, are pathogen recognition and triggering of the resistant phenotype fortuitous by-products of another physiological function? How many other plant loci, besides R genes, encode functions required for resistance? And how many of these correspond to previously analyzed biochemical functions of plant defense? Do common features of resistance genes exist among species, and if so, can they be effectively transferred? What is the distribution of resistance genes in wild populations? A saturation mutagenic approach, in a system where a broad range of diverse pathogens exists, is necessary to identify all loci meeting the genetic criteria of R genes, as well as genes required to effectively translate recognition into a resistant phenotype. One curse of the practical importance of breeding for disease resistance is that the use of many species has been necessary to achieve a practical goal. In order to genetically, and finally biochemically, understand the resistance response, a few model species should be thoroughly dissected. With particular respect to plant-pathogen interactions, a model system should fulfill certain criteria. First, the plant should be amenable to both Mendelian and molecular genetic analysis; ideally its pathogens should also be genetically tractable. Second, the plant (and, again, ideally, the pathogen) should be transformable and regenerable to allow gene identification by complementation of mutants or recessive alleles. This criterion also includes the ability to engineer trans-dominant mutations of cloned products to achieve specific disruption of a

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particular pathway. Third, mutagenesis, and recovery of mutants, should be relatively simple, such that true saturation mutagenesis screens can be performed. This criterion should also be expanded to include the possibility of screening for temperature-sensitive and conditional mutants. The importance of genetically identifying all steps in recognition and signal transduction pathways, as well as steps in response pathways, cannot be overemphasized. It is precisely experimental strategies of this sort that have been lacking in plant pathology to date. Fourth, the isolation of genes identified by mutant, or alternate allele, phenotypes must be fairly simple. Obvious techniques included under this rubric include insertion mutagenesis with transposons or the T-DNA of Agrobacterium species. A more generalizable strategy is to isolate genes identified by mutant or altered phenotype via “position cloning” (Botstein et al., 1980; Orkin, 1986; Koenig et al., 1987; Rommens et al., 1989). This collection of techniques relies on knowledge of the genetic position of the desired locus [predicating highly developed morphological and restriction fragment length polymorphism ( R E P ) maps] and the tools of “chromosome walking.” Fifth, there should be a large degree of genetic variability in both plant and pathogen populations, and there should be a large array of microbes pathogenic to the model plant. This criterion is required if we are ever to understand fully, at a mechanistic level, the rich diversity in plant-pathogen interactions in natural populations, as briefly introduced above. Sixth, paradigms discovered, and genes isolated, via adoption of the model should be transferrable to crop species.

6 . Why Arabidopsis? The adoption of A. thaliana as a model for nearly all aspects of plant biology has been responsible, in part, for the development of tools that have hastened its emergence as a model in plant-pathogen interactions. At the present time, A. thaliana now satisfies all of the criteria defined above. It is a member of the crucifer family; as such, many important classes of crucifer pathogens can be expected to either infect Arabidopsis species or have relatives that do. The species A. thaliana has been an object of plant genetic study for nearly a century, and a great wealth of information is available regarding all aspects of its biology (Redei, 1970, 1975; Estelle and Somerville, 1989; Meyerowitz, 1987, 1989; Meyerowitz and Pruitt, 1985; Koncz et al., 1992). It has a rapid life cycle, as little as 4 weeks from seed to seed, thus simplifying Mendalian genetic analyses. As well, each individual can produce thousands of seeds after self-fertilization, and outcrossing is trivial. Mutagenesis of seeds or pollen via chemical (ethylmethysulfate, diepoxybutyrate) and physical (X-ray, y ray, fast neutron bombardment) means is straightforward. Through traditional mutagenic approaches, saturation screens can be developed for traits of interest (a recent example for mutations affecting embryonic pattern formation is found in Mayer et al., 1991),

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and over 350 mutations leading to visible phenotypes have been used to construct a dense genetic map on five linkage groups (Koorneef, 1990). Arabidopsis thuliana is quite readily transformed using either Agrobacterium or naked DNA (Lloyd et al., 1986; Schmidt and Willmitzer, 1988; Valvekens et al., 1988). More recently, insertion mutagenesis with T-DNA has become routine, if laborious, and development of transposon tagging with heterologous transposons from maize is under intense investigation (Feldmann and Marks, 1987; Feldmann et al., 1989; Feldmann, 1991; Koncz et al., 1989,1990; Marks and Feldmann, 1989; Valvekens et al., 1988; Yanofsky et al., 1990; Dean et al., 1992; Masterson et al., 1992). The isolation of genes known only through their mutant phenotype and map position is also highly advanced compared to nearly all other plant species. A compelling argument for further development of the tools necessary for position cloning in A . thaliana was the original observation that the nuclear genome was very small compared to other plant species, approximately 70 megabase (Mb) pairs, and nearly devoid of repetitive DNA (Leutwiler et al., 1984; Pruitt and Meyerowitz, 1986). More recent estimates of its genome size suggest that it is probably no bigger than 150 Mb (Arumuganathan and Earle, 1991). The development of two RFLP maps was of major importance, as cosegregation of an RFLP marker with a trait of interest is the first important step in map-based cloning (Botstein et al., 1980; Lander and Botstein, 1986; Chang et al., 1988; Narn et al., 1989). These maps now consist of a total of around 300 markers, dispersed over a map distance of 600 cM, thus giving an approximate density of one marker each 0.5 map unit. As well, several complementary yeast artificial chromosome (YAC) libraries exist, containing inserts from 50 to several hundred kilobases (kb) (Ward and Jen, 1990; Grill and Somerville, 1991a; J. Ecker, personal communication). YAC libraries allow simplified chromosome walking from a near or cosegregating RFLP marker, using end probes derived from YAC clones identified via hybridization the nearest flanking RFLP marker (Grill and Somerville, 1991b). There is a large, multinational project under way to complete a physical description of the A . thaliana genome by the turn of the century. A critical first goal is the construction of a physical map consisting of contiguous cosmid and YAC clones. Physical mapping of contigs covering 90% of the genome (contained on over 20,000 cosmid clones) is finished (Hauge et al., 1991). Recently, YAC clones covering around 30% of the genome have been identified using the RFLP probes (Hwang et al., 1991). As well, Lazo et al. (1991) have created a library of genomic Arabidopsis DNA that is maintained in plant transformation competent Agrobacterium. Combined utilization of these tools will hasten the ability of gene identification via mapping and complementation of mutations and recessive alleles. Cloning strategies based on identification and isolation of sequences deleted at a locus of interest have also recently been developed for use with A . thaliana (Strauss and Ausubel, 1990; Wieland et al., 1990; Sun et ul., 1992). Intense scrutiny of A . thaliana genome will revolutionize our understanding of many basic plant processes.

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Of particular interest with respect to plant-pathogen interactions is the availability of over 100 land races (ecotypes) of A . thaliana from various locations around the Northern Hemisphere. Since the species is almost always selffertilizing, any local genetic variation can be expected to be rapidly frozen after several generations. Thus, evolution in the presence of absence of a particular pathogen can be expected to lead to the same natural variation as described above for crop and other natural species. As detailed below, the available A . thaliana genotypes certainly contain different resistance gene functions directed against a range of pathogens. Further exploitation of the available gene pool, and investigation of other natural isolates, will allow analyses of evolution of resistance in wild populations, much as described above for S. vulgaris. A preliminary analysis of molecular relatedness among 33 land races, using RFLP probes from the available collection and random amplified polymorphic DNA (RAPD) mapping oligonucleotides, is in progress and will help to define the level of molecular variation within the species (T. Debener et al., unpublished observations). Finally, in the last 3 years, many groups have addressed the question whether Arabidopsis is a host for phytopathogens. Two approaches were taken to establish useful pathosystems. The first was to identify naturally occurring associations of pathogens with A . thaliana. The second was to ask whether pathogens isolated from other species can colonize A . thaliana successfully and cause disease symptoms similar to those found on their natural hosts. In each case, an obvious corollary is the need to find A . thaliana land races resistant to each pathogen isolate. A third approach, reliant on the first two, is to isolate plant mutants unable to generate a normal resistance response when challenged with the appropriate pathogen. The current state of these approaches is the focus of the remainder of this review.

IV. Arabidopsis Pathogens: Interaction Phenotypes and Genetics

A. Fungi Several groups have isolated naturally infected A . thaliana plants over the last few years. Many classes of fungal species infect Arabidopsis, and there is an immense potential for further development of select systems. Both specialized obligate biotrophs and rather unspecialized nonobligate fungi have been found to infect Arabidopsis. Koch and Slusarenko (1990a) and Mauch-Mani et al. ( 1992) described a battery of fungal species isolated from infected plants under both glasshouse and field conditions. Several species, Rhizoctonia solani, Botrylis cinerea, Pythium spp., and Chromelosporium spp., are known to be general, broad host-range pathogens with no observed race structure on other plant

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species. Several obligate biotrophic species, with agronomically important relatives, were also isolated from infected Arabidopsis. These include Puccinia thlaspeos, Albugo candida, Plasmodiophora brassicae, and Peronospora parasilica (Koch and Slusarenko, 1990a,b; Holub et al., 1992; Dangl et al., 1992a; Mauch-Mani et al., 1992). Although most obligate biotrophs are highly specialized for life on a particular host species, Koch and Slusarenko (1990a) were also able to coax an isolate of Erysiphe cruciferarum from Brassica napus through its life cycle on Arabidopsis. Erysiphe cruciferarum is known to have race-specific interactions on its normal host (Lucas et al., 1988). This latter observation suggests that test inoculation with fungal isolates from other Brassica species may uncover specific resistance against them in Arahidopsis. This would open the way to exploitation of A. thaliana as a “mine” for fungal resistance genes of immediate agronomic interest. This strategy is already being used in analyses of resistance against phytopathogenic bacteria, as detailed below. By far the most important development with respect to fungal pathogens has been the phenotypic characterization of interactions between a range of A. thaliana genotypes and several P. parasitica isolates, mostly by Crute and Holub (Holub et al., 1992; Dangl et al., 1992a). Koch and Slusarenko detailed the interaction of the first fungal isolate (1 990b) at the microscopic level and found that resistance in their incompatible interaction was accompanied by a typical HR. Rapid collapse of a few epidermal cells surrounding the penetration hyphae, indicative of HR, was observed, although the fungal hyphae continued to penetrate futilely into the mesophyll. Differences in resistant phenotypes are a hallmark of this system (as, in fact, are differences in susceptible phenotypes). Holub e f al. (1992) discuss various levels of flecking and pitting in the incompatible interactions of one fungal isolate with two different A. thaliana genotypes. As well, they describe various levels and speed of sporulation in compatible interactions. The genetic control of these responses is beginning to be dissected. The current hypothesis of Crute and Holub (Dangl et al., 1992a) suggests at least six distinct resistance specificities uncovered in the interactions of only five fungal isolates with six plant genotypes. Segregation analysis for several combinations suggests that single plant loci control resistance in the majority of cases. Interestingly, Mauch-Mani et al. (1992) state that resistance in the interaction they are analyzing is codominant, and that they can distinguish heterozygotes from homozygote-resistant F, individuals by virtue of an intermediate phenotype. This finding has practical implications with respect to subsequent RFLP mapping of the resistance locus, since scoring of each genotypic class can be reliably performed in the F, generation. There are certainly functional implications as well, since codominance, characteristic of many plant-pathogen interactions, argues against a simple all or none triggering of the resistance reaction. In another instructive example, Holub and co-workers have shown that both

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a “strong” and a “weak” gene in one plant genotype condition resistance to a particular fungal isolate. Molecular mapping of each of these loci is in progress. The mapping and ultimate isolation of the genes responsible for different phenotypic manifestations of resistance will help answer many outstanding questions. Are there strong and weak alleles, or does genetic background play a determinative role in R-gene function? What is the nature of the heretofore mysterious “genetic background” and can it be dissected using the various resistance phenotypes in this system as tags? The use of a mutagenic approach to answer this question will be particularly useful, since at least four of the R-gene loci will soon be mapped. The finding of codominant resistance, and of strong and weak genes in A . thaliana, is also consistent with race-specific interactions between fungal pathogens and crop plants, further supporting the use of A . thaliana as a model for understanding these processes (Crute and Norwood, 1986; Crute, 1985).

B. Bacteria Given the extent of genetic knowledge regarding bacterial phytopathogens, and the ease of their manipulation, it is no surprise that their application to A . fhaliana came very early. Although it is only very recently that a pathogenic bacteria, Xanthomonas campestris pv. campestris, was isolated from a natural infection (Tsuji and Somerville, 1992); the first successful inoculation experiments with both pseudomonads and xanthomonads were done several years ago. It should also be noted that both Agrohacterium tumafaciens and A . rhizogenes can also cause tumors and hairy root syndrome on A . thaliana (this is, obviously, the basis of transformation using T-DNA transfer). A recent report begins exploitation of A . thaliana genetics to analyze plant control of the infection process by these two pathogens (Lincoln et al., 1992). 1. Pseudomonas syringae Several groups have shown that some isolates from various pathovar groups of pseudomonads can colonize some A . thaliana land races (Schott et al., 1990; Ausubel et al., 1991; Bent et al., 1991; Dangl e f al., 1991; Davis et al., 1991; Debener et al., 1991; Dong et al., 1991; Whalen et al., 1991). These bacterial pathogens were isolated from a range of species, mostly various brassicas (pv. maculicola) and tomato (pv. tomato). They can multiply and cause symptoms (necrosis and spreading chlorosis) associated with their effect on the nominal host species. These results demonstrated that there is variability in the interaction between P. syringae and A . thaliana that is dependent on both host and pathogen genotype. Thus, some P. syringae isolates are pathogenic on all tested

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Arahidopsis genotypes, some on none, and some on only a subset. On the basis of findings of “differential responses,” we and others began to ask whether single loci in both plant and pathogen controlled the generation of a HR as predicted by the gene-for-gene hypothesis. Resistance of various land races of A. thaliana to these bacterial isolates is often accompanied by a classical HR. Two bacterial avr genes have been defined and cloned using plant genotypedependent conversion of bacterial virulence to avirulence on various A . thaliana land races (Debener et al., 1991; Dong et al., 1991; Whalen et al., 1991). As well, several previously cloned avr genes, defined through the interactions of other P. syringae pathovars with other plant species, detect resistance specificities in A. thaliana. These are the avrPpiAl gene from pathovar pisi (Vivian et al., 1989; Dangl e f al., 1992b) and avrB cloned from pathovar glycinea (Staskawicz et al., 1987; R. Innes and B. Staskawicz, personal communication). Although not unexpected in light of findings discussed above (Kobayashi et al., 1989; Whalen et al., 1988). these results have important implications for understanding the number of bacterial and plant determinants involved in gene-forgene recognition, their respective functions, and their distribution and evolution among bacterial isolates, as well as within and between plant species. It is also apparent, and will be detailed below, that these results also have consequences with respect to cloning genes of immediate agronomic utility from a model species. Experiments relating to biochemical events induced by bacterial infiltration are discussed in Section IV. The R gene (RPMI) corresponds to one of the avr genes defined using A . thulium, avrRpml. RPMl was defined in the land race Col-0 in a cross with the susceptible land race Nd-0 and has been localized by RFLP mapping (Debener e f al., 1991). Recently, we isolated several YAC clones, containing large Arahidopsis DNA inserts, which hybridize to the RFLP markers genetically closest to RPMI. One of them, with a 270-kb insert, is thought to contain RPMl. This conclusion is based on three pieces of data. First, an RFLP marker telomeric to RPMI hybridizes to the 270-kb YAC clone, whereas while an RFLP centromeric to RPMl hybridizes to two other YAC clones. Second, an end-specific probe from the 270-kb YAC hybridizes to both of the other YAC clones, and end probes from one of them cross-hybridizes to the 270-kb clone. Thus, the genetic interval known to contain RPMl is physically overlapped by this set of YAC clones. Finally, the end probe from one of the two centromeric YACs, which detects the overlap, was used as an RFLP probe. It is genetically closer to RPMl than the RFLP probe used to isolate it, thus proving that our chromosome walk is proceeding in the correct direction. R P M l , then, is contained on less than 270 kb of Arahidopsis DNA, encompassing roughly 5 map units. The RPMl region is, therefore, highly recombinogenic, a trait often associated with disease resistance loci in crop plants. Subcloning of the YAC and gene identification via complementation of the recessive allele are in progress (T. Debener et al., unpublished observations).

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Localization of RPMI has also hastened progress in understanding how the avr gene PpiAI from pathovar pisi is recognized by A . thaliana. The first interesting observation was that the avrPpiA I gene generated resistance and susceptible phenotypes identical to that defined by avrRpmI on a series of 15 A . thaliana land races. We have recently shown, in fact, that avrRpmI and the avrPpiAI gene encode nearly identical proteins, and that resistance to both cosegregates to RPMl (Dangl et al., 1992b). Thus, the A . thaliana RPMl gene has functional, if not structural homologs in pea, and presumably in bean, where both the avrRpm 1 gene and the avrPpiAI gene detect a novel R-gene function (Vivian et al., 1989; Dangl et al., 1992b; Fillingham et al., 1992). This example highlights the use of A . thaliana to “mine” genes of potential agronomic interest. Resistance to the other avr gene defined on A . thaliana, avrRpt2, is conditioned by two separate, interacting genes from Col-0, defined in a cross with the susceptible land race Po-0 (A. Bent et al., personal communication). The function encoded by avrRpt2 is also recognized by an R-gene specificity in soybean (Whalen et al., 1991). They recently isolated a mutant plant that is unable to recognize avrRpt2, but is still capable of recognizing avrRpmI and avrB. This mutation segregates as a recessive in a backcross. It must, therefore, lie in one of the two genes required for resistance to avrRpt2, or in a gene required specifically for the action of one of them. Preliminary RFLP mapping suggests that the mutation is not at RPMI; thus at least two different genes in Col-0 have been functionally identified (B. Kunkel and B. Staskawicz, personal communication). At least two other plant mutants of the same class were isolated by another group (F. Ausubel, personal communication). Allelism tests show that these mutations map to the same locus, named RPS2 (B. Kunkel, A. Bent, B. Staskawicz, and F. Ausubel, personal communication). Mutant screens, of several kinds, for loss-of-recognition mutants utilizing all available avr genes are in progress. Combined with the genetic definition of several R-gene specificities, they will ultimately lead to a detailed understanding of both recognition and signal transduction mechanisms used to generate the resistant phenotype. As well, the results derived from bacterial systems will be merged with those from the fungal system outlined above. Genetic combination of loss-of-function or altered function mutants from each pathosystem will provide us with a circuit diagram of how, and indeed whether, signal transduction pathways from each R gene impinge on one another. Finally, it should be mentioned that many isolates from other pathovar groups and the species P. cichorii have been tested on a battery of A . thaliana land races (Dangl et al., 1991; Davis et al., 1991). All P. cichorii generate varying degrees of HR and are incapable of sustained in planta growth. No compatible interaction was observed, limiting the utility of these strains in further analysis of specificity. A converse outcome was seen with several pathovar tahaci and pathovar phaseolicola isolates, which gave essentially null phenotypes (our unpublished observations). We used one P. cichorii strain giving a good HR, and

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a P. syringae pv. tabaci strain that makes a null reaction to screen for altered response plant mutants. One was found, in response to the pathovar tahaci isolate, which repeatedly makes chorotic lesions and supports 30- to 50-fold more in planta bacterial growth than the parent land race. Genetic analysis shows that this mutation is codominant (T. Debener et al., unpublished). The locus identified in this screen is probably not involved in specific recognition, but may define a gene whose product is necessary in generation of nonhost resistance.

2. Xanthomonas campestris Several groups showed that nearly all tested X. campestris pv. campestris isolates grew several orders of magnitude and caused typical black rot symptoms on A. thaliana (Tsuji et al., 1991; Simpson and Johnson, 1990; Parker et al., 1992; Whalen et al., 1991; A. Horrichs, M. Arnold, and J. L. Dangl, unpublished). Simpson and Johnson showed that several infection routes gave similar results, and that some land race variability existed in response to two X. campestris pv. campestris isolates based on differential growth and symptom development. No genetic analysis of the basis for this difference was reported. In contrast, Tsuji et al. (1991) showed that a clear differential reactivity between land races Pr-0 (chlorosis and symptoms) and Col-0 (no symptoms) was conditioned by a single, dominant nuclear gene in the Col-0 land race. Interestingly, this phenotypic difference is due to tolerance in Col-0, as the bacterial isolate grows equally well in both land races. Until recently, no X . campestris pv. campestris strain had ever been reported to make a clear HR on any A. thaliana genotype, and the reliance on qualitative differences in bacterial aggressiveness may make this system problematic. Lummerzheim et al. (1993) did, however, identify a Brazilian isolate which triggers an HR in a spray assay, and other X. campestris pathovars (pv. raphani and pv. amor-aciae) were found that caused no symptoms and were unable to grow on various land races, but could trigger a weak, HR-like phenotype (Parker et al., 1992). They have isolated an avr gene from an X. campestris pv. raphani strain (avrXca),and identified an A. thaliana land race reproducibly susceptible to a virulent X . campestris pv. campestris strain carrying avrXca. This system is also made problematic by the fact that the in planta growth differences between virulent and avirulent bacteria are not more than 100-fold. This is in contrast to the differences observed using P. syringae isolates described above (see Debener et al., 1991; Dong et al., 1991; Whalen et al., 1991).

C. Viruses Viruses that are pathogenic on a range of Brassica species are also able to replicate and cause symptoms on A. thaliana. The first demonstration was that

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cauliflower mosaic virus (CaMV) caused typical chlorosis, stunting, and mosaic symptoms on a small number of plant genotypes (Susnova and Poljak, 1975). Melcher (1989) greatly expanded on these first studies by including virions from six CaMV isolates, as well as two recombinant virus plasmids derived from one of them. He observed that different quantities of viral particles were necessary to generate strong symptoms among tested isolates. Isolate-specific differences in symptom development were also noted, as shown for the interaction of CaMV with its more common hosts. The mechanism of CaMV spread through the plant is known in some detail, and a directed search for mutants or genotypes defective in this process has begun (S. Leisner and S. Howell, personal communication). Their previous work suggested a close relationship between developmental stage in the plant and the kinetics of viral movement. Thus, it is expected that some combinations of plant genotype and viral isolate are well matched for rapid virus spread, and other combinations are not. They analyzed four CaMV isolates, giving a range of phenotypes on the normal host (turnip), on a spectrum of A. thaliana genotypes, and report one ecotype that is resistant to one CaMV isolate. As well, they have observed plant-genotypespecific differences in tissue distribution of virus using a novel whole-plant blot technique. Genetic dissection of these differences will lead to refined understanding of viral movement and tissue-specific components controlling symptom development. The majority of current analysis of viral pathogens of A. thaliana concentrates on single-stranded RNA viruses representing a wide range of viral families (F. Ponz, personal communication). Using sensitive ELISA assays to detect viral replication, they found that only a subset of tested viruses is able to colonize A. thaliana. The most highly advanced pathosystems are the interactions of A. rhaliuna with turnip crinkle virus (TCV) and turnip yellow mosaic virus (TYMV). Li and Simon (1990; A. Simon, personal communication) first showed that six tested plant genotypes were killed within 22 days postinoculation with TCV isolate M. As well, they were unable to find any mutants showing altered phenotype after screening 7000 seedlings from an EMS-mutagenized population. Perseverance, and an expanded battery of 26 plant genotypes, uncovered one land race, Dijon, which was resistant to TCV-M. Resistance is manifested as a delay of symptom onset, and a drastic reduction in TCV-M replication. Preliminary genetic analysis shows that resistance is mediated by a single, codominant gene in the Dijon ecotype. Two A. thaliana genes, encoding novel glycine-rich proteins preferentially activated in the incompatible interaction, have also been isolated (A. Simon, personal communication). They may represent a class of glycine-rich proteins known to bind RNA, since they contain a characteristic RNA-binding domain. Use of another particularly nefarious virus, TYMV, has led to the isolation of a large number of putative A. thaliana mutants expressing high levels of toler-

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ance (M. Skotnicki, personal communication). The viral isolate used causes severe lesions, chlorosis, and plant death on land race Col-0. From 250,000 EMSmutagenized M, seedlings, over 20 putative mutants that were stably tolerant to TYMV infection in subsequent generations were isolated. The putative mutants are divided among six phenotypic classes. Three classes have no visible phenotype, but support high, medium, or no virus replication. Two classes express weak symptoms, and also support either high or low virus levels. The final class is leafless, but still allows high levels of viral replication. Future genetic analyses should provide valuable information regarding the life cycle of TYMV, and the number and nature of plant genes whose normal function results in disease symptoms. As mentioned in the introduction, one highly evolved state of plant-parasite interactions would be commensalism, tolerance of the pathogen by the plant. The fascinating preliminary evidence from the TYMV system may lend critical understanding to the phenomenon of tolerance. It will be of immense interest to see whether any of the TYMV-tolerant plant mutants have altered behavior with respect to other pathogens. Several viroids have been tested for their ability to replicate and cause symptoms on A. rhaliana, but all are replication negative (F. Ponz, personal communication). Ponz and colleagues have, however, potentially uncovered a way to analyze host factors responsible for repressing viroid replication. They cloned a potato spindle tuber viroid (PSTV) dimer, in both orientations, between the 35s promoter of CaMV and the GUS reporter gene. These constructs were transferred into tobacco and A . thaliana. In tobacco, a natural “symptomless” or tolerant host, no GUS activity is observed with the PSTV dimer in either orientation, although the viroid replicates. Surprisingly, the A. thaliaria plants with the viroid in the negative orientation are GUS positive. This suggests that A. thaliana, and by implication all nonhosts, may contain a factor (putatively a transcription regulator) that normally binds PSTV sequences, thus preventing viroid replication. A search for GUS-minus mutants, presumably viroid positive, is under way. These preliminary examples suffice to show that A . thaliana is an appropriate model for plant-virus interactions. A particular strength of current work is the complementary approaches taken by the various groups. As well, as described above for bacterial interactions, the analysis of both naturally occurring resistance and tolerant mutants will provide genetic tools necessary to address the relationship between these phenotypes.

D. Nematodes Several species of nematodes are very destructive plant pathogens. Beyond agronomic considerations, the control of various nematode species life cycles on their hosts is a fascinating example of highly specialized biotrophic plant-pathogen interactions (Dropkin, 1989; Hussey, 1989). Specific resistance

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controlling nematode invasion is known in some systems, and the isolation of resistance genes from tomato and potato via position cloning is in progress (Barone et al., 1990; Klein-Lanthorst et al., 1991; Messeguer et al., 1991). As well, the usual approaches to isolate plant genes specifically involved in resistance reactions have been applied to plant-nematode interactions (HammondKasock et al., 1989, 1990). Recently, Wyss, Simons and collaborators (Sijmons et al., 1991) established that A . thaliana is a host for several important nematode species: Heterodera schachtii, H . trifoli, H . cajani (sedentary cyst-forming nematodes), Pratylenchus penetrans (a migratory species), and Meloidogyne incognita (a root knot-forming species). They described optimization of culture conditions allowing routine completion of the life cycles of H . schachtii and M. incognita. The nematodes used were isolated from a range of plant species, and all discernible stages of the life cycle of each species were observed. In fact, the small, thin, and essentially transparent nature of A . thaliana roots allowed detailed observation of late developmental stages of H . schachtii for the first time. Seventy-four A . thaliana genotypes were screened with one H . schachtii isolate in order to assess variability in this interaction. Although a quantitative gradient of susceptibility was observed, no clear resistance could be demonstrated. It is hoped that analysis of a broader spectrum of both nematode isolates and plant genotypes will reveal natural resistance. Since each nematode species has critical, plantdependent requirements for colonization, it may be possible to test known hormone and root morphology mutants of A. thaliana for altered susceptibility. Of course, screening of mutagenized plant populations will also be used to isolate mutants with an altered response to nematode infection. One particularly attractive aspect of this system is the ability to define critical junctures in the nematode life cycle. Each stage of nematode development, then, could be interrupted or perturbed by mutations in the host plant. Although laborious, screening for mutant plants interrupting particular stages of the nematode life cycle would be clearly rewarding.

V. Biochemical Responses Enough preliminary analysis has been completed to state that A . thaliana responds to attempted pathogen ingress in ways closely analogous to those of other species. Based on previous work in a plethora of species (Crute et al., 1985; Collinge and Slusarenko, 1987; Hahlbrock and Scheel, 1989; Lamb et al., 1989; Dixon and Harrison, 1990; Dangl, 1992b), the first investigations focused on analysis of genes involved in phenylpropanoid metabolism and degradative enzymes. A small family of genes encodes phenylalanine ammonia lyase (PAL) in A . thaliana (Oh1 et al., 1990; Davis et al., 1991). The expression of at least

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one of them is induced, in cultured cells, by the addition of a nonspecific bacterial elicitor, PGA lyase (Davis and Ausubel, 1989). This study also demonstrated that activities for other enzymes implicated in plant defense were also induced. Unfortunately, cultured cells do not retain race-specific recognition, limiting their utility in analysis of these events. The use of gene-specific PAL probes, in combination with virulent and avirulent Pseudomonas strains, suggested that the PAL2 gene was more rapidly, and more transiently, induced than PAL1 during incompatible interactions (Davis, 1992). PAL mRNA induction during compatible interactions is typically delayed, and of lower magnitude. Interpretation of these results is confounded, however, by the differences in bacterial strains. Thus, the use of cloned avr- genes in isogenic, normally virulent, bacteria should give more succinct results. Yet, in such an experiment, using the avr-Rpt2 gene described above and resistant and susceptible A. thaliana land races, no clear correlation of PAL1 mRNA accumulation with specific incompatible interactions could be discerned. Appearance of PAL1 mRNA in both resistant and susceptible land races was, however, more rapid in the presence of the UVY gene (Davis et al., 1992). Many other genes whose products have been implicated in the defense response in other species have been cloned from A. thaliana. Dong et al. (1991) identified three tightly linked P-glucanase genes. Transcription of these genes is differentially regulated: mRNA from all three gradually accumulates after challenge with virulent bacteria, whereas only the BGL2 transcript is weakly induced by avirulent bacterial strains. In the presence of the avrRpr2 gene, in an otherwise virulent bacterial strain, none of the P-glucanase genes is specifically activated. Promoter fusions to the GUS reporter also suggest a spatial control of P-glucanase expression. The BGL2 promoter is expressed in a zone surrounding the site of virulent bacterial infiltration, whereas the BGL3 promoter directs expression diffused throughout the infected leaf (X. Dong and F. Ausubel, personal communication). Two genes encode the committed step in aromatic amino acid biosynthesis, 3-deoxy-~-arabinoheptulosonate 7-phosphate synthase (DAHP). Keith et al. (1991) showed that one of these genes, D H S I , was more rapidly induced in an avRpt2-mediated incompatible interaction than in the corresponding compatible interaction. This is particularly intriguing, as the structure of an A. thaliana phytoalexin (see below) may be derived from aromatic amino acids. Genes encoding enzymes involved in oxidative bursts have also been cloned from A . thaliana, including superoxide dismutase (SOD),lipoxygenase (LOX), and glutathione S-transferase (GST) (F. Ausubel, personal communication). The Ausubel group has preliminary evidence suggesting at least two distinct, or branching, signal transduction pathways, based on mRNA accumulation kinetics after challenge with strains either containing or lacking avrRpt2. Further mutagenic dissection of the processes leading to activation of these defense genes, described briefly below, could help to unravel the complicated series of events culminating in resistance.

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Other defense-related genes recently cloned from A. fhaliana include several encoding various classes of chitinase (Samac et al., 1990; Samac and Shah, 1991) and a series of PR proteins, examples of which have homology to either glucanases or osmotin (Metzler et al., 1991; Uknes e f al., 1992). Samac and Shah (1991) used promoter-GUS fusion expression in transgenic plants to show that the single A. thaliana gene encoding acidic chitinase is induced by fungal pathogens in both A. thaliana and tomato. This acidic chitinase promoter also displays developmentally regulated modes of expression: GUS was detected in roots, leaf vascular tissue, anthers, guard cells, and hydrathodes. These results are consistent with observations made with essentially all plant defense-related genes (Dixon and Harrison, 1990; Dangl, 1992b) and PR proteins from other systems (Lotan et al., 1989; Memelink et al., 1990). It is now hoped that use of these target promoters, in combination with mutagenic analyses in A. thaliana, will help unravel their role in establishing either localized or systemic resistance. We have taken a slightly different approach to the involvement of induced gene expression in generation of the resistant phenotype. Our work relies on cosegregation of pathogen-induced gene expression with R-gene-mediated resistance. Somssich ef al. (1989) isolated a series of parsley clones whose expression was induced by a fungal elicitor. Although some encode proteins of known function, several encode novel proteins. These workers have shown that several of the so-called ELI (elicitor inducible) genes are conserved in other species including A. thaliana (Trezzini et al., 1992). We screened for induced expression of 16 A. thaliana ELI homologs following infiltration with virulent and avirulent P. syringae pv. maculicola ( Psm) strains. Other than the expected, but low, activation of PAL, we found that only one gene, encoding the EL13 homolog, was strongly activated (Kiedrowski ef d.,1992). This induction is more rapid, and prolonged, during incompatible interactions than compatible interactions involving a particular bacterial strain, m2, from which avrRpml was isolated. The RPMl resistance gene, described above (Debener et al., 1991), is directed against this strain. Our most interesting observation is that the activation of EL13 in m2 incompatible interactions is dependent on the presence of a dominant allele at RPMl. We used the homozygous F, families from a Col-O(RPMI1RPMI) X Nd-O(rpmllrpm1) cross described above to prove this point. At the time point after infiltration with Psm isolate m2 chosen to maximize the differential induction of EL13 gene activity observed in Col-0 and Nd-0, leaves from the F, families were harvested and RNA was isolated. In RNA blot experiments, all 15 RPMIIRPMI families had high levels of EL13 mRNA, as did the resistant Col-0 genotype; all 15 rpmllrpml families had low levels, as did the susceptible Nd-0 genotype. This is strong evidence that EL13 activation requires RPMl function (P= lo-”). Two highly related full-length cDNA clones from A. rhaliana have been sequenced, and it seems that only two genes are present in A. thaliana. Other than the expected high homology to the parsley EL13 gene, no other similarities were found in the various databases. Thus,

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the function of this conserved plant defense gene remains unknown. We are now constructing antisense and sense constructs in order to make phenocopy mutants of EL13 and determine whether the expected mutants have an altered phenotype after infiltration with P. syringae pv. maculcola isolate m2 and other incompatible pathogens. The utilization of families homozygous for a known resistance function, but essentially randomly assorted at all other loci, is a powerful tool for addressing the role of any defense-related gene activity in establishment of resistance. A functional definition of an A. thaliana phytoalexin, 3-thiazol-2’-yl-indole, was achieved by S. Somerville and co-workers (Tsuji et al., 1992). Neither phytoalexin activity nor the phyoalexin molecule was present in healthy tissue. Both were induced by infiltration of leaves with an avirulent P. syringae pv. syringae bacterial strain that causes an HR, but not by a virulent X . campestris strain, a hrp mutant of the P. syringae pv. syringae strain, or buffer inoculation. This molecule is identical in structure to camalexin, isolated from the crucifer species Camelina sativa (Browne et al., 1991). Eight phytoalexins have now been isolated from various crucifer species, all sharing similar structural motifs based on an indole core. This is an exciting finding, since the power of A . thaliana genetics can now be brought to bear on the issue of camelexin biosynthesis through the use of auxotrophic mutants defective in aromatic amino acid biosynthesis (Last and Fink, 1988; Last at al., 1991). It should also be possible now to succinctly determine whether the accumulation of camalexin is a necessary component of specific resistance responses by analyzing whether mutants in its production are impaired in the ability to generate a race-specific HR. Another example of how the genetics of A. thaliana can be utilized to address long-standing issues in the physiology of plant-pathogen interactions is provided by Bent et d . (1992). Synthesis of the plant hormone ethylene has long been known to be induced during plant-pathogen interactions; many of the plant defense genes described above are ethylene inducible (see Samac et al., 1990). Is ethylene production and/or perception a necessary part of the resistance reaction? Several mutants defective in ethylene biosynthesis and perception are now available (Bleecker et al., 1988; Guzman and Ecker, 1990). Bent and colleagues showed that two mutants in ethylene perception, einl and ein2, are not altered in their ability to generate an HR against any of three tested P. syringae avr genes. This result suggests that at least the perception of ethylene, as defined in these mutants, is not required for a resistance response. It should be noted, however, that these two mutants were isolated using a loss-ofperception screen on young seedlings. There could be independent modes of ethylene perception operating in mature leaves during pathogen attack. Interestingly, the eiti2 mutant is nearly symptomless after infiltration with a number of virulent bacterial isolates, although it supports normal growth of these bacteria. Thus, the wild-type ein2 product is necessary for development of the chlorotic necrosis associated with compatible interactions in this system. This observation

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may have practical implications in the engineering of tolerance. As well, this finding also separates einl from ein2 functionally, implying the existence of different downstream targets from the products of these loci.

VI. Current Impact of Arebidopsis as a Model A. Definition of Resistance Genes This topic is where the first expected major breakthroughs using A . thaliana as a model can be expected, although the "first" cloned R gene may not come from any of the systems described here. Ultimately, however, a concentrated effort on Arahidopsis will provide a wealth of information about the breadth of potential structures and functions that can be utilized as R genes. Using the interactions with the downy mildew fungus P. parasitica (see III,A) and Pseudomonas (see III,B) as illustrations, will all of the R genes postulated, or demonstrated, to date in A. thaliana be molecularly related? Or will all R genes against the known Pseudomonas avr functions be related, with another structurally distinct set aimed at the mildew isolates? An answer to this question can come fairly quickly on the heels of the first cloning. One can then clone all molecular homologs via lower stringency hybridization and locate them on the RF'LP map. Since the map positions are or will soon be known for several R genes in both pathosystems, comapping of the homologs to a position containing another known specificity will be highly suggestive. How many such homologs are in the A. fhaliana genome, and do they also function as R genes against either the same or different pathogens? The mapped R-gene homologs may encode new specificities that we have not yet functionally defined. This argues for continued expansion of both the number of informative pathosystems and the number of resistance specificities within each. Naturally, it could also be the case that each R gene encodes a unique product, suited to its role in recognition of a distinct avr function. In this case, the concerted effort on A. thaliana will also yield valuable information regarded the range of structures and functions employed to recognize pathogens.

B. Genetic Dissection of Plant Defense The impact of the model pathosystems introduced above on the thorough definition of all loci necessary for a resistance response will be enormous. It is important to recognize that the mutagenic approaches described in sections III,A, IKB, and III,C, and also in this section, will yield information about signal transduction functions, R-gene function, and other communication networks within the plant leading to resistance. Loss-of-recognition mutants described so

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far (against the avrRpt2 gene discussed above) are specific for that avr function. Thus, recognition of other cloned avr functions is maintained. These analyses are just beginning, and a true saturation screen, for loss of the ability to respond to several cloned avr functions, will be invaluable. Screens for altered, or lost, recognition of P. parasitica isolates in the same plant genotypic background will also begin in the near future. Together, these screens also allow estimation of the interconnectedness of specific recognition and transduction pathways. Moreover, we may be able to divine a minimum number of mutable genes required for triggering the resistant phenotype. Strategies aimed at isolating mutants incapable of activating the transcription of particular plant defense genes will also be informative. This strategy (Davis, 1992) involves construction of transgenic plant lines carrying one (or more) promoter-reporter gene fusion. Candidate promoters are, in this case, genes involved in known aspects of the overall plant defense mechanism such as PAL, GST, CAM, and P-glucanase described above (Dong ef al., 1991; Davis et al., 1991). Seed from appropriate transgenic target strains is mutagenized, and M2 plants are assayed for altered expression of the reporter gene following virulent or avirulent pathogen challenge. This approach is complicated by the probability that the promoters in question have very complex expression modes, probably dictated by complex, potentially interacting, sets of cis-acting promoter elements. Thus, mutations that fail to abolish completely the transcriptional activation of the target promoter may be difficult to identify, as they would express only a lowered quantity of reporter activity. Most current reporter systems must be laboriously screened, although several selective reporters are being developed (Karlin-Newmann er al., 1991). On the other hand, identification of mutations quantitatively affecting activation of the defense system may prove quite useful. This is especially true when one considers that it appears that different pathways are used during induction of the target genes mentioned above. This approach is also powerful since it works “backward” from a particular promoter, through the steps required to activate that promoter. As such, it is clearly complementary to loss-of-recognition screens described above. It will be of great interest to see whether mutants found via such a “promoter-directed‘’ screen are connected to those isolated by loss or altered pathogen selections.

C. Induced Systemic Acquired Resistance The phenomena known as induced resistance, immunization, or systemic acquired resistance (SAR) have a long history in the literature of plant pathology (Chester, 1933). The establishment of a systemically immunized state in plants requires triggering of a local HR by a pathogen. Abiotic necrosis does not induce SAR, nor do pathogens that do not cause necrosis. As well, the immunized state persists for varying times and spreads for varying distances through the

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plant (Kuc, 1982; Ward et al., 1991). It has recently been shown that immunomodulators such as 2,6-dichloroisinicotinic acid (INA) can trigger SAR in the absence of necrosis (MCtraux er al., 1991). It is also becoming clear that salicylic acid acts, either directly or through an unidentified intermediate, as an internal messenger that spreads the initial immunizing signal throughout the plant. Since SAR can be induced under field conditions, a basic understanding of its genetic control would contribute to rational designs for its implementation. Uknes et al. (1992) have recently established that A . rhaliana can be immunized using INA, and thereby protected against infection by either Pernospora parasitica or Pseudomonas syringae pv. tomato. Phenotypic observations in the fungal system show that INA triggers SAR up to 7 days postapplication. The cellular stage at which immunized resistance is operative is dose dependent; single-cell HR is observed at higher INA doses. Protection against the bacterial pathogen was observed as a diminution of chlorotic symptoms. Systemic acquired resistance was accompanied, as in other systems, by the systemic accumulation of PR proteins, including a glucanase and an osmotin-like protein. This result sets the stage for a genetic dissection of the establishment of S A R . Future results will illuminate our understanding of not only disease resistance mechanisms but also basic physiological processes such as long-distance communication between plant cells.

Acknowledgments The exploitation of A . fhaliuno in understanding plant-pathogen interactions is very new. Thus far, cooperation among the groups working in this area has been extremely generous. For me, the personal interactions with those involved have been both rewarding and, importantly, fun. Without this degree of good will, exploration of the complex issues detailed above loses much of its attractiveness. Therefore, I thank the many individuals, and their colleagues, who provided me with infonnation summarized in this review: Andrew Bent, Barbie Kunkel, and Brian Staskawicz, University of California, Berkeley; Eric Ward, Scott Uknes, and John Ryals, CIBA-GEIGY; Fred Ausubel, Massachusetts General Hospital; Keith Davis. Ohio State University; Jane Parker, Sainsbury Laboratory; Ian Crute and Eric Holub. Horticulture Research International; Alan Slusarenko. University of Zurich; Shauna Somerville, Michigan State University; Anne Simon, University of Massachusetts; Mary Skotnicki, Australian National University; Steve Howell, Cornell University; Fernando Ponz, CIT-INITA, Madrid. The participants in the first ARAPANET (Axhidopsis &thology &work) Workshop. held in Koln. including those named above, who responded to my requests for unpublished data, are also thanked. Mostly, I am indebted to the members of my group, who are responsible for the work cited from our lab, and whose critical reading helped improve the manuscript. Research in our laboratory is funded by the German Ministry for Technology (BMFT), the German Research Society (DFG). and the European Community Arabidopsis BRIDGE program.

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Toward a New Concept of Cell Motility: Cytoskeletal Dynamics in Amoeboid Movement and Cell Division Yoshio Fukui Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 6061I

I. Introduction The purpose of this article is to review and reevaluate past and current studies relevant to cytoskeletal dynamics. I define cytoskeletal dynamics as the cytoskeletal reorganization that occurs as a consequence of monomer-polymer transition and protein-protein as well as protein-membrane interactions. Dynamics is best exhibited when it occurs concomitant with specific physiological activities, including chemotaxis, cell division, capping surface receptors, and substrate exploration. Therefore, in this article, I intend to review historical as well as current studies on cytoskeletal dynamics relevant to those physiological activities. It is obvious that all different nonmuscle systems have unique activities and components. Regardless of this diversity, most components and mechanisms seem to be very similar. Therefore, rather than trying to review all those systems, I will focus on Dirryostelium cytoskeletons. In some cases, similar components are termed differently in other systems, so I will suggest representative papers for those systems. Although conventional wisdom suggests that the force-producing machinery in nonmuscle cells is similar to the “sliding-filament’’ mechanism in muscle, current knowledge suggests a few new mechanisms of force generation for the former. These mechanisms are emerging largely from the area of modem cell biology, and some aspects of it will be reviewed in this chapter. Particularly, the mechanism of force generation seems central to cytoskeletal dynamics, and therefore, these problems will be discussed in detail in Section VI. Since this review cannot be comprehensive in all aspects, specific references that are directly relevant to particular subjects will also be suggested. 85

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II. Conventional Theories Actin constitutes as much as 10% of the total protein in nonmuscle cells. This is equivalent to about a 1% hydrophilic protein solution, half of which is filamentous polymer. The dynamism of cytoplasm exhibits as a thixotropic transition with polymerization and crosslinking of the filaments through regulation of pH, calcium, and associating proteins. Most biological gelation is reversible. Conventionally, gelation-solation has been believed to be responsible for cell motility. Although gelation does not involve highly efficient mechanochemical transduction, it still can generate enormous motive force.

A. Amoeboid Movement 1. Anterior vs Posterior Contraction The dynamics of giant, free-living amoeba has been providing a model for studying the force behind amoeboid movement since Dujardin (1835). For instance, Hyman (1917) proposed that “the ectoplasm is an elastic tensile gel which exerts a tension upon the more fluid endoplasm.” She anticipated that there is a dynamic “sol-gel transition,” and that the cytoplasm consists of a liquid that can be transformed into a gel, forming an ectoplasmic tube. The reverse process is liquefaction or solation. Pantin (1923) followed this idea and proposed that tail contraction is responsible for the movement of limax-type amoeba. He contributed the suggestion that gelation occurs posteriorly and liquefaction occurs anteriorly in cells. Osmotic pressure and gelation-solation are still considered to play significant roles in cell movement. Mast refined the above hypothesis and proposed the posterior contraction theory (1926). Mast’s theory was based on the hydrodynamic gradient of the cytoplasm, as was Hyman’s, but he also proposed the significance of the contraction of the plasmagel. He applied external force to a single amoeba and observed that the direction of the flow of plasmagel immediately reversed (1931). This was a simple paradigm of cellular dynamics brought about by cytoskeletons (Conklin, 1917). After Conklin, the term cytoskeleton became gradually established as a name for the structure responsible for the motile nature of cells (Camp, 1937; Seifriz, 1938). Kamiya (1950a,b) later established the force required for the reversion of cytoplasmic flow (balance pressure), being as much as +20 cm of the hydrostatic pressure. This value is consistent with the force generated by a single Dictyosteliurn amoeba as well as with a theoretical value of the force generated by the conventional myosin included in a single cell, as will be discussed in Sec-

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tion VI. For reviews on classic theories of amoeboid locomotion, refer to De Bruyn (1947), Goldacre (1952) and Wohlfarth-Botterman (1964). In 1954 Huxleys and co-workers proposed that a single molecular mechanism is responsible for the contraction of cytoplasm (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954). This theory, called the sliding-filament theory, was a revolutionary concept of the mechanism of contraction. It was applied to primitive motile systems by Kamiya and Kuroda (1958), who observed shearing force generated at the interface between the ectoplasm and the endoplasm of Nitella internodal cells. The in vitro motility assay technique by Sheetz and Spudich (1983) opened a new avenue in analyzing the force-generation mechanism. The history of the conceptual transition from the conformational changes (Szent-Gyorgyi, 1951) to the sliding mechanism was recently reviewed (Fukui and Yumura, 1986). The term conformational change is used in a strict sense for changes in molecular structure, occumng in the order of angstroms, whereas the term classical conformational change is used to imply the overall transition in cellular structures, observed in the range of micrometers. The allosteric changes in “walking” proteins (Alberts et al., 1989, p. 130) represent the former concept, whereas folding and unfolding of ectoplasm (Goldacre and Lorch, 1950; Goldacre, 1952; Bovee, 1952) represents the latter. Although the possibility remains that the folding and unfolding of some of the cytoskeletal proteins may contribute to exertion of force, the dynamic nature of the cytoplasm is largely due to a transient, rapid reorganization of the actomyosin cytoskeleton (Pollard, 1976). The reorganization includes assembly/disassembly and perhaps transport and association with other cytoplasmic components. Allen proposed the frontal zone contraction hypothesis for amoeboid movement (Allen, 1961; Allen and Allen, 1978). This hypothesis located the site of motive force just behind the leading edge where the endoplasm exhibits a fountain-like stream. Earlier, Lewis (1942) had observed “a steady fountain streaming,” as described in his review. The idea central to Allen’s hypothesis is the viscoelastic property of the endoplasm, which theoretically can drag the rest of the endoplasm forward. The distribution of actin and conventional myosin in Amoeba proteus (Gawlitta et al., 1980; Stockem et al., 1982; Stockem and Klopocka, 1988) is not favorable to this hypothesis, presuming that most of the force is generated by actomyosin-I1 (actin and conventional myosin). It is possible that the swelling pressure of actin gel (Nossal, 1988; Oster, 1988) and presumed projectile force by actomyosin-I (actin and a small, single-headed myosin) (Fukui et al., 1989) contribute to the exerting force at the frontal lamella. The projection of the leading edge, irrelevant of the mechanism, in turn, could propel the rear. This dragging force obviously derives from the viscoelastic property of the cytoplasm (Allen and Allen, 1978; Oster, 1988).

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2. Solation-Contraction Coupling The gelation of nonmuscle cell extract is similar to the superprecipitation of muscle extract that is induced with ATP (Szent-Gyorgyi, 1951; Nonomura and Ebashi, 1974). These two terms, gelation and superprecipitation, however, must be used separately. Gelation is the process occurring in the system concomitant with changes in the structure/organization of the filaments. This process is usually slow and change is measured as an increase in viscosity; the change can take hours if no excessive external force is applied. In other words, the actin gel, crosslinked with actin-binding proteins, has a property similar to a nonNewtonian fluid, which exhibits two different sets of resistance upon application of external force. This property was experimentally demonstrated by Pollard and colleagues (Sato et al., 1987). When actin+-actinin gel is forced to deform rapidly, it produces 40 times more force than slowly deforming gel. This time-dependent force is due to changes in the network organization of the system. The energy stored in the cytoplasmic gel appears to play a role in exerting motile force (Oster, 1988). On the other hand, superprecipitation is a rapid process that occurs in seconds, and the interaction of actin with myosin as well as ATP hydrolysis is necessary. Obviously, this latter process is identical to the actomyosin-11-based “contraction” in nonmuscle cells. Hereafter, myosin-I and -11 refer to a singleheaded, low-molecular-weight myosin and the conventional two-headed myosin, respectively. In this regard, the solationxontraction coupling hypothesis (Hellewell and Taylor, 1979; Taylor and Condeelis, 1979; Taylor and Fechheimer, 1982) contributed to the establishment of a new model for the mechanism of nonmuscle contraction. It also played a major role in establishing experimental paradigms to assess the significance of actin-binding proteins (ABP) and Ca2+ in Amoeba and Dictyostelium. The essence of this hypothesis is the idea that solation, but not gelation, is a prerequisite for contraction. It was demonstrated that contraction occurs concomitant with local dissolution of the gel (Taylor and Condeelis, 1979). This process was recently described as a self-destructive process for cytochalasin-induced contraction of fibroblasts (Kolega et al., 1991; Janson et al., 1991). The evidence that a 120-kDa actin-crosslinking protein inhibits actinactivated Mg2+-ATPase(Condeelis et al., 1984) is favorable to this model. Although the mechanical force can be transmitted by either gel or sol to the other domains of the cell, the maximal contractile force appears to be generated coupled with solation.

3. Cortical Expansion The cortical expansion model proposes that directional formation of pseudopods toward a chemotactic stimulation results from the expansion of actin gel

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(Condeelis ef a/., 1990). The model hypothesizes that the first crucial event is the activation of actin polymerization with a peak by 10 sec after stimulation with cAMP (Hall et al., 1988). The second event is the incorporation of actincrosslinking proteins into the actin network. One of the key proteins responsible for this event is proposed to be ABP-120 (Condeelis et al., 1988). The third event is the swelling of the gel. The swelling occurs by expansion of the gel by swelling pressure (Oster, 1988) subsequent to further development of the actin network. Oster defined the swelling pressure as the balance between osmotic pressure and elastic pressure. The osmotic pressure is brought about by the flow of positive charges into a gel’s negatively charged matrix, whereas the elastic pressure is largely due to bending and writhing of actin fibers. The logic of the latter hypothesis is based on Flory’s network theory (Flory, 1953) for hydrophilic colloid, which established a relationship between the average length of filaments and the number of crosslinkers (Nossal, 1988). The model also presumes a drop in free energy accompanying actin polymerization and an increase in entropy at a focal point (Hill and Kirschner, 1982). This model is not mutually exclusive with either anterior or posterior contraction hypotheses. Rather, it suggests a mechanism for a local force generation, independent from interaction of actin with myosin.

4. Compaction-Extension-Traction The compaction+xtension-traction hypothesis is based primarily on highfidelity localization of actin and myosin in Dictyosrelium by agar overlay immunofluorescence (Figs. 1 and 2) (Fukui et a/., 1986, 1987). This technique shows that actomyosin-I1 forms bipolar filaments and exhibits dynamic changes in localization in response to stimulation with the chemoattractant cAMP (Yumura and Fukui, 1985). Actin cytoskeleton also undergoes a dynamic translocation between Triton X- 100-soluble and -insoluble fractions upon cAMP stimulation within 5 sec (McRobbie and Newell, 1983, 1984). The former change occurs in less than 30 sec at 4°C in a wild-type Nc4 strain (Yumura and Fukui, 1985) and slower in axenically grown Ax3 cells (Nachmias et al., 1989). According to these studies, myosin-I1 forms bipolar filaments, 0.6 pm long, which are aligned parallel to each other at the posterior end. There is also a sarcomere-like linear array along the side wall of actively migrating monopodial amoeba (Fukui and Yumura, 1986). The authors proposed a multistep mechanism for amoeboid movement based on the dynamic recruitment of myosin-I1 (molecular maneuver mechanism: Fukui and Yumura, 1986). The theory hypothesizes that the assembly of myosin filaments is brought about by the vectorial translocation of monomers along Factin, and amoeboid locomotion is the consequence of three-step mechanism occurring in a spatially and temporally regulated manner: ( a ) compaction of the posterior cell body, ( h ) extension of leading lamella, and (c) traction of the cell

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body toward anterior pseudopods. Much of our current knowledge substantiates this theory. Apparently there is a diversity in the pattern of cell locomotion that has been brought about by evolution. The compaction+xtension-traction theory represents only the paradigm, and there are numerous exceptions in which this mechanism is not established. For instance, spermatids of Ascaris mum do not exhibit this mechanism. When treated with an extract from male glandular vas deferens, the sperm crawl actively on coverslips under anaerobic conditions. It has been proposed that two independent processes (continuous extension at the leading edge and continuous shortening at the base of pseudopod) are responsible for the observed rapid locomotion (speed of 70 pm/min) (Sepsenwol and Taft, 1990). It appears that projection and retraction are the major mechanisms in this organism, but no compaction is apparent.

B. Cell Division The term cytokinesis is usually used for a mode of cell division that is coupled with mitosis (Wilson, 1928). However, cells occasionally divide in a different manner, not coupled with mitosis. This mode of cell division is called cytofission (Chalkley, 1935). Chalkley proposed that locomotion on a substratum is a prerequisite for cell division of A . proreus. Although this hypothesis has been experimentally ruled out by Rappaport and Rappaport (1986), it appears that there is no significant difference between cytokinesis and cytofission in terms of cytoskeletal dynamics and force generation. Therefore, I use the term cell division to refer to both cytokinesis and cytofission in this review. There may be a substantial overlap in mechanisms of amoeboid movement and cell division. Particularly, the cortical flow hypothesis (Bray and White, 1988) should be considered a model feasible for general cell movement.

1. Polar Relaxation The beauty of cell division is the harmony of temporal and spatial dynamism of events occurring in a relatively short period of time. The rheological facet of cytokinesis was extensively studied in sea urchin eggs (Mitchison and Swann,

FIG. 1 Cytoskeletal organization in locomoting Dictyosfelium discoideum amoeba. Indirect immunofluorescence micrographs prepared using the agar overlay method (Fukui er a/.. 1986, 1987). The amoebae are polarized with their anterior lamella to the left. (a) Actin stained with a monoclonal antiDicryostelium actin. (b) Myosin-I stained with a polyclonal anti-Dictyostelium myosin-I. (c) MyosinI1 stained with a monoclonal anti-Dictyosfelium myosin-11. (d) a-Actinin stained with a monoclonal anti-Dictyosfelium a-actinin (mAb-18 provided by Dr. Michael Schleicher, Max-Planck Institute for Biochemistry). (e) Tubulin stained with a monoclonal anti-yeast a-tubulin. Scale bar, 10 pm.

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1954a,b, 1955; Hiramoto, 1970). It was found that the initial rounding-up at prometaphase is associated with an increase in the cortical tension (“stiffness”). The increase in stiffness is substantial: from 3.4 to 57 dyn/cm2 per micrometer deformation, or 17-fold (Mitchison and Swann, 1955). In most cells, the rounding-up is followed by accompanying elongatiordconstriction. The major issue has been to determine whether the force is primarily due to relaxation at the poles or contraction at the furrowing region. The polar relaxation (Chalkley, 1935, 1951) and astral relaxation (Wolpert, 1960) theories supposed that forces responsible for the cleavage derive from contraction of the cortical gel. Taking the measured increase in surface area, 80% of which occurs at the polar regions, and a decrease in stiffness, occumng mainly at the poles, this theory postulated the differentiation of the poles (astral differentiation) which, in turn, results in the relaxation in tension at the poles, relative to the furrow. The polar relaxation theory was reevaluated (Schroeder, 1981; White and Borisy, 1983). The core assumptions for this latter model are (a) effects of the mitotic aster to activate the cortex, (b)presence of filamentous tension-generating elements, and (c) free movement of the elements in the plane of the cortex (White and Borisy, 1983). These assumptions theoretically lead to a sequential change in organization of the elements, based on computer simulation, and ultimately give rise to a circumferential ring at the equator. The key feature of this model is the prediction of a local relaxation of the cortex at the poles prior to contraction at the furrow. This initial relaxation is stimulated with activation of the aster, and subsequent rearrangement of filamentous components occurs in such a way that more tension can be generated at the equator. 2. Equatorial Contraction The theory opposing polar relaxation is the equatorial stimulation hypothesis (Marsland and Landau, 1954; Marsland, 1956). The evidence to support this mechanism has been substantiated by Rappaport and colleagues (Rappaport, 1986, 1990). This theory was also attested to by a recent theoretical study (Devore et al., 1989). The theory hypothesizes the stimulation of furrowing at the equator by a diffusible factor(s). This idea was supported by various experimental data, most of which were derived from manipulation of marine eggs and A. proreus. As mentioned above, this theory speculates the presence of diffusible factors emerging from the aster, and transport along the astral rays (Devore et al., 1989), which unequivocally stimulates the contraction of the equatorial cortex.

FIG. 2 Cytoskeletal organization in dividing D.discoideurn. (a) Actin, (b) myosin-I, (c) myosin-11, (d) a-actinin, and (e) a-tubulin. Note that myosin-I and a-actinin are not located at the cleavage furrow (b, d). Refer to legend of Fig. 1 for antibodies. Scale bar, 10 pm.

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A similar function of interphase centrosomes for cell division has been indicated in Dictyostelium (Kitanishi-Yumura et al., 1985). In this study, the multinucleated cells induced by microtubule inhibitors exhibited multipolar division on a substratum. The authors suggested a major role for the microtubulecentrosome complex in the determination of the cellular unit. The primary conclusion was the implication that actin and myosin are reorganized into each division plane to form an apparently contractile structure. It should be noted that myosin organization seems to be secondary to the primary event of actin organization as demonstrated by recent gene disruption studies discussed below. The organization of the actin cytoskeleton at the furrowing regions appears normal in the myosin-I1 mutant (Fukui et al., 1990). In this mutant, a myosin heavy chain gene was disrupted by homologous recombination and the cell synthesized only heavy meromyosin (HMM-140), which did not assemble into filaments (De Lozanne and Spudich, 1987; Fukui et el., 1990). It is now clear that the events for actin dynamics precede those of myosin. The myosin-I1 mutants can divide on a substratum by traction-mediated cytofission, and this division is accompanied by lamellipodal activity at the poles. The actin organization appears normal at the polar lamellae (Fukui et al., 1990). In suspension, this mutant periodically displays active formation of the polar lamella. Consequently the cell exhibits bipolar morphology typical to anaphase-telophase cells. Nevertheless, the cell does not complete division in suspension (Spudich, 1989). The division requires attachment to a substratum. The presumptive stimulus appears to induce assembly of the actin cytoskeleton at both the poles and the equator, and myosin organization follows.

111. Current Concepts The cortical-gel contraction hypothesis predicted that actomyosin ATPase produces the energy of contraction in nonmuscle systems (Marsland and Brown, 1942). An equally significant conceptual revolution followed, in which the sliding-filament mechanism theory achieved popularity. The latter theory dominates as the current concept of the force generation mechanism for either cell locomotion or cell division in nonmuscle cells (Spudich, 1974; Fukui and Yumura, 1986; Clarke and Baron, 1987).

A. Contraction The broad band-like area encircling the equator (Marsland and Landau, 1954) was later called the contractile ring and many of its properties were studied by

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Schroeder (1968, 1972). The major filamentous component was first determined to be actin in newt eggs (Perry et al., 1971) and in HeLa cells (Schroeder, 1973). Myosin-I1 was identified as the main component of the contractile ring by indirect immunofluorescence localization in HeLa cells (Fujiwara and Pollard, 1976) and microinjection of antibodies into starfish blastomeres (Mabuchi and Okuno, 1977). The myosin filaments aligned parallel to each other and to the plane of constriction (Yumura and Fukui, 1985; Schroeder and Otto, 1988). A high-resolution visualization with a confocal microscope attested to the equatorial orientation of filamentous myosin-I1 at the furrow (Fig. 3). Recent gene disruption studies demonstrated directly that myosin-I1 is a prerequisite for cell division in D. discoideum (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987). Only a few pieces of evidence are available for the “filamentous” organization of myosin-I1 in cell locomotion (Nachmias, 1968; Gawlitta et al., 1980;

FIG. 3 A confocal fluorescence micrograph of myosin-I1 at the furrowing region of a dividing D. discoideum amoeba. The cell was stained with a monoclonal anti-Dicfyostelium myosin-I1 followed

by FITC-labeled anti-mouse IgG, and observed under a Zeiss LSM using a 63X planapo objective. The image was z-sectioned with a 0.3-pm increment, digitized, and processed for contrast enhancement. This image represents the optical section at about 1.5 pm from the dorsal surface of the furrow. Scale bar, l pm.

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Svitkina et al., 1989). Nevertheless, under optimal conditions, myosin filaments are arranged in parallel, manifesting a pseudo-sarcomeric structure in migrating Dictyostelium amoeba (Fukui and Yumura, 1986). The reason for this difficulty is probably due to rapid disassembly of the filaments by either phosphorylation of the heavy chain (Kuczmarski and Spudich, 1980; Kiehart and Pollard, 1984; C8tC and Bukiejko, 1987; Kuczmarski et al., 1987) and/or artifactual disruption with fixatives (Yumura and Fukui, 1985). The alignment of myosin filaments, parallel to each other and to F-actin, is the organization advantageous to the sliding-filament mechanism, and there is no evidence available to dispel this mechanism. However, the sliding filament model does not exclude other possible mechanisms for force generation. Other cytoskeletal components associated with the contractile ring have been localized at the furrowing region. These components include actincrosslinking and barbed-end capping proteins. The former proteins comprise a-actinin and filamin, which have been localized at the cleavage furrow in chick embryonic cells (Fujiwara et al., 1978) and echinoderm eggs (Hamaguchi and Mabuchi, 1986), and in chick embryonic cells (Nunnally et al., 1980). In contrast, a recent immunofluorescence localization in Dictyostelium demonstrated that a-actinin appears to be excluded from the cleavage furrow of this organism (Fig. 2d). The barbed-end capping proteins include an 82-kDa actin-modulating protein that was recently localized at the constricting region of rat fibroblasts (Sato et al., 1991). As in the contractile machinery of skeletal muscle (Pollack, 1990), it seems that nonmuscle contractile systems are also fabricated with numerous components. Rationally, those components should possess actin-, myosin-, and/or membrane-associating properties. There are apparently many others that have not been identified. The reason for this shortfall is the difficulty in isolating specific motile organelles from actively moving nonmuscle cells because of their delicacy, divergence, and dynamism. Fibroblastic stress fibers and intestinal brush border are two systems that have been studied extensively in the last two decades (Bryers et al., 1984; Mooseker, 1985). The contractile ring is probably likewise suitable for biochemical characterization (Mabuchi, 1986). Schroeder and Otto (1988) demonstrated that 600 mM KCl and ATP did not alter the organization of actin and myosin in an isolated contractile ring of sea urchin eggs. They also showed that the breakdown of actin with the actinsevering protein gelsolin did not affect the association of myosin with an isolated contractile ring. This evidence indicated that actin and myosin are attached to the plasma membrane by self-supporting mechanisms. Mabuchi et al. (1988) isolated the cleavage furrow of newt eggs. The isolated cleavage furrow exhibited an arc-like structure containing several unique proteins. This study also revealed that the binding site of F-actin with plasma membrane bears an electron-dense globular structure, indicating association with other components.

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B. Projection The dynamics of nonmuscle cell motility also manifests as projection rather than contraction. Evidence for this mechanism has been accumulated from a broad range of organisms, including echinoderm, mollusk, and arthropod sperm, and protozoan and vertebrate cells (Tilney, 1985). When triggered, the acrosoma1 process elongates to 90 pm in less than 10 sec. This rapid elongation results from polymerization of actin. Tilney estimated that it would take only 12 sec to reach 90 pm, given that actin concentration is 4 mM in the acrosomal vesicle before fertilization. Actin is under the control of ( a ) polymerization by subunit addition to the plus and minus ends at different rates, (b) nucleation of the trimer, and ( c ) actin-modulating proteins (Craig and Pollard, 1982; Pollard and Craig, 1982; Pollard and Cooper, 1986). However, all acrosomal processes are not equipped with the same structural motif. In the mussel Mytilus, the elongation is a slow process, taking a few minutes to reach the maximum length of 2-5 pm (Tilney, 1985). It has been revealed that 45-65 actin filaments are already present in the vesicle of unfertilized sperm as tightly cross-bridged, hexagonally packed paracrystals. It has been suggested that this relatively slow elongation may be due to “a biased one-dimensional stochastic walk” introduced with unidirectional, ratchet-like action of the actinmembrane linkage (Tilney et al., 1987). Although the polymerization of actin itself appears to be able to generate sufficient force for nonmuscle motility (Cooper, 1991), this latter mechanism has not been fully elucidated. Myosin-1’s appear to be a good candidate for the mechanoenzyme responsible for the projectile force (Kom and Hammer, 1990; Pollard et al., 1991). Their heavy chain (-130 kDa) consists of an N-terminal domain (-80 kDa) highly homologous to the head domain of the conventional myosin and a small (-50 kDa) tail domain. The tail domain is unique compared to that of myosin-I1 and many protozoan myosin-1’s contain a membrane-binding site. Myosin-1’s have been identified in a wide variety of organisms including yeast, Acanthamoeba, Dictyostelium, Drosophila, chicken, and mouse. The MY02 gene product of Saccharomyces encodes a type of myosin-I whose tail domain has calmodulinbinding, a short a-helical coiled-coil, and globular domains (Johnson et al., 1991). Mouse dilute and chicken PI90 are a class of myosin-1’s identified from brain, whose amino acid sequences are similar to that of yeast MY02 (Mercer et al., 1991; Larson et al., 1990). Nina C gene products from photoreceptor cells of Drosophila are putative myosin-1’s that have a unique N-terminus resembling a catalytic domain of protein kinases (Monte11 and Rubin, 1988). A 110kDa-protein-calmodulin complex of chicken intestinal microvilli forms spirally arranged bridges between F-actin in the core and its lateral membrane (Matsudaira and Burgess, 1979), is an integral membrane protein (Glenney and Glenney, 1984), and has actin-activated Mg2+-ATPase (Mooseker, 1985; Conzelman and Mooseker, 1987).

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Protozoan myosin-1’s have been studied most extensively (Kom and Hammer, 1988; Kom, 1991; Pollard et al., 1991). Four and five different myosin-I genes have been identified from Acanthamoeba (AMIA, AMIB, AMIC, AMID) and Dictyostelium (DMIA, DMIB, DMIC, DMID, and DMIE), respectively (Hammer, 1991; Kom, 1991). Their head domains are highly homologous to myosinI1 and among these myosin-I’s, -65% of them are an exact copy of muscle myosin subfragment-I. The tail domain of five of those myosin-I isofoms (AMIB, AMIC, AMID, DMIB, DMID) has similar sequences, referred to as membrane-binding, ATP-insensitive actin-binding, and tail homology regions. DMIA and DMIE have sequences similar to those of the above five myosin-Is, but lack the region of the ATP-insensitive actin-binding site (Titus et al., 1989). They designated this class of myosin-1’s as abm A for DMIA, and abm B and abm C for DMIB and DMID, respectively. More recently, it was found that Acanthamoeba has a high-molecular-weight putative myosin-I whose tail domain is unique relative to all other myosins (Horowitz and Hammer, 1990). Among the unique properties of myosin-1’s is the membrane-binding region that is located at the tail. Purified Acanthamoeba myosin-1’s (MIA, MIB) bind to isolated membrane and synthetic anionic phospholipid vesicles by chargecharge interactions (Adams and Pollard, 1989; Miyata et al., 1989). The movement of myosin-I-coated membrane vesicles was also demonstrated (Adams and Pollard, 1986). There are an ATP-sensitive actin-binding site, ATP-binding as well as catalytic sites, and light chain-binding sites located in the conservative head domain (Wanick and Spudich, 1987). Immunolocalization studies have shown that AMIC is associated with plasma membrane and membrane of the contractile vacuole (Baines and Kom, 1990). Dictyostelium myosin-I (most likely the mixture of DMIB and DMID) is also located at the leading edge of the lamella (Fig. lb) (Fukui et al., 1989). A recent study (Zot et al., 1992) demonstrated that isolated Acanthamoeba myosin-I bound to a pure phospholipid layer can support movement of F-actin at the rate of 0.2 pm/sec. They also showed that myosin-I supported movement on a planar membrane of high (5-40%), but not low (0-2%), phosphatidylserine. This suggests that the tail domain of myosin-I can recognize a biological membrane and might be targeted to a specific region of membrane in vivo by virtue of its unique sequence. Some of the myosin-1’s might support the projection of lamella (Fig. 4c). This idea is based on the above-mentioned localization of Dictyostelium myosin-1’s which have (a) the membrane-binding domain at the tail and (b) the second ATP-insensitive actin-binding domain near the head. Therefore, it is logically valid that (a)myosin-I-bound membrane moves relative to F-actin and/or (b)Factin slides relative to another F-actin. If actin filaments at the leading edge are uniformly oriented with their barbed ends bound to the membrane, the only direction of movement, relative to F-actin, would be toward the tip of pseudopods or lamella. If actin filaments are crosslinked in such a way that their movement

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relative to substratum is stabilized, the membrane could be propelled forward. Although this possibility has been speculated (Fukui et al., 1989; Pollard et al., 1991), its feasibility remains open for study (Hammer and Jung, 1991).

C. Cortical Flow The membrane flow model (Bretcher, 1984, 1988) is an extreme example of the theory that proclaims membrane recycling as a single mechanism for cell locomotion. The proponents of this model postulate that a continuous flow of plasma membrane from the front toward the rear, both on the dorsal and the ventral surface, propels the cell forward relative to the substratum. The appeal of this model is its simplicity, but it is not supported by some experimental data, which favor the cytoskeletal model discussed below. This latter model accounts for the attachment of plasma membrane to the underlying cytoskeleton. Studies by Jacobson and colleagues (Ishihara et al., 1988) allowed the direct measurement of phospholipid or membrane-bound glycoproteins. They analyzed the dynamics of a major murine membrane glycoprotein (GP80), labeled with rhodamine-conjugated antibody. The study showed that newly forming lamella are devoid of GP80. This result ruled out the insertion of membrane at the leading edges. A recent study further demonstrated that, in human polymorphonuclear (PMN) leukocytes, the fluorescent lipid analog moved forward with the same velocity as the cell movement (Lee e f al., 1990). This evidence does not agree with the above-mentioned continuous retrograde membrane flow model. The lipid flow model was also declined by Sheetz and colleagues (Sheetz et al., 1989). An analysis of the motion of Con-A-coated colloidal gold particles in mouse macrophages provided evidence against the lipid flow model and in favor of a model suggesting the participation of the actin cytoskeleton. Further evidence against the membrane flow model was provided when the force needed to move the gold particles was monitored using a single-beam optical gradient trap method (Kucik et al., 1991). This is a recently invented technique that allows manipulation of small objects by laser radiation pressure (the optical tweezers) (Ashkin and Dziedzic, 1987). It was found that the particles are preferentially attached to the cell surface at the leading edge of the lamella with a force of l o p 6 dyn (Table 111) and transported to the rear of the cell in a cytoskeleton-dependent manner. It seems that the actin-based cytoskeleton is the component responsible for the centripetal flow of the plasma membrane. There is also consistent flow of an actin-containing structure underlying the plasma membrane. At present, it is not conclusive whether actin filaments transport other components by the sliding mechanism or by their own centripetal movement. Leading edges of most cells, including pseudopods, lamella, and neuronal growth cone, consist of F-actin,

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and F-actin is believed to bind to the plasma membrane at the barbed or fast growing end (Small et al., 1978). This actin polarity predicts that the steadystate incorporation of actin subunits should predominantly occur at leading edges of lamella and there must be a continuous flow of actin from the leading edge toward the base of lamella. Wang (1985) demonstrated that the actin subunit moved at a constant rate of 0.79 pm/min relative to substratum in gerbil fibroma cells. The centripetal flow of actin subunits at the lamella occurs in Swiss 3T3 fibroblasts at rates of -0.26 pm/sec (Fisher et al., 1988). The rate of transport of surface beads was also measured to be -0.21 pm/sec, a value that correlates well with that of actin. Note that the above flow rate indicates the transport of vesicular bodies, whereas Wang’s rate showed the flow of actin subunits. Based on in vitro measurements, the flux rate of actin filaments is much slower than 0.11 pm/min (Pollard and Mooseker, 1981), and, therefore, whether the centripetal flow of actin is solely derived from “treadmilling” or from other mechanisms remains to be solved. Centripetal flow of myosin-I1 was also suggested in mouse 3T3 fibroblasts (McKenna et al., 1989). In this study, smooth muscle myosin labeled with tetramethylrhodamine iodoacetamide was microinjected and the fluorescent image was recorded every 2-5 min. Incorporated myosin exhibited filamentous rods, 0.73 pm long with 1.10 pm space, and was located along with stress fibers. The significance of this study was the observation of a continuous assembly of myosin into rods at the edge and subsequent centripetal flow of the rods. They suggested that this flow was primarily due to the dislocation of the rods rather than continuous assembly and disassembly. It is unlikely that this is a direct sliding movement over F-actin, presuming uniform polarity of F-actin with its barbed end pointing away from the edge (Small et al., 1978). Moreover, the average speed of this translocation, relative to the substratum, was much slower than that of actin (0.18 pm/min vs 0.79 pm/min). Therefore, it is difficult to reconcile this myosin movement with the centripetal flow of actin. Nevertheless, it might be possible that myosin moves toward the edge relative to F-actin at a higher speed and the observed dislocation simply represented the net translocation. Axonal transport provides another system for studying the dynamism of cytoskeleton. In this system, the term retrograde transport is used instead of centripetal flow. The growth cone exhibits dramatic dynamics in organization upon target recognition. Up to now, the growth cone from cultured Aplysia neurons has been studied most (Forscher and Smith, 1988; Smith, 1988). The authors demonstrated that the leading edge contains a dense F-actin meshwork and exhibits a ruffling movement. Ruffling of the membrane was exhibited as waves that were formed at the leading edge and then transmitted rearward at a rate of 3-6 pm/min (Forscher and Smith, 1988; Smith, 1988). With cytochalasin B as a probe, the dynamics of actin and microtubule cytoskeletons was explored

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using digital video microscopy. The study indicated that assembly of F-actin at the leading edge is essential for the generation of retrograde waves. A possible mechanism of dynamic actin recruitment during cell division has been studied by Cao and Wang (1990a). They observed the movement of actin in living cells by fluorescence cytochemistry. In normal rat kidney cells, only a small population of actin at the cleavage furrow fluoresced, indicating that de novo assembly takes place elsewhere. The organization of the contractile ring actin appeared to be brought about by movement of the preexisting filaments. They also demonstrated that injected actin moves toward the cortex during anaphase and telophase and is then transported toward the furrow by lateral movement (Cao and Wang, 1990b). The mechanism for this transport along the cortex has not been established. The recently observed axial cortical actin filament sheet encompassing the equator of the furrow region in Dictyostelium might be a candidate for the path of actin transport (Fukui, 1990; Fukui and InouC, 1991, 1992). Retrograde actin transport was also studied in rapidly moving epithelial keratocytes of goldfish using photoreactivation of a caged actin (Theriot and Mitchison, 1991). This technique allowed the measurement of the rates of actin transport relative to the substratum and relative to the cell margin, and the rate of filament turnover. The study demonstrated that the activated actin band moved rearward at a speed roughly equal to the speed of the cell, regardless of the cell speed (0.06-8.0 pm/min). It was also shown that, in the lamella, actin subunits were exchanged at a rate as rapid as t,,* = 23 sec. In bipolar “tethered” cells, however, they did not observe any centripetal movement. On the basis of these observations, the authors proposed a nucleation-release model featuring a rapid turnover of unit actin filaments with random orientation. The critical mechanism of the cortical flow of cytoskeletal components seems to be one of the most significant issues yet to be elucidated.

D. Signal Transduction Many signal transduction events relate significantly to cytoskeletal dynamics. For instance, chemotactic stimulation as well as growth stimulation causes transient changes in cell shape, cell motility, and/or cell division. The most visible milestone reached in the last decade in this area is the implication that organization of the actin cytoskeleton is probably regulated by the factors involved in the signal transduction cascade. Among the best-studied factors is profilin, a low-molecular weight G-actin-binding protein first purified by Lindberg and colleagues from calf spleen (MW 16,000) (Carlsson et al., 1977). In this study, they estimated that -50% of the actin is present as monomers, not as filaments. In nonmuscle cell cytoplasm, actin constitutes as much as 10% of the total

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protein or 200 pM concentration. In vitro, most actin can polymerize into filaments, whereas in the cytoplasm, as much as 100 pM actin is present as monomers. In a steady state, the critical concentration of actin polymerization at the barbed and pointed end is 0.1 and 0.5 pM,respectively. In other words, in vitro, the monomer concentration is in the range 0.1 to 0.5 pM. For details of actin polymerization, refer to reviews by Kom (1982), Pollard and Cooper (1986), Cooper (1991). For these reasons, it has long been suggested that profilin might play a significant role in regulating actin polymerization in the cytoplasm by binding to G-actin. Supporting this assumption is its high binding constant with actin (Kd = 2-10 pM) (Pollard and Cooper, 1986). In fact, profilin was purified as a 1:l complex with actin (profilactin) (Carlsson et al., 1977). A clue to the physiological function of profilin in regulating cytoskeletal dynamics was found by Lassing and Lindberg (1985). They found that profilin can interact directly with anionic phospholipids. This interaction concomitantly caused dissociation of profilin from G-actin and ultimately resulted in the polymerization of actin. This reaction was most prominent in interaction with phosphatidylinositol 4,5-bisphosphate (PIP,). It is known that PIP, serves as one of the key substances in the signal transduction pathway in which phospholipase C (PLC), activated with a receptor and G-protein, split PIP, into diacylglycerol (DAG) and 1,4,5-inositol triphosphate (IP,). The DAG stimulates protein kinase-C (PKC), whereas IP, acts to release calcium from cytoplasmic storage, and hence they are called second messengers (Nahorski, 1988; Forscher, 1989). A recent study by Goldschmidt-Clermont et al. (1991) indicated that most PIP, interacts with profilin at the molar ratio of 8:l in the resting state in vivo. This interaction appears to protect PIP, from being hydrolyzed by PLC. The receptor-ligand interaction results in the activation of PLC to a level that hydrolyzes some of the PIP, bound to profilin. The breakdown of one or two PIP, in the PIP,-profilin complex may drop the affinity of the rest of the PIP, to profilin and thereby progressively accelerate breakdown into DAG and IP,. Profilin, now free from PIP,, can interact with actin and cause disassembly of F-actin. Three signal transduction compounds have been identified in cellular slime molds: cAMP (Bonner, 197l), pteridines (Tillinghast and Newell, 1987), and the dipeptide glorin (Shimomura et af.,1982). Glorin is thought to be a slime mold counterpart of leukocyte stimulation factor met-Leu-Phe (Schiffmann et al., 1975). Among those, the cAMP cascade has been best studied, which includes G-proteins (Kumagai et al., 1989), phospholipids (Van Haastert et af., 1991), and Ca2+ (Newell et al., 1990). For reviews, refer to Gerisch (1982) and Devreotes (1989). Of particular interest are the downstream events that ultimately lead to actomyosin dynamics. Newell and colleagues identified a rapid association of actin

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with the Triton-insoluble cytoskeleton after stimulation with cAMP (McRobbie and Newell, 1983). This CAMP-induced interaction of actin with cytoskeleton occurred as rapidly as 5 sec, and this peak, as well as a second and a third peak, was postulated to correspond to the CAMP-elicited pseudopod formation and shape changes (Futrelle et al., 1982; McRobbie and Newell, 1984). Newell’s group reported that, in vivo, cAMP caused a rapid accumulation of IP, with a peak at 5 sec (Europe-Finner and Newell, 1987), and this was a result of the activation of membrane-associated PLC (Lundberg and Newell, 1990). Recent purification of two profilin isoforms (profilin I, 11) from Dictyostelium (Haugwitz et al., 1991) seems very important in studies of the downstream events of signal transduction in this system. The transient activation of actin-activatable Mg2+-ATPase of conventional myosin coupled with filament assembly has long been a subject of study (Spudich et d.,1981; Fukui and Yumura, 1986). It has been suggested that this activation occurs by reversible phosphorylation of the heavy chain (Kuczmarski and Spudich, 1980). and the regions responsible for the phosphorylation and filament assembly have been determined (Pastemak et al., 1989a). Transient phosphorylation of the light chain with cAMP and its role in stimulation of the Mg2+-ATPase was also demonstrated (Berlot et a[., 1985). It seems plausible that the dynamic changes in the myosin organization upon CAMP stimulation is under the control of the signal transduction pathway. Supporting this idea is the evidence for (a) relevance of the myosin organization to the CAMP-elicited increase in phosphorylation of the heavy chain (Berlot el a/., 1987) and (b)regulation of the association of myosin with the cytoskeleton through inhibition of the heavy chain phosphorylation by cyclic GMP (cGMP) (Liu and Newell, 1991). In Dictyostelium, the myosin assembly appears to follow that of actin as described in the preceding sections. Therefore, it is crucial to identify the factor that participates in the above-mentioned signal transduction pathway by regulating actin polymerization. In this regard, recent identification of the Src homology-3 (SH3) domain in the C-terminus of ABP,, in S. cerevisiae, which is closely related to the ATP-insensitive actin-binding domain of Dictyostelium myosin-I, is provocative (Drubin et al., 1990; Koch et al., 1991). Another signal transduction domain, SH-2, has been identified in the actin-binding protein tensin, which appears to participate in anchoring actin filaments to the focal contacts in chicken fibroblasts (Davis et al., 1991).

IV. Cytoskeletal Components Dictyostelium discoideum is one of the best-studied nonmuscle systems in which actin and myosin have been characterized (Clarke and Spudich, 1974), major

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actin-binding proteins have been isolated (Table I), and genetic engineering has been made possible (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987). This is also the system that allows a high-resolution visualization of cytoskeletal structures in situ (Fukui et al., 1986, 1987; Fukui and Inout, 1991).

TABLE I Cytoskeletal Proteins and Genes of DictyosteliunP Actom yosin system [Actin] LABPI

Crosslinking

Capping

Membrane-associating

Side binding Monomer-binding [Myosins]

Component Actin (P) (G) ABP-120 (P) (G) a-Actinin (P)

(G) p30a (P) (G) p30b (p) ABP-240 (P, I) ABP-50 (P) (G) Severin (P) (G) Cap 32/34 (P) (G) p24 (P) (G) Ponticulin (P) Hisactophilin (P, G) ABP-220 (P, I) profilin-I, I1 (P, G) myosin-I1 (P) mhcA (G) EMLC ( G ) myosin-I (P) ahmA ( G ) DMIB ( G ) DMlD (G)

[Microtubule system]

[IF system]

a , P-tubulins (I) kinesin (P, I) dynein-like (I) not yet identified

Reference Woolley ( 1972) Romans and Firtel( 1985) Condeelis e t a / . (1981) Noegel er al. (1989) Condeelis and Vahey (1982) Fechheimer er a/. (1982) Noegel era/. (1987) Fechheimer and Taylor (1984) Fechheimer er a/. (1991) Brown (1985) Hock and Condeelis (1987) Demma et a/. ( 1990) Yang ef a/. (1990) Brown e t a / . (1982) Andrd et a/. (1988) Schleicher ef a/. (1984) Hartmann et al. (1989) Stratford and Brown (1985) Noegel e t a / . (1990) Wuestehube and Luna (1987) Scheel e t a / . (1989) Bennett and Condeelis (1988) Haugwitz era/. (1991) Clarke and Spudich (1974) De Lozanne et a/. (1985) Pollenz and Chisholm (1991) CBtd ef a/. (1985) Titus e r a / . (1989) Jung er a/. (1989) Jung e r a / . (1992) White ef a/. (1983) McCaffrey and Vale (1989) Koonce and McIntosh (1990)

T h e list is arranged chronologically. (P), (G),and (I) indicate protein, gene, or immunological identifications, respectively.

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A. Actomyosin System 1. Actin An actin-like protein was first purified from a wild-type Nc4 strain by Woolley (1972). The cellular actin concentration was estimated as 200 pM (-10 mg/ml) with MW 42,000 in an axenic strain Ax3 at growth phase (Spudich, 1974; Spudich and Cooke, 1975). Spudich (1974) reported that the vegetative cells have only a single species of actin, which comigrates with skeletal muscle actin in gel electrophoresis and shares most common properties with muscle actin. At the genetic level, the Dictyostelium actin gene represents a multigene family consisting of 17-20 genes of which at least 15 are expressed (McKeown et al., 1978; Romans and Firtel, 1985). The sequence analysis indicated that Dictyostelium actin genes are more similar to those of mammalian cytoplasmic actins than to muscle actins. Romans and Firtel (1985) suggested that the high multiplicity in Dicfyostelium actin might be subject to a “narrow range” regulation, rather than to different major functions. A recent biochemical study showed that, in vegetative Ax3 cells, the concentration of total cellular actin is 93 p M , and roughly half of that is polymerized in the cytoplasm with an average length of 0.2 pm or about 76 subunits per filament (Podolski and Stech, 1990). They also demonstrated that there are three distinctive classes in the length of the polymer, very likely representing different populations in the cytoplasm.

2. Actin-Binding Proteins a. Crosslinking Proteins

a-Actinin is the most conservative actin-binding protein and is found in a wide variety of muscle and nonmuscle cells. Dictyosrelium a-actinin comprises the 95-kDa gelation factor isolated from an actin-enriched fraction (Condeelis and Taylor, 1977). It was later purified and the native protein was shown to be a rod-shaped, homodimer, 38-40 nm long. a-Actinin crosslinks F-actin to form bundles. The optimal actin crosslinking activity occurs at a pH range between 6.8 and 7.0 and in the presence of submicromolar Ca2+ (Condeelis and Vahey, 1982; Fechheimer et al., 1982). Its cytoplasmic concentration and the molar ratio of the dimer to actin were estimated as 1.2% and 1:30 (Brier et a[., 1983). Dicfyostelium has a single aactinin gene, and its sequence showed a high homology to chicken nonmuscle a-actinin with the typical calcium-binding loop of the calmodulin superfamily as well as a putative actin-binding N-terminal domain (Noegel et al., 1987). ABP-I20 was originally identified as a gelation factor that exhibited a rodshaped homodimer with M , 120 kDa in SDS-gel electrophoresis (Condeelis et ai., 1981). In vitro, it increases the viscosity of actin solution, inhibits myosin

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Mg2+-ATPase,and induces formation of side-to-side and end-to-side interactions between F-actin (Condeelis et af.,1982). This protein shows no Ca2+ sensitivity and seems to be a network-forming protein rather than a bundling protein. Complete cDNA sequencing (Noegel et al., 1989) showed that this protein has 857 amino acids providing a calculated M, 92.2 kDa, and it has a high homology to Dictyostelium and chicken nonmuscle a-actinins and also to human dystrophin, a 400-kDa gene product of human Duchenne muscular dystrophy locus, and demonstrated to be located at the triad junctions in skeletal muscle (Hoffman et al., 1987a,b). This homology among different proteins most likely represents the actin-binding domain shared by these actin-binding proteins. A 30-kDa actin bundling protein was purified from low-salt extract of Ax3 (Fechheimer and Taylor, 1984; Fechheimer, 1987). The cytoplasmic concentration was estimated to be 0.04% of the total protein, and it binds to F-actin at a molar ratio of 1:lO in vitro. The association of the 30-kDa protein with actin was inhibited by either Ca2+ or Mg2+. However, the calcium sensitivity was over a hundred times greater than that of magnesium. Equilibrium sedimentation showed that it is an elongated protein with a native MW of 31,700 and Stokes radius of 3 nm (Fechheimer and Taylor, 1984). A recent study demonstrated that Dictyostelium has a single gene encoding this protein, and the cDNA sequence indicates that it consists of 295 amino acids with a predicted MW of 33,355 (Fechheimer et al., 1991). In fact, its molecular weight has been claimed to be 34,000 by Johns et al. (1988). The latter group also demonstrated that rat kidney fibroblast contains an immunologically identical protein. A recent study by Fechheimer et al. (1991) indicated that this protein shares an actin-binding and Ca2+-bindingsequences with ABP- 120 and a-actinin, respectively, and also has a homologous domain with human cytovillin. Cytovillin is a 75-kDa microvillus membrane protein isolated from cultured human cells (Pakkanes and Vaheri, 1991). This calcium-sensitive 30-kDa protein of Dictyostelium was named p30a (Brown, 1985). Another actin-bundling protein (p30b) was isolated from Dictyostelium, which was similar to p30a in electrophoretic mobility, Stokes radius (3.5 nm), and ability to bundle F-actin (Brown, 1985). It showed a maximum actin crosslinking activity at a stoichiometric ratio of 1:30 as shown by low shear viscometry. This protein appears different from p30a based on peptide mapping and lack of immunological crossreactivity and calcium sensitivity in interacting with F-actin. Two high-molecular-weight actin crosslinking proteins have been purified from Dictyostelium. ABP-240 is an asymmetric dimer with a native M, 434 kDa and 142 nm long, which is similar to chicken gizzard filamin (Hock and Condeelis, 1987). It increased the low shear viscosity of F-actin and showed immunological crossreactivity with filamin. The second high-molecular-weight protein (ABP220) showed an actin side-binding, rather than crosslinking, property.

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ABP-50 is a recently identified actin-binding protein with M, 50 kDa (Demma et al., 1990). This protein exhibits a high affinity binding to actin (Kd 2.1 pM) at a molar ratio of 1 5 , with or without calcium, and induces the formation of F-actin bundles in vitro. The same laboratory showed that ABP-50 is similar to a mitotic apparatus-associated 5 1-kDa protein of sea urchin eggs, which was shown to be identical to yeast elongation factor-la (EF-la) (Ohta et al., 1990). The characterization of the ABP-50 gene has been performed based on a complementary sequence analysis of two cDNA clones (Yang et al., 1990).

b. Capping Proteins The 40-kDa protein severin is an actin-severing monomeric protein with a Stokes radius of 2.9 nm (Brown et al., 1982; Yamamoto et al., 1982). It provoked a loss of sedimentability of F-actin in the presence of 0.1 mM Ca2+.The severing effect was exhibited in a stoichiometric fashion and the maximum effect was observed at a molar ratio to actin higher than 1:10. Severin represents a class of capping proteins that binds to the barbed end of F-actin (Brown et al., 1982). This property most closely resembles that of fragmin, a Ca2+-sensitive ABP comigrating with actin from Physarum polycephalum (Hasegawa et al., 1980). The sequencing study demonstrated that severin is highly homologous to villin and gelsolin from vertebrates (Andr6 er al., 1988). The homologous region probably serves as the actin-severing domain, and gelsolin, whose M, is about 90 kDa, was possibly evolved by tandem duplication from a severin-like ancestor (Andre er al., 1988). A barbed-end-binding, Ca2+-insensitive capping protein has been purified from the membrane-rich fraction of Ax2 cells (Schleicher et al., 1984). This protein (Cap 32/34) is a 1:l complex of two subunits with apparent M, 32 and 34 kDa, and the native protein has a Stokes radius of 3.5 nm with M, 65 kDa. The purified Cap 32/34 inhibited actin polymerization in a Ca2+-independent manner and bound to the barbed end (Schleicher et al., 1984). It has been verified that the 32 and 34-kDa subunits are encoded by a single, distinctive gene and there is no homology between these peptides and severin, fragmin, gelsolin, or villin (Hartmann et al., 1989). c. Membrane-Binding Proteins A protein ( M , 24 kDa) that binds both Gand F-actin has been purified (p24) (Stratford and Brown, 1985). This protein was isolated as a member of the membrane-associating proteins. They found that the interaction of p24 to actin is specific with Kd 1.8-3.5 X M. Although the predicted amino acid sequence of p24 has no obvious transmernbrane domains (Noegel et al., 1990), its C-terminus region appears to share homology to Octopus rhodopsin and vertebrate synaptophysin, a major integral membrane protein of synaptic vesicles (Sudhof et al., 1987). Ponticulin (ponticulus = small bridge) is a glycoprotein of M, 17 kDa purified from membrane cytoskeleton of Ax3 (Wuestehube and Luna, 1987). This is the major membrane protein, constituting 0.4-1 .O% of the total membrane protein,

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and is responsible for most of the actin-membrane binding. It is suggested that this protein mediates a lateral association of F-actin with membrane and probably initiates nucleation of an actin triplet (Shariff and Luna, 1990). Hisactophilin (M,17 kDa) is another membrane-associating protein, which appears to be different from ponticulin (Scheel et al., 1989). This protein binds to actin at a molar ratio of 1.4:1, and stimulates its polymerization. This interaction is insensitive to Ca2+ or Mg2+ and is effective only within a pH range 6.5-7.5. The predicted sequence does not indicate the presence of a transmembrane domain but demonstrates an unusually high content of histidine (3 1 residues of 118 amino acids).

d. Actin-Side-Binding Proteins The second high-molecular-weight actin binding protein (ABP-220) was identified as a peptide that crossreacted with an antibody against chicken brain fodrin (Bennett and Condeelis, 1988). The isolated ABP-220 did not show actin crosslinking activity. However, rotary shadow electron microscopy revealed that this protein binds to the side of Factin in vitro. Like spectrin, native ABP-220 was suggested to be a dimer of M, 500 kDa and a 118-nm-long rod-shaped protein with Stokes radius of 13 nm. e. Monomer-Binding Proteins Two profilin isomers (I, 11) have been purified. These profilins have M, approximately 13.1 kDa (PI 5.2) and 12.8 kDa (PI 6.8), and single genes, 0.7 and 0.6 kb, respectively, have been identified (Haugwitz et al., 1991). Deduced amino acid sequences indicated the presence of putative actin-binding domains, and the purified profilins actually caused a delay in elongation of F-actin. The affinities of profilin I and I1 with actin were estimated as 1.8 X l o p 6 and 5.1 X 10V6 M ,respectively. 3. Myosins a. Conventional Myosin (Myosin-ZZ) Dicfyostelium myosin-I1 contains two heavy chains of 210 kDa, and two light chains of 16 and 18 kDa (Clarke and Spudich, 1974). It forms bipolar thick filaments in v i m (Clarke and Spudich, 1974) and in vivo (Yumura and Fukui, 1985), and its Mg2+-ATPaseactivity is enhanced with actin. A single cDNA encoding heavy chain of myosin-I1 (DDIMYHC) was cloned (De Lozanne et al., 1985) and its complete amino acid sequence, consisting of 21 16 residues, has been determined (Warrick e f al., 1986). A most conspicuous property of this myosin relevant to its dynamics is its ability of reversible assembly into bipolar filaments, which is regulated by heavy chain phosphorylation (Kuczmarski and Spudich, 1980; Cat6 and Bukiejko, 1987). The domains necessary for filament assembly have been determined to be the 20-nm-long tail, extending 90 to 110 nm from the head-tail junction (Pastemak et al., 1989a). Refer to Warrick and Spudich (1987) for a review. An

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essential light chain (EMLC) gene has been shown to have only 28% homology with human and chicken nonmuscle sequences (Pollenz and Chisholm, 1991).

b. Small Myosins (Myosin-I’s) Dictyostelium myosin-I was first isolated by C6te et al. (1985). This myosin has a native M , 150 kDa and consists of a single heavy chain of 117 kDa. The light chains have not been purified yet. Dictyostelium appears to have at least five myosin4 genes, and four of them have been characterized (Jung et al., 1989; MIB; Titus et al., 1989, MIA, MIE; and Jung and Hammer, 1992, MID). The most conspicuous feature of this class of myosin is the lack of a tail domain necessary for assembly into filaments (Kom and Hammer, 1988; Hammer, 1991). This myosin exhibits high ATPase activity in the presence of K+ and EDTA, which is essentially negative in the conventional myosin. See Section 111 for more details.

6. Microtubule System Tubulin has not been isolated from Dictyostelium. It was characterized by White et al. (1983), who identified a- and P-tubulins by two-dimensional gel electrophoresis probed by a polyclonal antibody against sea urchin a-and P-tubulins, and monoclonal antibodies against yeast a-tubulin. Peptide mapping indicated that Dictyostelium tubulins (M, 55 kDa) were more basic (PI 6.2-6.7) compared to the brain tubulins (PI 5.7-6.0). Dictyostelium a-tubulin was also identified with a monoclonal anti-chick brain a-tubulin (Kitanishi-Yumura et al., 1985). It has been shown that this tubulin resists being depolymerized with calcium or cold temperature and is largely insensitive to colchicine (White et al., 1983; Kitanishi-Yumura et al., 1985). Ethyl-N-phenylcarbamate and thiabendazole have been shown to disrupt Dictyostelium microtubules effectively (Kitanishi-Yumura et al., 1985). Difficulty in purification of Dictyostelium tubulin appears to be primarily due to low concentration (0.5-0.05% of cellular protein) (White et al., 1983). A microtubule-based, anterograde motor protein, kinesin-like protein, has been partially purified from Ax3 (McCaffrey and Vale, 1989). This protein was composed of a single 105-kDa polypeptide (9s) and showed a microtubuleactivated ATPase activity. This protein also induced fast microtubule movement in vitro (2 pm/sec). This velocity is nearly fourfold faster than that generated by kinesins from other systems, but compatible with that generated by Acanthamoeba kinesin. Dictyostelium kinesin did not crossreact with antibodies against squid or bovine brain kinesins, and most significantly, did not exhibit nonhydrolyzable rigor-like binding to adenylyl imidodiphosphate (AMP-PNP), which had been considered a general property of all kinesins. A cytoplasmic dynein-like protein has been partially purified (Koonce and McIntosh, 1990). The native protein of 20s showed ATP-sensitive binding to

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microtubules and UV-vanadate-sensitive photocleavage upon CTPase activity, typical of dyneins. It exhibited only infrequent and slow (<1 pm/sec) microtubule motility in the assay using bovine brain microtubules. The dynein-like protein with a diameter of 11 nm was also suggested to associate with the isolated microtubule organizing center complex (MTOC) (Omura and Fukui, 1985). Purification and characterization of this system are awaiting future studies.

C. Other Systems Other cytoskeletal components including intermediate filament proteins have not been identified yet.

V. Dynamic Changes of Dictyostelium Cytoskeleton A. Actomyosin System 1. Chemotaxis McRobbie and Newel1 (1983, 1984) first identified changes of actin associated with Triton-insoluble cytoskeleton after stimulation with cAMP or folk acid. Rapid polymerization of actin and association with the cytoskeleton occurred within 3 sec and were followed by depolymerization. This depolymerization is probably correlated with “cringing” (Futrelle et al., 1982). Following this event is a second actin polymerization occurring between 45 and 80 sec. The second polymerization was suggested to be correlated with pseudopod extension (Condeelis et al., 1988). Rapid and reversible changes in organization were also identified in myosinI1 (Yumura and Fukui, 1985). They found that the myosin filaments disappeared from the endoplasm and accumulated at the cortex within a few seconds, and the original distribution was spontaneously recovered in 2-3 min at low temperatures. This transition was so rapid that they could not follow the sequential changes at the room temperature in the wild-type Nc4 strain. This chemotacticelicited translocation of myosin was verified to be correlated to the CAMPinduced rounding-up of the cell body (Nachmias et al., 1989). The immunofluorescence localization of ABP- 120 showed that this protein was incorporated into newly forming pseudopods (Condeelis et al., 1988). Similar to actin, ABP- 120 associates with the Triton-insoluble cytoskeleton within 30-40 sec following stimulation with cAMP (Dharmawardhane et al., 1989). A mutant that has no detectable ABP-120 was found to be able to form pseudopods in a manner similar to that of the wild type (Brink et al., 1990). A recent study using a motion analysis system for targeted gene disruption mu-

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tants of ABP-120 demonstrated that those mutants are rounder than the wild type and exhibit a slow rate of pseudopod extension (Cox et al., 1992). Severin has been found throughout the nascent pseudopod (Brock and Pardee, 1988). In the fully developed pseudopod, however, severin appeared to be concentrated in the hyaline cap. It was also localized in the cytoplasm surrounding phagocytic vesicles. It was suggested that a role of this protein is dynamic disruption of the actin network during pseudopod extension and phagocytosis. Preferential incorporation of p30a into filopods has been demonstrated (Fechheimer, 1987). In vegetative cells, p30a was shown to accumulate into folic acid-induced filopods within 5 min (Johns et al., 1988). The localization of this protein is consistent with its in vitro characteristic of inducing formation of actin bundles, and appears to participate in the dynamic formation of filopodal projections in response to physiological needs. ABP-50 has been demonstrated to exhibit dynamic transcompartmentalization after stimulation with CAMP (Dharmawardhane et al., 1991). With peak at 90 sec, ABP-50 was incorporated into filopods, and at the same time, the amount of ABP-50 associated with the Triton-insoluble fraction increased. Ponticulin has been found over the entire submembraneous regions of plasma as well as cytoplasmic vesicular membranes, but there were no specific accumulations indicated (Wuestehube et al., 1989). 2. Cell Division

The dynamic organization of Dictyostelium cytoskeleton has been studied for actin and myosin by immunofluorescence during cell division (KitanishiYumura and Fukui, 1989). They observed that both actin and myosin-I1 showed a cell cycle-dependent recruitment during prophase and telophase. In prophase, actin and myosin-I1 were dissociated from the cortex, and they returned to the cortex in anaphase. Whereas actin redistributed uniformly into the cortex, myosin-I1 relocalized specifically at the anticipated cleavage furrow. The filamentous nature of actin and myosin-I1 at the contractile ring has been demonstrated (Yumura and Fukui, 1985). This dynamism has been recently validated in living cells by video microscopy assisted by digital image enhancement (Fukui, 1990; Fukui and Inoue, 1991, 1992). This study revealed the occurrence of ( a ) a new F-actin array, parallel to the polar axis and perpendicular to the contractile ring and ( b )a gradual transformation of the contractile ring into a posterior cortical network in daughter cells. This evidence indicated that the contractile ring does not disassemble at the end of cell division, but is recruited to the daughter cells. Myosin-I also appears to exhibit a dynamic translocation, as shown by the immunolocalization of cells in division (Fukui et al., 1989). a-Actinin has been localized at the pseudopod of polarized cells (Brier et al., 1983). It is also located at a cortical a-actinin network (Y. Fukui and

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M. Schleicher: Fig. Id). During cell division, a-actinin exhibits an interesting dynamics of localization (Fig. 2d). It is still associated with F-actin at the polar lamella, but no longer localized at the contractile ring. The absence of aactinin in the contractile ring is not consistent with localization reported for other systems (Fujiwara et al., 1978; Hamaguchi and Mabuchi, 1986; Sanger et al., 1987). The above observation implies the presence of a cell cycledependent regulatory mechanism for interaction between different cytoskeletal components.

3. Capping Actin, myosin-11, a-actinin, and ABP- 120 have been shown to exhibit dynamic organizational changes during capping of surface receptors (Carboni and Condeelis, 1985). Although all of those components accumulate into patches, only ABP-120 is removed from the cap during the capping process. This observation supports the above-mentioned implication of the presence of a regulatory mechanism of type-specific association of those components with unique actin-based cytoskeletal domains. By virtue of the mutant that does not synthesize normal myosin-I1 (De Lozanne and Spudich, 1987), the role of actomyosin-I1 for capping, but not patching, has been directly demonstrated (Fukui et al., 1990).

4. Substrate Exploration Cell behavior is the integration of all the locomotive events occurring over a certain period of time, triggered by either endogenous or exogenous stimulations. Actin and myosin-I1 have been shown to exhibit dynamic changes in organization concomitant with substrate exploration (Fukui et al., 1991). The amoeba stopped locomotion upon contact with the smooth-etched substrate interface and exhibited lateral scanning motion of their filopods. They behaved as though they were equipped with a mechanosensory device and preferred to stay on the same substrate area. This behavior was described as a decision-making, learning process, and this experience appeared to be imprinted into the cytoskeleton as memory. During surveillance of the substrate, actin was localized at the pseudopod exploring the substrate, and myosin-I1 showed a unique localization different from that of actin. This study also revealed a lattice-like organization of myosin-I1 filaments, which probably was transient (Fukui et al., 1991).

5. Morphogenesis The dynamics of the cytoskeleton during the developmental stage has only rarely been studied because of technical reasons. Using the agar overlay method, Yumura et al. (1984) observed distinctive localizations of actin and myosin41 in aggregation streams. Actin showed an intensive accumulation at the pointed tip, whereas myosin-I1 exhibited lateral and posterior localizations.

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In spirally aggregating cells, they observed that myosin-I1 showed an extensive staining at the outermost cortex, suggesting that it may be responsible for generation of the centripetal force. Recent study by Eliott et a / . (1991) demonstrated the differentiation of an epithelium-like layer at the periphery of slugs. Myosin-I1 was also found to be localized extensively at the lateral and posterior cortex, exhibiting U- or C-shaped patterns. The developmental defect of myosin-I1 mutants (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987), indeed, suggests a role of this myosin in establishing a mechanical basis for cellular polarity during development.

6 . Microtubule System 1. Locomotion An interphase cell has about 30 microtubules, as demonstrated by immunofluorescence (Fukui el al., 1986, 1987). In these cells, the microtubules exhibit a network emerging from a MTOC which is usually located in front of the nucleus (Yumura and Fukui, 1983). This position of the MTOC is obviously a natural consequence of cell division that is endowed with a precise bipolarity. The transition of microtubules from interphase to mitotic cycle has been recently studied by high-resolution polarization microscopy (Fukui, 1990; Fukui and Inoue, 1991, 1992).

2. Cell Division The dynamics of the spindle microtubules was first studied by electron microscopy (Moens, 1976). At metaphase, the spindle is 2 pm long and it elongates to 10 pm in anaphase. Morphological changes observed in overlapping domain of the pole-pole microtubules suggest the occurrence of a sliding mechanism responsible for spindle elongation (Moens, 1976; McIntosh et al., 1985; Kitanishi-Yumura and Fukui, 1987). The temporal separation of the spindle elongation (anaphase-B) from the chromosome segregation (anaphase-A) has been suggested (Roos and Camenzind, 1981; McIntosh et al., 1985; KitanishiYumura and Fukui, 1987). In living cells, however, anaphase-A was not resolved by high-fidelity video microscopy (Fukui and InouC, 1991, 1992).

3. Capping It has been shown that 10V5 M tubulozole inhibits capping of Con-A receptors (De Priester et al., 1990). This drug brought about the shortening of the peripheral part of microtubules and concomitant disorganization of F-actin as demonstrated by fluorescence microscopy. Real time resolution of the microtubule dynamics corresponding to this process has not been reported. (See Table I1 for a summary of the cytoskeletal dynamics of Dictyoste/ium.)

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TABLE II Cytoskeletal Dynamics of Dictyosteliurn Component Actin

Activity Chemotaxis

Cell division

Myosin-I Myosin-I1

Capping Substrate exploration Locomotion Cell division Chemotaxis Cell division

Severin p30a

Capping Substrate exploration Slug migration Capping Cell division Capping Chemotaxis Pseudopod Filopod

ABP-50 Microtubules

Chemotaxis Locomotion

a-actinin ABP- 120

Cell division

Capping

Reference McRobbie and Newell (1983) Yumura and Fukui (1985) Condeelis et a/. (1988) Kitanishi-Yumura and Fukui (1989) Fukui and InouC (1991) Carboni and Condeelis ( I 985) Fukui era/. (1991) Fukui e t a / . (1989) Fukui e t a / . (1989) Yumura and Fukui (1985) Nachmias et al. (1989) Kitanishi-Yumura and Fukui (1989) Fukui and InouC (1991) Carboni and Condeelis (1985) Fukui et al. (1991) Eliott et a / . (1991) Carboni and Condeelis (1985) Y. Fukui and M. Schleicher (unpublished) Carboni and Condeelis (1985) Dharmawardhane et al. (1989) Brock and Pardee (1988) Fechheimer (1987) Johns er al. (1988) Dharmawardhane et al. (1991) Yumura and Fukui (1983) Fukui et a / . (1986, 1987) Moens ( 1976) Roos and Camenzind ( 1981 ) McIntosh et a / . ( 1985) Fukui and InouC (1991) De Priester er a/. (1990)

VI. Critical Evaluation of the Mechanism A. Biomechanics of Cell Motility The cortical tension of a dividing sea urchin egg was measured as the stiffness to resist suction with a pipet (Mitchison and Swam, 1955). The value oscillated in the range 5-60 X 10V8 dyn/pm */pm deformation after correction for a 100pm diameter egg sucked with a 50 pm pipet. The average tension for the

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isometric contraction of the cleavage furrow was estimated in the order of 2.5 X dyn in cleaving echinoderm eggs (Rappaport, 1967). In this study, flexible glass needles were inserted through poles, and the tension was calibrated by measuring the movement of the needles by constriction of the furrow. The cyclic changes in the tension were studied in echinoderm eggs for a period of 60 min with the compression method (Yoneda and Dan, 1972). They showed that the maximum tension of equatorial constriction was about 6 X l o p 3 dyn, a value consistent with that of Rappaport. The cortical tension was also measured in sea urchin eggs as the viscoelastic force to resist compression in the range 2-4 X l o p 6 dyn/pm compression (Hiramoto, 1976). Balance pressure, regarded as an absolute value equal to the motive force responsible for the cytoplasmic streaming of P.polycephalum, was measured to be as much as 20 cm of water (2 X dyn/pm2) (Kamiya, 1940). Kamiya's double chamber method also allowed him to measure the more delicate force generated by a single Chaos chaos amoeba, giving rise to less than 15 mm of water (1.5 X l o p s dyn/pm2) (Kamiya, 1964). Actual force derived from the cortical tension of a single neutrophil has been measured by indenting the cell contour with a flexible glass needle of known bending constant (Worthen et al., 1989). The value was between 0.05 and 0.23 x l o p 3 dyn/pm indentation for the resting or stimulated cells, respectively. Forces necessary to resist motion of fibroblastic lammela and Dicryostelium cortex have been measured using the same technique, providing the values 1.5-3.0 X l o p s dyn/pm displacement and 4.1 X dyn/pm indentation, respectively (Felder and Elson, 1990; Pastemak et al., 1989b). Although these measured values vary depending on cell types, activities, and method, it is clear that the cell does generate an extreme amount of force. For instance, the measured cortical tension of a single Dictyostelium cell is so great that it can lift 10,OOO entire cells based on the following estimation:

(a) Assume a spherical Dicryostelium cell with a 5-pm diameter. The cellular volume (V,) = 6.6 X cm3. (6) Assume that buoyant density ( d ) = 1.07 g/cm3 (Fukui, 1976); gravity (g) = 981 (g X cm X secp2). Force necessary to lift a single cell to its diameter (F,) = V, X d X g = 7 X lo-* dyn. (c) Assume that the cell generates cortical tension (T,) = 7 X l o p 4 dyn. The number of cells that can be lifted by the measured cortical tension ( N , ) = T,/F, = 10". Similarly, Albrecht-Buehler ( 1 990) estimated that the contractile force of a single sarcomere (-6 X l o p 6 dyn) can lift 60 entire myofibers. Evidently, the cellular mass or weight is so small that the inertia and friction are negligible compared to the chemical energy inherent in the cell.

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6. Biomechanics of Motor Proteins The realistic nature of the cytoskeletal dynamism, how the components move, interact, assemble, and create meaningful force, is largely unknown. According to a recent in vitro measurement, an interaction of a single myosin head with F-actin generates about one piconewton (pN) force per one ATP hydrolysis (Ishijima el al., 1991). Force was calibrated on the basis of the displacement of a glass microneedle attached to a single F-actin. The F-actin was pulled by interaction with a small number (<5-150) of myosin molecules, and the force was resolved at less than a piconewton with a time resolution of sub-milliseconds. This study also indicated that at velocities greater than 1 pm/sec, myosin appears to manifest multiple-step interactions with actin per ATP hydrolysis as demonstrated by the disappearance of the force fluctuations. The evidence led to the suggestion that the myosin head might be able to propagate the free energy liberated by hydrolysis of a single ATP to multiple working strokes. The proportion of the duration of the power stroke to the total ATP hydrolysis cycle was quantized by Uyeda et al. (1991) as the duty ratio, being 0.05. In this study, the unitary velocity of F-actin moving over a single heavy meromyosin was measured in a solution consisting of 1.8% methylcellulose, which provided an environment where a short F-actin did not rapidly dissociate from myosin by Brownian motion. Multiplication of the unitary velocity by the duty ratio gave rise to a myosin step size of about 10 nm for a single ATP hydrolysis which is very consistent with Huxley's ( 1969) swinging cross-bridge model. For simplicity, let us apply the 1 pN force (Ishijima et al., 1991) to estimate the total cellular force. The cellular protein is 10% (w/v), equivalent to 100 mg/ml. Dictyostelium has about 0.5% myosin-I1 per total cellular protein (Clarke and Spudich, 1974). The myosin concentration is about 1% ( I mg/ml), crediting a 50% loss during purification. We already estimated the cellular volume (V,) as 6.6 X 10- I 1 cm3, or 66 pm3. The cellular weight W, = V, X d = 7.1 X lo-" (g), which contains 7.1 x (g) of myosin. This corresponds to about lo5 myosin molecules per cell, taking the Avogadro number of 6 X and the native M, 500 kDa. The native myosin-I1 has two heavy chains, therefore, a single Dictyostelium cell contains about 2 X lo5 myosin heads. If 10% of the myosin heads actively interact with F-actin and generate the force, the total cellular myosin should generate as much as 2 X 10W3 dyn (FM).This force is about three times greater than the measured cortical tension (T,) or strong enough to lift about 30,000 cells up to 5 prn. The force potentially developed by the treadmilling of actin polymerization has been estimated as 1-6 x dyn for a single addition of subunit (Albrecht-Buehler, 1990), or 30 pN (3 X dyn) for the barbed-end addition on a bundle of 10 actin filaments in a typical microvillus (Cooper, 1991). The force of F-actin network to resist stress has been measured to be as much

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as 14 dyn/cm2 for a gel made of 24 pA4 actin (Sato et al., 1985). All these estimations are summarized in Table 111.

C. Biased Friction? As estimated above, the measured cortical tension of Dictyostelium amoeba is only a fraction of the sum of the force potentially generated by cellular myosins. In responding to a chemotactic stimulation, the cell’s cytoskeleton exhibits an overall reorganization in an extremely dynamic fashion (Section V). Actin filament networks are collapsed and reassembled into a new organization in less than 30 sec. Myosin filaments, each of which includes hundreds of subunits, are also collapsed. Two hundred thousand myosins are now vulnerable to the physical world and exposed to a violent thermal environment. They rotate at frequencies of 106/sec and collide with each other and with other molecules about a million times per second (Alberts et al., 1989). Although the diffusion of small molecules such as ATP in cytoplasm is as rapid as 100 pmlsec (Alberts e f al., 1989), the movement of macromolecules such as myosin must be much slower because of its intrinsic diffusion constant and collision with other components. The molecular folding of myosin with phosphorylation of the heavy

TABLE 111 Biornechanical Forces System Echinoderm egg

Contractile ring Physarum plasmodium Chaos amoeba Actin gel Neutrophil Dictyostelium amoeba Fibroblast Sarcomere Actin subunit Actin bundle Membrane protein Myosin head Dicryosielium myosin

Forcea

Reference

5-60 X dyn/pm2/pm 6 X lOW3dyn/egg 2-4 X dyn/egg 1.5 X 10-3dyn/egg 2 X 10-4dyn/pm2 1.5 X dyn/pm2 1.4 X lo-’ dyn/pm2 0.5-2.3 X 10Wsdyn/pm 4.1 X 10-4dyn/pm 1.5 X 10-sdyn/pm 6 X 10W6dyn/unit 1-6 X 10-6dyn/unit 6 X 10-6dyn/bundle 3 X dynbundle dynlparticle lo-’ dynhead 10 - dynlcell

Mitchison and Swann (1955) Yoneda and Dan ( 1972) Hiramoto (1976) Rappaport ( 1967) Kamiya (1940) Kamiya (1964) Sat0 ei al. (1985) Worthen et a/. ( 1989) Pasternak et a/. ( I 989b) Felder and Elson (1990) Albrecht-Buehler (1990) Albrecht-Buehler (1990) Albrecht-Buehler (1990) Cooper ( I99 I ) Kucik et a/. (1991) Ishijima et al. (1991) This chapter

9 o m e of the original values were measured or calculated in different units and computed into dynes per micrometer.

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chain (Kuczmarski et al., 1987) is obviously beneficial for faster movement. In reality, most myosin molecules favor accumulating into a specific site, rather than diffusing randomly, and assemble into filaments at the posterior cortex (Fukui and Yumura, 1986). The filament assembly appears to be a consequence of conformational changes with dephosphorylation (Fukui and Yumura, 1986) and lateral association (Pasternak et al., 1989a). The directional translocation might be mediated by either a linear translocation along F-actin or a stepwise transient association with the actin network. If all the F-actin filaments bind to the plasma membrane at the barbed end (Small et al., 1978), the only possible movement of myosin is anterograde. Although no evidence for the former mechanism is available, the latter mechanism has been postulated by Pardee and colleagues (Mahajan et al., 1989). By fluorescence energy transfer and light-scattering assembly assays, they demonstrated that the self-assembly into filaments was accelerated by association with F-actin, and this process did not require ATP hydrolysis. In this study, for technical reasons, dilute protein solutions (1.3 pM actin and 0.05 pM myosin) that are considerably lower than the estimated cellular concentrations (200 p M actin and 2 p M myosin; Section IV) were used. In addition, in this in vitro assay, the reaction was much slower (tl,* = 1-5 min) than the observed in vivo response (less than 30 sec) (Section V). Therefore, at this point, we cannot rule out either of these mechanisms. Actin organization is definitely the primary event occurring concomitant with dynamic restructuring (Fukui et al., 1990) (Section 11), which is followed by the association of myosin and other components in a temporally and spatially regulated manner (Section V). Not only the hydrolysis of ATP but also all the chemical bonds can store and produce equally high level of energy (Section V). The mechanism I suggest in this review is that of the directional translocation accompanied by production of a “biased” frictional force. This hypothesis requires a conceptual agreement that the association of myosin head with actin is such that the allosteric changes occur in a strictly biased fashion, and the rod portion interacts with some other components to provide frictional support; therefore, myosin could “pull” F-actin rather than “walk.” This agreement is necessary since the frictional force derived by a movement of the molecule per se is negligible because of its size. F-actin, bound with the membrane at the barbed end, is probably capped, and, therefore, at steady state, elongation is minimal (Section IV). Myosin, now bound to actin, walks along the F-actin cable, at a speed of 1 pm/sec, hydrolyzing eight ATPs every second, and whips its head in the order of 10-20 nm per hydrolysis (Uyeda et al., 1991). The myosin might be monomeric or oligomeric. The demonstration that the pair of heads can bind to a single F-actin (Craig et a]., 1980) suggests the possibility of walking of a monomer or low-level oligomer along F-actin. Now, whereas a single motion produces 1 pN force, there are some 200,000 myosins (Section V). This hypothetical force should, in turn, induce an anterograde

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e

+=lQ

FIG. 4 Diagram illustrating postulated multiple force-generation mechanisms in nonmuscle cells. (a) Polymerization force (Albrecht-Buehler, 1990; Cooper, 1991); (b) gel swelling pressure (Oster, 1988); (c) projectile force (Fukui cf a/., 1989); (d) biased friction (this chapter); (e) sliding-filament mechanism (Spudich, 1974; Fukui and Yumura, 1986; Clarke and Baron, 1987).

cytoplasmic flow (Fig. 4d) that could be secondarily boosted by the contraction of the posterior body by the observed pseudo-sarcomeric actomyosin structure (Fukui and Yumura, 1986). This latter mechanism is energized by the sliding of filaments (Fig. 4e). If the above considerations are correct, it is now clear why genetically engineered mutants that synthesize abnormal myosin (De Lozanne and Spudich, 1987) or little or no myosin (Knecht and Loomis, 1987; Manstein et d., 1989) are still motile. The abnormal motility of those mutants is brought

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about by the intrinsic force generated by interaction between actin-based cytoskeletons. The alternative force is so great that, in some cases, we cannot detect any apparent defect in motility due to lack of a particular component (Brink et al., 1990). In this regard, it is important to admit that we do not necessarily have sufficient resolution and sensitivity in assays to determine cell misbehavior.

VII. Summary and Perspective The role of cytoskeletal components can be assessed by studying genetically engineered mutants. Lack of a component, however, does not always manifest impaired motile activities. This is very likely due to multiple compensation mechanisms that deliver enormous mechanical force. These alternative forces include subunit addition, gel osmosis, projection, and biased friction as described in this review (Fig. 4). Properly regulated contractile machinery, however, is a prerequisite for the cell’s perfect life. The question is whether the defect is household or luxurious. Luxurious implies the phenotypes that are not obviously manifested at ordinary cellular-level activities. A defect in the luxurious activities, however, should cause behavioral or communicative impairments. Our current knowledge in this area remains far short of objectives. At most, this is an extremely interesting, challenging area awaiting future studies. Highfidelity video microscopy (Inout, 1986), motion analysis of individual cell behavior (Soll, 1988), quantum-level analysis of motor proteins in virro (Ishijima ef al., 1991), and direct visualization of molecular dynamics in vivo (Sheetz er al., 1989; Lee et al., 1990; Cao and Wang, 1991a,b; Kolega et al., 1991; Theriot and Mitchison, 1991) should reveal fascinating answers to questions about the nature of cytoplasm. Genetic manipulation most obviously exhibits misbehavior of cells during development (Knecht and Loomis, 1987; De Lozanne and Spudich, 1987; Witke et al., 1992). This fact indicates that even subtle damage at the cellular level causes severe communicative errors between the cells, which ultimately results in impaired development. Studying their developmental defects should unveil the actual physiological functions of the cytoskeletal components.

Acknowledgments I thank many of my colleagues for providing valuable information for writing this review. Special thanks go to Dr. G. Albrecht-Buehler, who energized my writing activity, and to Angela Traben for proofreading the manuscript. This work was supported by NIH GM39548.

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Fine Structure, Innervation, and Functional Control of Avian Salt Glands Rijdiger Gerstberger and David A. Gray Max-Planck-Institut fur Physiologische und Klinische Forschung, W. G. KerckhoffInstitut, D-6350 Bad Nauheim, Federal Republic of Germany

1. Introduction To maintain cellular functions, both the intracellular fluid volume and the composition of (non)-ionic solutes have to be controlled within narrow limits. Although single-celled organisms are separated from their mostly aqueous environment by only a plasma membrane endowed with specific transport systems, channels, and energy-driven pumps, multicellular organisms use their extracellular fluid space, with sodium and chloride as the major osmotically active ionic solutes, as a regulated exchange system between the intracellular compartment and the “external world.” In the vertebrate kingdom, various strategies to maintain body fluid homeostasis have evolved with regard to extracellular fluid volume (ECFV) and tonicity (ECIT), according to the axiom of Bernard (1865), which is still a central tenet of comparative physiology. With the kidney and its transport systems representing the main osmoregulatory organ in mammalian salt and water balance, accessory tissues highly specialized for the transport of sodium, chloride, or divalent ions in combination with osmotically induced water fluxes are described for submammalian vertebrates and include the gills of teleost fish (Foskett, 1987), the rectal gland of elasmobranchs (Solomon er a/., 1984a, 1985a), the skin of amphibians (Lindemann and Voute, 1977), the gut system of fish and birds (Skadhauge, 1981; Kirsch et al., 1985), and the salt-secreting glands of marine reptiles and birds (Peaker and Linzell, 1975; Dunson, 1976). The unusual position of the avian salt-secreting glands in the head region of marine birds and their accessibility have stimulated scientific examination in the past with first reports appearing as early as 1665 (Technau, 1936). With detailed descriptions of the exact anatomical localization of these glands including their duct systems in various families of the bird kingdom, Jacobson (1813) and Inr~rnorronalRewen, OJ Cyrologr. Vol. 144

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Nitzsch (1820) summarized the knowledge of their time without presenting information concerning the fine structural aspects of the glands, or any indication of the putative physiologic function of the glands. Nitzsch only stated that “the possibly oily nature of the fluid can be excluded as it quickly evaporates when soaked up by paper” and that “the secreted fluid might resemble drops of tears.” Ontogenetic studies concerning the development of glandular structures in the head region of various vertebrate classes then created the false idea that the salt gland was homologous to the organ of Jacobs, involved in chemoreception (Kolliker, 1860; Mihalkovics, 1898). It was not until the early twentieth century that Cohn (1903) managed to rule out the participation of the salt gland in sensory processes. Numerous observations then described the close relation between the size of the salt glands and the salinity of the water in the respective habitat for a variety of primarily marine, but also terrestrial, avian species (Marples, 1932). Fast increases in the specific organ weight of the salt glands in ducklings (Anus platyrhynchos) adapted to 3% saltwater as drinking fluid as well as the reduction of salt gland size in Eider ducks (Somateria rnoflissima) kept on freshwater strongly supported this relationship (Heinroth and Heinroth, 1928). More than 100 years after the pioneering studies by Nitzsch and Jacobson, Technau (1936) published a detailed analysis of the avian salt-secreting gland, emphasizing the whole spectrum of exact localization, macromorphology, and formation of the duct system of salt-secreting glands obtained from 106 genera of birds, including such rare species as Urubu, South American Phytotoma, and islandic anatides. With regard to the physiology of the glands, however, Technau (1936) only dedicated a few words to its function, supporting the idea of Heinroth and Heinroth (1928) and Schildmacher (1932), who reported that the salt glands of the Eider duck secrete a fluid that “protects the nasal mucosa against irritating influences of the salty seawater.” The often reported observation of fluid droplets at the tip of the beak during open-sea feeding of truly marine birds such as petrels, albatrosses, and penguins (Peaker and Linzell, 1975), and the urgent need to eliminate the excessively ingested salt from their extracellular fluid compartment at the restricted concentrating capacity of their kidneys, finally led to the discovery of the important role that avian salt glands play in maintaining body fluid homeostasis (Schmidt-Nielsen et al., 1958; Schmidt-Nielsen, 1960).

II. Secretory Tissue of the Avian Salt Gland A. Zoology of Salt Glands The gross anatomy, fine structure, secretory mechanism, and hormonal control of the avian salt-secreting gland have been thoroughly revised over the last two

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decades (Peaker and Linzell, 1975; Van Lennep and Young, 1979; Butler, 1984; Holmes and Phillips, 1985; Komnick, 1985; Butler et al., 1989, Simon and Gray, 1989; Gerstberger, 1992), following the pioneering work of SchmidtNielsen (1960). In the present review on various aspects of fine structure, innervation, and blood supply, and also afferent and efferent control mechanisms of avian salt gland function, new insights gained within the last 10 years are incorporated with well-known observations that now can be looked upon from a different point of view. These recent findings should shed light on (a) the functional cellular architecture, ( 6 )the osmo- and volume-receptive system involved in salt gland control, (c) the efferent effectors such as neurotransmitters and hormones, and (d) the intracellular signal transduction pathways. Functional salt glands have been demonstrated in at least 10 avian orders including Charadriiformes (gulls and plovers), Procellariiformes (albatrosses, petrels) (Fig. I), Pelicaniformes (pelicans, cormorants), Sphenisciformes (penguins), Gaviiformes (divers), and Anseriformes (ducks, geese), as well as Falconiformes (eagles) and birds living in arid land zones (Peaker and Linzell, 1975; Thomas and Phillips, 1978; Mahoney and Jehl, 1985; Sonawane, 1987; Conway et al., 1988). The exact location of the avian salt-secreting gland in the head region differs depending on the species and/or its habitat. The salt glands of wading birds, plovers, gulls, and petrels are found in the crescent-shaped depressions in the frontal bones above the eyes (Fig. 8), whereas the salt glands of ducks and geese are located along the edge of the frontal bone in the upper orbital membrane. The salt glands of falcons and woodpeckers are in the upper maxillary cavity and beneath the orbital membrane, respectively. Concerning the embryological origin of the salt glands, Marples (1932) stated that the gland is eventually formed by branching of the two main ducts arising from the rudiment of the nasal cavity, and then growing backward to the final position. Histological studies performed with the Adelie penguin (Pygoscelis adeliae) allowed the first rudiments of supraorbital salt glands to be traced back to “solid crescent structures on either side of the nasal cartilage,” growing posteriorly to develop dorsal to the eye (Herbert, 1975). Thus, the glandular matrix develops from the ducts as branched tubules radiating from a central canal. Salt-excreting glands evolved in marine birds to eliminate excess ions, mainly sodium and chloride, from the extracellular fluid compartment to maintain body fluid homeostasis even under severe osmotic stress (Bemdge and Oschman, 1972; Kirschner, 1980). Reptiles living in marine, estuarine, and arid zones and marine elasmobranchs also developed “extrarenal glandular organs whose main function is to excrete a hypertonic salt solution” (Van Lennep and Young, 1979). The salt-secreting glands of reptiles comprise lingual glands in crocodilians, lacrimal glands in chelonians, sublingual glands in snakes, and lateral nasal glands in lizards, whereas the nonhomologous shark rectal gland represents a rectal appendix in the dorsal mesenteries of the peritoneal cavity (Bulger, 1963; Dunson, 1976; Komnick, 1985). Whenever appropriate, comparative aspects of

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FIG. 1 Marine birds such as this adult New Zealand albatross possess supraorbitally located saltsecreting glands (salt glands), with their main ducts opening into the nasal cavity, to maintain body fluid homeostasis in their salty environment.

salt-secreting gland structure and function in elasmobranchs or reptiles have been taken into consideration in this review. The salt glands of marine and estuarine birds, or of saltwater-acclimated freshwater birds bearing salt glands, are able to actively extract sodium and chloride from the extracellular fluid compartment, represented by the perfusing bloodstream, and to excrete up to 1.O ml/min/g gland weight of a hypertonic salt solution against a marked concentration gradient. Sodium is accompanied by chloride in equimolal concentrations, whereas potassium concentrations in the secreted fluid remain low. Depending on the species, sodium concentrations range from about 500 meq/liter, as described for the saltwater-acclimated duck or some cormorants, to more than 1200 meq/liter, as reported for petrels and albatrosses (Schmidt-Nielsen, 1960; Peaker and Linzell, 1975). Both sodium and chloride therefore must be actively transported against a marked concentration gradient of 1:3 to 1 :8, compared to the mere ratio of 1:1.5 for the shark rectal gland. Like the avian salt gland, most reptilian salt glands are able to highly concentrate sodium and chloride from the perfusing bloodstream, thereby elim-

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inating excess salt from body fluids at low water losses. In herbivorous lizards, potassium and bicarbonate constitute a major portion of the excreted electrolytes (Dunson, 1976; Van Lennep and Young, 1979). In many exocrine glands, the rate of secretion is closely correlated to the concentration of solutes in the secreted fluid. For the avian salt gland, however, this aspect has been discussed controversially. With salt gland secretion rates in the duck varying from 0.2 to 0.8 ml/min, there appeared to be no correlation between secretion rate and sodium concentration of the fluid with ion concentrations being at their maximal level (Butler et al., 1989). At secretion rates lower than 0.1 ml/min, however, the concentrations of sodium and chloride were concomitantly reduced (Deutsch et al., 1979; Gerstberger et al., 1984a). A comparable relationship was reported for the Kelp gull (Gray and Erasmus, 1989b). In the goose, either a slightly positive or a negative correlation was observed, depending on the salt and volume loading of the animals (Hanwell et al., 1971a).

6 . Fine Structure of the Secretory Cell The avian salt gland represents a compound tubular gland, with its main excretory ducts branching into primary and secondary ducts forming the central canals of secretory lobes (or lobules) (see Section II,D), which are composed of numerous radially arranged secretory tubules with small lumina merging into these central canals (Van Lennep and Young, 1979) (Fig. 2). Different from the salt glands of the gulls, plovers, or the oyster catcher, where secretory lobes running parallel all drain into one of the two main ducts leaving the secretory tissue, the duck’s salt gland is composed of spherical secretory lobes. Central canals of these lobes, receiving inflow from all the lumina of radiating secretory tubules, empty into primary ducts, which finally merge with the medial or lateral segmental ducts (Butler et al., 1991). Sheaths of peritubular connective tissue and a dense interlobular connective tissue matrix containing nerve fibers and blood vessels embed single secretory tubules and lobes, respectively. The first detailed morphologic description of the avian salt gland at the electronmicroscopic level was camed out by Doyle (1960) using specimens of the Great Black-Backed gull (Larus marinus) and the petrel (Oceanodroma leucorrhoa). Subsequently, the salt glands of L. argentatus, L. ridibundus, and the saltwateracclimated Pekin duck (A. platyrhynchos) were subjected to basic light- and electron-microscopic examination (Komnick, 1963a,b,c, 1964; Dulzetto, 1965; Ernst and Ellis, 1969). 1. The Principal Cell

The typical secretory tubule of the avian salt gland comprises five to eight cells radially arranged around a central lumen of 1-1.5 pm in diameter, thus forming

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a monolayered epithelial sheath (Fig. 2 ) . The secretory tubules radiating from each central canal are lined by a cuboidalxolumnar epithelium of principal secretory cells at the proximal, and so-called terminal or peripheral cells at the blind-ending distal segments (Emst and Ellis, 1969). Morphometric analysis allowed the total surface area of the secretory tubule lumina to be determined as 3.65 cm2 in the saltwater-acclimated Pekin duck (Marshall et al., 1987). The peripheral cells revealing rather high mitotic activity (Fig. 5) are characterized by simple, unspecialized cytoarchitecture with high numbers of unbound ribosomes, but low density of basally located mitochondria. The basolateral membranes appear unfolded with only a few plicae detectable. Furthermore, the cells are equipped with large Golgi apparatus (Ernst and Ellis, 1969). The fully specialized principal cells constituting the major portion of the secretory tubules in salt-acclimated animals (Fig. 5 ) possess a large spheroidal nucleus located in the center of the cell or slightly above it. These cells reveal maximal surface membrane amplification due to marked interdigitations at the basal, but also lateral, cell borders (Figs. 2 and 3). The basal lamina embedding a secretory tubule remains straight and does not follow the plasma membrane invaginations that enwrap a great number of mitochondria (Komnick, 1963a,c; Emst and Ellis, 1969). These mitochondria are of elongated shape, characterized by densely packed cristae within their inner matrix, and situated within the basal infoldings of the plasma membrane (Komnick, 1963c) (Fig. 3). Purified mitochondria1 preparations enabled the calculation of ADP utilization per molecule cytochrome c, as well as of sodium transport with maximal values of eight sodium ions (Na+) per molecule ATP (Chance et al., 1964). “Secretion canaliculi,” once proposed by Doyle (1960), are fully absent from the electrolytetransporting epithelium, which is composed of the avian principal secretory cells (Van Lennep and Young, 1979). Different from the basolateral membrane, the apical border shows only small microvilli protruding into the small lumen of the secretory tubule. Adjacent principal cells are apically interconnected via junctional complexes comprising zonulae occludentes, zonulae adherentes, and desmosomes (Fig. 4). To outline the basolateral membrane surface with all its plicae, interdigitations, and complex foldings, horseradish peroxidase, ruthenium red, and lanthanum salts were used

FIG. 2 The secretory parenchyma of the functional avian salt gland. (a) Light-microscopic cross section through a lobe of an Eider duck salt gland (Masson Goldner staining), with secretory tubules (ST) radially arranged around the central canal (CC). Bar, 30 pm. (b) Light-microscopic cross section through secretory tubules (ST) of an Eider duck salt gland (Azan staining) with five to eight principal cells surrounding the tubular lumen (L). Countercurrent arrangement of secretory tubules and capillaries (CA). Bar, 10 pm. (c) Electron micrograph of a cross section through one secretory tubule of a Pekin duck salt gland revealing intercellular luminal tight junctions (TJ), basolateral infoldings (IN), and nuclei (N) of seven secretory cells. Bar, 1 pm. (Unpublished micrographs, courtesy of Prof. Dr. W. Kiihnel.)

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FIG. 3 The basolateral plasma membranes of avian salt gland secretory cells. (a) Extensive basolateral infoldings (IN) of a salt gland secretory cell (Pekin duck) as demonstrated by lanthanum treatment. BL, basal lamina; N, cell nucleus; L, lumen. Bar, 1 pm. (b) Basal membrane infoldings (IN) of a salt gland secretory cell (Japanese swan goose) with numerous cristae-type mitochondria (M). The base of the secretory cells is in contact with a continuous basal lamina (BL). Bar, 1 pm. (c) Freeze-fracture replica of the basal infoldings from a secretory cell of a saltwater-acclimated Pekin duck. Mitochondria (M) are densely packed within the intracellular compartments. BL, basal lamina; P, particle-rich fracture faces; E, particle-poor fracture faces. Bar, 0.5 pm. [(a) From Berridge and Oschman. 1972, with permission; (b) Unpublished material, courtesy of Prof. Dr. W. Kiihnel; (c) from Riddle and Ernst, 1979, with permission.]

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to mark the extracellular interstitial space between neighboring secretory cells in saltwater-acclimated ducklings (Martin and Philpott, 1973, 1974; Hootman et al., 1987) (Fig. 3). Using these extracellular space tracers, both the extent of the space and the permeability of the junctional complex could be examined in actively secreting salt glands. Ruthenium red reacts with anionic components of the cell surface. Horseradish peroxidase could be found filling the matrix area of the peritubular connective tissue and the spaces formed by the basolateral cell membrane infoldings and in the junctional complexes and even the luminal space. Penetration into the lumen of the secretory tubules was also described for lanthanum, suggestive of rather leaky cell-to-cell contacts (Hootman et d., 1987).

FIG. 4 The luminal junctional complex in the avian salt gland secretory tubule. (a) Narrow lumen (L) of a secretory tubule from a Pekin duck salt gland bounded by six secretory cells joined lumiSurface specializations such as short microvilli (MV)protrude into the nally by tight junctions (TI). lumen. Bar. 0.5 pm. (b) High magnification of the junctional complex between adjacent principal cells in the Herring gull salt gland. The intercellular gap (I) is bridged by a zonula occludens ( O ) , zonula adherens (A), and desmosome (D). Bar, 0.1 pm. (c) Freeze-fracture replica of the zonula occludens of secretory cells from a Pekin duck salt gland. A junctional douplet consisting of two strands is visible with the fracture running from the P face (PF) of the luminal membrane to the P face (P) of the lateral membrane. The two-stranded junction can be locally widened (80 nm) with interconnecting cross bars (asterisk). Bar. 0.1 pm. [(a) from Ernst and Ellis, 1969, reproduced with permission from J . Cell.. B i d . (1979). 40. 231-305; (b) from Komnick and Kniprath, 1970, with permission; (c) from Riddle and Ernst, 1979, with permission.]

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To elucidate cellular mechanisms involved in the ion transport processes and their control systems, the intact avian salt gland proved to be of limited use, whereas the isolated secretory cell represents an ideal model. Barmett and colleagues (1983), however, opposed the use of long-term cell cultures of salt gland secretory cells due to the dedifferentiation including the loss of polarity and basolateral infoldings. Some of the latter problems could possibly be overcome using confluent sheets of dissociated cells seeded on a porous collagen support that expressed polarized features such as apical microvilli, tight junctional complexes with transmural resistance, and basolateral membrane infoldings (Lowy et al., 1985a,b) (Fig. 14). Enzymatic and mechanical dissociation of salt gland tissue gave high yields of viable principal cells readily identified in suspension by their large size, cytosomal granularity, and retained cell surface plicae. Apical polarity of these cells might, however, be lost (Hootman and Emst, 1980). 2. Apical Tight Junctions As mentioned, leakiness of the secretory epithelium was indicated by extracellular space markers and also the retrograde injection of India ink or colloidal thorium via the excretory duct of the gland. This procedure resulted in penetration of the ink from the tubular lumen to the basement membrane (Doyle, 1960). By transmission electron microscopy (EM) of the apical junctional complex, the zonula occludens in the secretory cells of the avian salt gland, possibly proteinaceous in nature, appeared to be a component of the epithelial junctional complex, which includes the zonula adherens (intermediate junction) and the desmosomes (Fig. 4). In freeze-fracture EM, the tight junction appeared as continuous, intramembrane strands in the P-face (outwardly facing cytoplasmic leaflet) with complementary grooves in the E-face (inwardly facing extracytoplasmic leaflet). In general, tightness of an epithelium is represented by the correlation of freeze-fracture strand number and transepithelial resistance (Claude and Goodenough, 1973). Freeze-fracture studies in the nasal salt gland of the Herring gull demonstrated apical cell junctions of the single-stranded type (Ellis et al., 1977). Freezefracture replicas of principal secretory cells of the duck salt gland, obtained from the particle-rich (P) face of the luminal membrane to the particle-rich (P) face of the basolateral membrane, also revealed shallow luminal intercellular junctional complexes consisting of only two closely juxtaposed junctional strands on the P face of the lateral membrane. With the exception of short focal widenings, two sets of doublets were rarely seen, each doublet having a width of 20-25 nm (Riddle and Emst, 1979) (Fig. 4). The simplicity of the junctional morphology in the avian salt gland indicates leaking of ions and other solutes, quite in contrast to the complex network of anastomosing strands, described for high-resistance epithelia such as frog skin or endothelial cells forming the blood-brain banier

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(Claude and Goodenough, 1973; Simons and Fuller, 1985; Gumbiner, 1987). The peripheral, transport-inactive cells possess zonulae occludentes composed of a loose meshwork of interconnecting strands at a junctional width of about 100 nm. Interestingly, principal cells from salt glands of osmotically unstressed ducks showed zonulae occludentes of slightly higher complexity, the strands often being made up of two sets of doublets (Riddle and Emst, 1979). Although direct access to the glandular tubules has not been possible in vivo, transepithelial resistance can be used as a measure for tightness of junctional complexes between cells, determined from confluent layers of primary salt gland cell culture systems (Lowy et al., 1985b) (Fig. 14). Values in the range 300 R X cm2 represent only modest resistance compared to those obtained from brain endothelial cells or frog skin (2000 R X cm2) (Bradbury, 1985).

3. Comparative Aspects Reptilian salt glands such as those found in the desert lizard Uromastyx acanthiniirus are composed of two types of electrolyte-transporting principal cells, called “light” and “dark” cells according to the electron density of their cytoplasm, mainly due to the packing of large numbers of mitochondria. Whereas the light cells are ovoid shape, the dark cells are triangular in shape. The apical membranes of the principal cells of the salt gland reveal short microvilli, as in the avian salt gland, except for a few cells endowed with apical brush borders (tuft cells), whereas the lateral membranes are strongly plicated (Van Lennep and Young, 1979). In addition, cells containing a well-developed rough endoplasmic reticulum and secretion granules in the apical zone, stained with periodic acid-Schiff’s reagent (mucoid cells), and so-called “basal cells” because of their location at the base of the secretory tubules have been described (Van Lennep and Komnick, 1970). The posterior sublingual salt glands of all sea snakes investigated so far lie beneath the tongue in the lower jaw and partially enclose the tongue sheath, into which they secrete. The ultrastructure of the sublingual salt gland in sea snakes resembles that of other reptilian salt glands with their highly irregular principal cells being rich in mitochondria and plasma membrane (baso-) lateral digitations. Quite different, however, adjacent apical cell surfaces appear to be joined by zonulae adherentes and not occludentes, implying a continuity between the interstitial space and the tubular lumen. Systemic salt loading in the poisonous pelagic sea snake Pelumis induced secretion from its large single posterior sublingual salt gland with sodium (“a+]) and chloride ([Cl-]) concentrations as high as 700 mM at low potassium ([K+]) concentrations of 15 mM, results comparable to those observed with the avian salt gland (Dunson and Taub, 1967; Dunson, 1968, 1976; Dunson et al., 1971; Dunson and Dunson, 1974). The dogfish rectal gland is composed of a central canal surrounded by an inner layer of transitional epithelium, a broad layer containing the secretory

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tubules, and the outer capsule. The principal cells of the secretory tubules, described in detail by Bulger (1963), reveal pronounced membrane interdigitations at the basal as well as lateral cell borders. Comparable to the avian secretory cell, mitochondria of the cristae-type are densely packed in the basal cellular compartment, and endoplasmic reticulum is found only in small amounts. The Golgi apparatus can be recognized as a separate unit close to the nuclear membrane. Comparable to the shallowness of the junctional complex between neighboring salt gland cells in birds, occluding junctions between secretory cells of shark and stingray rectal glands showed an average strand nurnber of 2 and 3.5, respectively. Apical boundaries of the cells are quite tortuous, giving rise to a long linear junction and large paracellular conductance/unit apical surface area (66 m/cm2). The measurements are similar to those obtained for the avian salt gland (S. A. Emst, personal communication). As emphasized by electrophysiologic studies, these epithelia might also be of the leaky-type (Emst et al., 1981; Greger et al., 1986).

C. Cellular Aspects of Salt Acclimation 1. Glandular Hypertrophy To become functionally active, the salt glands of newly fledged marine or young freshwater-acclimated birds must undergo a complex adaptive process, finally leading to the fine structural aspects described for the secretory principal cell. The most obvious signs of salt acclimation are represented by increased organ weight and augmented secretory capacity. This could be described for adult Pekin ducks, Australian chestnut tails (A. castanea), and Glaucous-winged gulls (L. glaucescens) during long-term salt acclimation (Holmes et al., 1961; Ellis et al., 1963; Fletcher et al., 1967; Deutsch et al., 1979; Baudinette et al., 1982). Acclimation of newly hatched common gulls (L. canus), Glaucous-winged gulls, or ducklings to saline in their food supply also induced glandular hypertrophy (Schmidt-Nielsen and Kim, 1964; Spannhof and Jiirss, 1967; Gerstberger et al., 1984a; Hughes, 1984) (Fig. 5). Deadaptational processes resulted in opposite responses. Quantitative morphometric analysis of structural differences between salt glands of Hemng gulls acclimated to freshwater compared to those maintained on seawater yielded a decrease in whole organ size to 60% of the control values, with the intercellular fluid volume and the number of cytosolic mitochondria both diminished by half (Komnick and Kniprath, 1970). Deadaptation also resulted in a fast decline of Na-K-ATPase activity (see Section V,A), movement of the mitochondria toward the cell nucleus, and, importantly, loss of plicated plasma membranes. Clusters of filamentous osmiophilic material were found in the apical cytoplasm, and both acid phosphatase and peptidase activities were stimulated (Hossler et al., 1978).

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Variations in size and function of the salt glands have been reported for Franklin’s gulls (L. pipixcan) breeding in freshwater habitats and wintering in marine habitats (Burger and Gochfield, 1984), and for female Eider ducks (S. mollissima) during egg incubation, lasting for 26 days, when not drinking saltwater. The nasal salt glands concomitantly underwent regression and reduction in Na-K-ATPase activity and secretory capacity, thus representing a naturally occumng phenomenon of deadaptation (McArthur and Gorman, 1978). Another important aspect of salt acclimation and its role in avian salt gland function is the occurrence of alterations in body fluid distribution (see Section VI). Studies have been restricted to some gulls species, Canada geese, and the Pekin duck (Roberts and Hughes, 1984; Gray et al., 1987; Brummermann. 1988; Gray and Erasmus, 1989b; Hughes, 1989; Brummermann and Simon, 1990). During the course of salt acclimation, plasma electrolyte concentrations of Pekin ducks increased at constant blood volume, whereas the apparent volume for inulin distribution diminished, reflecting a reduced extracellular, especially interstitial, fluid volume (Gray et al., 1987). With regard to the size of the extracellular fluid compartment, however, data have been conflicting with bromide spaces identical for adapted and nonadapted birds (Ruch and Hughes, 1975), and with exchangeable sodium pool size being either of equal value (Gray et al., 1987) or increased after salt acclimation (Roberts and Hughes, 1984; Hughes, 1989). In the gull L . glaucescens, saline drinking did not influence the sodium pool size (Roberts and Hughes, 1984), whereas the gradual adaptation of Kelp gulls to 100% seawater as the drinking fluid resulted in a change of blood hematocrit indicative of extracellular fluid depletion, with a mild increase in plasma electrolyte concentrations but unchanged colloid osmotic pressure (Gray and Erasmus, 1989b).

2. Cellular Hypertrophy/Hyperplasia and Membrane Biogenesis Hypertrophy and hyperplasia of principal secretory cells during salt acclimation have been shown in purely morphometric studies performed on Pekin ducks, where short-term acclimation to a hypertonic saline regimen caused an increased average cell diameter of 11.6 pm compared to 10.4 pm under control conditions (Ballantyne and Wood, 1969). Cell densities per unit of tissue volume were reduced by half (Merchant et al., 1985). A fivefold increase in mitochondrial profiles and a sevenfold rise in the number of lipid droplets were also described as a result of acclimation to a hypertonic saline regimen (Merchant et al., 1985) (Fig. 13). In addition, enhanced cellular protein synthesis (Holmes and Stewart, 1968), increased cellular proteinDNA ratios (Hootman and Emst, 198la; Hossler, 1982), the ribosomal composition of principal secretory cells including augmented relative RNA content of the cell (Holmes and Stewart, 1968; Ballantyne and Wood, 1969; Stewart and Holmes, 1970; Knight and Peaker, 1979), and the stimulated incorporation of tritiated thymidine into

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FIG. 5 Morphologic indications of adaptation to a high salt regimen. (a) Schematic drawing showing the effects of a saltwater regimen on the development of avian salt gland secretory cells, and their distribution along a single secretory tubule. Partially specialized cells (Day 0, stippled) become endowed with basolateral infoldings and numerous mitochondria (Day 2, hatched), and develop pronounced basolateral membrane amplification and cellular polarity (Day 1I , cross-hatched). Fully matured principal secretory cells (hatched) are found near the merging point of the branched secretory tubule with the central canal. (b) Cross section through the salt gland of a freshwater-adapted (1) and a saltwater-adapted (2) Pekin duck (Azan staining). Bar, 1 mm. (c) Histochemical demonstration of acyltransferase activity as a marker enzyme of membrane biogenesis in salt gland secre-

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nuclear DNA (Knight and Peaker, 1979) were used as representative measures of cellular hyperplasia and hypertrophy. Geese with unilateral postganglionic denervation or surgical removal of one of their salt glands responded to shortterm salt acclimation with partially compensatory growth of the remaining gland, as judged from increased RNA concentrations per unit mass of tissue and an augmented RNADNA ratio in the intact gland (Hanwell and Peaker, 1975; Knight and Peaker, 1979). As suggested by the studies of Emst and Ellis (1969), the hypertrophic process commences at the blind end of the secretory tubules, where peripheral cells become mitotically active, as indicated by the 15-fold stimulated incorporation of radioactive thymidine after 1 day of salt acclimation. During the first hours of salt stress, radiolabeled thymidine was preferentially taken up by connective tissue cells, whereas at later stages, the label was concentrated in parenchymal cells of peripheral tubular origin (Hossler, 1982) (Fig. 5). These newly created cells would then transform to partially and finally fully specialized principal secretory cells (Fig. 5 ) . Plasma membrane biosynthesis in the salt gland secretory cells was markedly enhanced during salt acclimation of ducklings. Elucidation of the development of the fine structure of the secretory epithelium during salt acclimation at the ultrastructural level was facilitated by the marked amplification of the plasma membrane (Emst and Ellis, 1969; Hossler et al., 1978). The principal secretory cells of the avian salt gland revealed pronounced alterations in the basal and lateral plasma membrane with values of 1800, 9500, and 3500 pm2/cell before, during, and after saltwater acclimation in ducks (Fig. 13). In contrast, there were no detectable differences in the membrane surface area of the peripheral cells (Merchant et al., 1985). Stimulated synthesis of RNA and (g1yco)-proteins could be detected as early as 2 hr after the beginning of the salt stress, as determined from the incorporation of tritiated uridine, leucine, or fucose into salt gland tissue (Sarras et al., 1985). Pulse-chase experiments at the electronmicroscopic level indicated that [3H] leucine is initially taken up by the rough endoplasmic reticulum, transported to and concentrated in the Golgi apparatus, and finally found throughout the plasma membrane, with the three steps traceable after 5 , 10, and 60 min, respectively.

tory cells during saltwater acclimation is confined to some cisternae of the Golgi apparatus (G). Bar. 0.5 pm. (d) Autoradiograms of a salt gland secretory lobule of a Pekin duck salt-acclimated for 6 days following [3H]thymidine incorporation. In the darktield micrograph (top), labeled nuclei are indicated by double arrowheads, unlabeled nuclei by single arrowheads. In the lightfield micrograph (bottom), labeled parenchymal cells are indicated by single arrowheads, labeled connective tissue (CT) cells by double arrowheads. Bar, 20 pm. [(a) From Ernst and Ellis, 1969 (modified), with permission; (b) from Gerstberger e r a / . , 1984a. with permission of Springer Press, Heidelberg; (c) from Barmett e r a / . , 1983, with permission; (d) from Hossler, 1982. with permission.]

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Plasma membrane phospholipids increased by a factor of 1.7 during enhanced membrane biogenesis associated with cellular salt acclimation, and phospholipids demonstrated by Sudan Black or thionine were highly concentrated in the principal, but not peripheral, cells (Ellis et al., 1963). Acyl transferase activity, used as marker of acyl lipid biosynthesis, increased almost threefold during the first 24 hr of salt acclimation. The cytochemical localization of acyl transferase activity was restricted to some cisternal elements of the Golgi apparatus, indicative of phospholipid synthesis in this organelle of the principal cells with subsequent packaging and vesicular transport to the preexisting plasma membrane (Levine et al., 1972; Barrnett et al., 1983) (Fig. 5). Acid phosphatase, another enzyme possibly involved in membrane turnover, was found throughout the secretory tubules except in their peripheral blind-ending section, with highest densities of the enzyme in the basolateral region of the principal cells, which suggests local membrane degradation (Ellis et al., 1963; Spannhof and Jurss, 1967; Hossler et al., 1978). In contrast, alkaline phosphatase was moderately concentrated in the basophilic peripheral cells and glandular capillaries, but virtually absent from principal cells (Scothorne, 1958, 1960; Ellis et al., 1963; Spannhof and Jurss, 1967). As suggested, alterations in salt gland ultrastructure have often been associated with the osmoregulatory status of the animal. The volume fraction of some cellular organelles, such as mitochondria, and the size of the intracellular space have been used to draw far-reaching conclusions on “physiologic significance.” A detailed stereological analysis of the effects of buffer osmolality alone on the salt gland epithelium during processing of the tissue strongly questions whether the changes in intercellular space geometry reported “represent physiologic mechanisms in vivo or differences in osmotic behavior during processing for electron microscopy” (Cowan, 1986). Morphometric analysis of cellular structures thought to be most relevant for the secretory process, the mitochondria and the intercellular spaces, should therefore only be performed under standardized conditions of tissue perfusion and fixation, as indicated by Butt and colleagues (1985).

3. Metabolic Enzyme Activities Another important aspect of both salt acclimation of the principal cells and their energy-consuming transport activities is presented by the specific activities of various soluble enzymes involved in energy metabolism. When various soluble enzymes in the salt glands of the gull and Pekin duck were measured, those involved in cellular metabolic processes such as mitochondria1 respiration, glycolysis, and the Krebs cycle appeared to supply most of the energy (A”) needed for the ion transport activity (McFarland el al., 1965; Kiihnel et al., 1969a,b; Stainer et al., 1970). In addition, in vitro studies revealed that the presence of Krebs cycle substrates greatly increased cellular respiration (Borut and Schmidt-Nielsen, 1963).

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Acclimation to hypertonic saline as drinking water in the Pekin duck stimulated the specific activities of soluble enzymes involved in glycolysis and the hexose-monophosphate shunt such as glucose-6-phosphate dehydrogenase (G6-PD) and phosphofructokinase (PFK), which is often regarded as the ratelimiting enzyme in glycolysis. Hexokinase (HK) activity was slightly stimulated and lactic dehydrogenase (LDH) remained unchanged (Stainer et al., 1970). Enzyme analysis performed in salt gland tissue of saltwater-acclimated western gulls (L. occidentalis) resulted in high activities of G-6-PD and LDH, indicative of the importance of the hexose monophosphate shunt and rapid anaerobic glycolysis (McFarland et al., 1965). With regard to enzymes of the Krebs cycle, in the duck salt gland isocitric dehydrogenase (ICD) and malic enzyme (ME) activities remained unchanged during salt acclimation, whereas succinate dehydrogenase (SDH) activity was markedly enhanced (Ellis et al., 1963; Stainer et al., 1970). Succinate dehydrogenase activity also increased during the first days of salt acclimation in both proximal and distal components of the salt gland secretory tubules in young common gulls, whereas in nonadapted hatchlings the enzyme was restricted to the proximal aspects (Spannhof and Jurss, 1967). The moderate (duck) to very high (gull) activity of glutamic-oxaloacetic transaminase (GOT) and the low activity of glutamic-pyruvic transaminase (GPT) in the gull salt gland might be indicative of the potential use of the glutamate-aspartate shunt as possible source of energy (McFarland et al., 1965; Stainer el al., 1970). Adjusted calculations of energy yields from glycolysis and the Krebs cycle including the hexose monophosphate shunt were in the order of 300% of the energy needed for the secretory process.

D. The Duct System 1. Avian Salt Glands Jobert (1869) was the first to describe the avian salt gland as being formed of two distinct segments with separate drainage ducts. The putative role of these ducts in the modification of the transepithelially secreted fluid remains unclear to date (Marshall et al., 1985). In gulls and other Charadriiformes investigated, the secretory lobes running parallel all empty via their central canals directly into one of the two ducts leaving the salt gland at its anterior edge (Fange et al., 1958a,b). In ducks, the salt gland consists of distinct medial and lateral segments, each comprising spherical secretory lobes. The external duct of the medial segment empties into the nasal cavity at the base of the vestibular fold; the external duct of the lateral segment opens onto the surface of the nasal septum (Fig. 6). As revealed by microfil injections into both drainage ducts, the medial segment covers two-thirds of the salt gland (Butler et al., 1991) (Fig. 6). Also different from the situation in the gull, anastomoses between the lateral and the medial segments of

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FIG. 6 The duct system of the avian salt gland. (a) Schematic drawing of the nerve supply to the right nasal salt-secreting gland of a saltwater-acclimated Pekin duck (left), and the location of its medial and lateral drainage ducts in the nasal cavity (right). (b) Transverse section of a Pekin duck salt gland with medial (dark staining) and lateral (light staining) segments filled by retrograde injection of colored microfil into the medial (MD)and lateral (LD)duct. PD,primary duct merging into MD. Bar, 500 pm. (c) Lining of the medial duct (Azan staining) in an Eider duck salt gland. Bar, 30 pm. (d) Scanning electron micrograph of the columnar cells lining the medial duct of a Pekin duck salt gland. Small microvillar protrusions face the lumen of the duct. Bar, I pm. [(a,b) From Butler ef (11.. 1991; copyright 0 1991, reprinted by permission of Wiley-Liss, a division of John Wiley and Sons, Inc. (c) Unpublished material, courtesy of Prof. Dr.W. Kiihnel.]

the salt gland duct system appear not to occur (Hakansson and Malcus, 1970; Butler et al., 1991), although computer-aided three-dimensional reconstruction of the duct system in the duck’s salt gland revealed a single anastomosis of the two main ducts at the posterior end of the gland (Marshall et al., 1987). The epithelium of the central canals as well as of the primary and main excretory ducts is composed of two to three cellular layers, with the basal layer

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containing small, rather flat, cells and the superficial layer, large cylindrical ones (Komnick, 1964) (Fig. 6). The latter cells show an abundance of small osmiophilic mitochondria and reveal an enlargement of their lateral surface membranes due to membrane infoldings, and of their apical membrane due to numerous short microvilli (Fig. 6). Marshall and co-workers (1987) differentiated between the central canals lined by cuboidal cells, and the primary and main ducts characterized by columnar cells showing extensive Golgi bodies, lateral membrane interdigitations with apical junctional complexes, and apical microvillar protrusions. Morphometric analysis of the surface area in the duct system, in combination with X-ray microanalysis of intra- and extracellular ion concentrations, led to the hypothesis of the duct system acting as a transporting epithelium (Marshall et al., 1985, 1987). With regard to the soluble enzyme pattern found in the epithelial cells lining the central canals and main ducts in the salt glands of the duck or Herring gull, high activities of G-6-PD, SDH, NADPH-specific ICD, ME, P-hydroxybutyrate dehydrogenase (P-HBDH), and nonspecific acid phosphatase, but not alkaline phosphatase, are expressed. ATPase activity in the epithelium of the duct system proved to be of low activity, thus suggesting no transport function for this glandular compartment (Scothome, 1958, 1960; Ellis et d., 1963; Kuhnel et d., 1969a,b), as hypothesized by Marshall and colleagues (1985). Tubules and collecting ducts of the white fishing sea eagle (Haliaectus leucogaster) and some Falconiform birds are endowed with sulfated and nonsulfated acid mucopolysaccharides (MPS), whose role in secretory or tissue protective processes remains unclear (Sonawane, 1987; More and Sonawane, 1988).

2. Reptilian Salt Glands and Elasmobranch Rectal Gland The duct system of the lacrimal salt gland in the green sea turtle Chelonia mydas consists of central canals that drain the secretory lobes and join to become the main duct. The well-vascularized duct system consists of distal large columnar cells and a proximal pseudostratified epithelium with wide intercellular spaces containing mucocytes (Marshall and Saddlier, 1989). The lateral nasal gland of the desert lizard U . acanthinurus, situated on the ventromedial and lateral aspects of the nasal cavities, drains its secretory fluid via a short excretory duct. It continues as an irregularly branching collecting duct lined by a pseudostratified epithelium whose ultrastructure does not differ significantly from the one described for dark principal cells (Van Lennep and Komnick, 1970). The fine structure of the nonhomologous elasmobranch rectal gland was elucidated by Bulger (1963) and Komnick (1985). Depending on the species, the central duct is lined by a single-layered or pseudostratified epithelium containing a variety of cell types, including mucocytes and interdigitated duct cells, undifferentiated basal and intermediate cells, and granulated cells, possibly of endocrine function (Komnick, 1985). Neighboring cells of the stratified

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epithelium of the rectal gland excretory duct are connected by junctions comprising 12 interconnecting strands, resembling a typical tight epithelium (Claude and Goodenough, 1973).

111. Blood Supply t o the Salt Gland A. Salt Gland Microvasculature Adequate blood supply to the supraorbital salt glands, first mentioned by Jobert (1869) and described in more detail by Marples (1932), represents a necessary prerequisite for active transcellular ion transport. Besides gross morphologic observations and light- or electron-microscope techniques using (ultra)-thin tissue sections (Komnick, 1963b), vascular corrosion castings of the salt gland vasculature, first performed by Fange et al. (1958b) using methacrylate injections into the common carotid arteries and only recently refined (Hossler and Olson, 1990), have proved helpful in unraveling the glandular microvasculature (Fig. 7). Of the marine birds, the salt gland vasculature of only the Herring gull and the saltwater-acclimated Pekin duck has been thoroughly investigated, with some additional information available concerning the nasal salt gland of the Adelie penguin. The salt gland of duck species is supplied by branches of both the ophthalmic and the supraorbital arteries with final drainage of its vascular bed by the ethmoidal and ophthalmic veins (Peaker and Linzell, 1975; Hossler and Olson, 1990). All blood vessels feeding into the glandular parenchyma approach the duck salt gland from its medial surface. Whereas the ethmoidal arterial vessels reach mainly the central and superior portions of the gland, branches of the supraorbital artery supply its temporal portion. With arteries and veins closely intertwined, anastomoses occur between veins and arterioles originating from both the ethmoidal and the supraorbital arteries (Hossler and Olson, 1990). In gull species, the supraorbital salt gland appears to be supplied by the external as well as internal ophthalmic artery (Fange et al., 1958b), with both blood vessels originating from the internal carotid artery. The posterior branch of the internal ophthalmic artery anastomoses with the external ophthalmic artery supplying the caudal portion of the gull’s salt gland, whereas the anterior portion of the internal ophthalmic artery sends off numerous branches to the rostra1 portion of the gland. This arrangement of both arteries inspired Fange and co-workers (l958b) to postulate that “blood could probably bypass the gland via the arterial arch and permit a large reduction in glandular blood flow without reducing the blood flow to the upper beak when the glands are not functioning,” a hypothesis not yet supported by physiologic investigations. In the Adelie penguin, blood vessels probably originating from the supraorbital artery

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pass into the salt gland from the orbit through holes in the frontal bone (Herbert, 1975). Comparing the various anatomical descriptions of avian salt gland blood supply, Hossler and Olson (1 990) presented a scheme whereby the ethmoidal artery was synonymous with the cerebral carotid artery, and the supraorbital artery with the external ophthalmic artery. As mentioned before, the avian salt gland is embedded in a connective tissue capsule with septa diverging into the secretory parenchyma of the gland, thus separating single secretory lobes from each other. Arterial blood vessels proceeding within these interlobular connective tissue bundles merge into arterioles that extend to the connective tissue layers surrounding the central canal (Van Lennep and Young, 1979). A detailed description of these small interlobular arterioles at the electron-microscopic level excluded their participation in the exchange of metabolites between the secretory tissue and the plasma, thus restricting their main task to the sufficient supply of water and electrolytes for the secretory process of the salt gland principal cells (Komnick, 1963b). These arterioles then break up into capillaries winding along single secretory tubules with blood flowing in opposite direction to the flow of the intratubular secretory fluid (Figs. 7 and 8). Each secretory tubule is surrounded by a very dense network of three to seven capillaries (Fig. 2) running toward the periphery of the secretory lobe, equipped with fenestrations 30 to 40 nm in diameter, with short distances between the endothelial cells and the basement membrane of the secretory epithelium (Schmidt-Nielsen, 1960; Ellis et al., 1963; Komnick, 1963b). Near the periphery of the lobes, these capillaries finally pass over into a venous plexus drained by veins in the interlobular connective tissue (Fig. 7). In contrast, the vasculature supplying the epithelium of the central ducts is represented by a simple capillary network. Comparative studies in the salt-secreting gland of a reptile, the chuckwalla Sauromalus obesus, revealed a similar organization of its microvasculature with the stroma surrounding the main excretory duct being penetrated by many large arterioles branching into a dense network of capillaries. These capillaries are closely associated with the principal tubules, with three to six of them surrounding each tubule. The capillaries merge into large venous sinuses situated at the periphery of the gland (Barnitt and Goertemiller, 1985). With regard to the shark rectal gland, microfil injections of the vasculature showed that the single rectal gland artery arising from the dorsal aorta divides into numerous circumferential arterioles. These finally branch into a dense capillary bed running parallel to the secretory tubules to terminate in a large central venous sinus, quite different from the situation in the avian salt gland (Hayslett et al., 1974). Concerning local autoregulatory control mechanisms of avian salt gland microcirculation, fine structural analysis indicated possible local contractility due to the arrangements of collagen and elastic filaments and “passive transformation of endothelial cells” (Komnick, 1963b). Using scanning electron microscopy of vascular corrosion castings, sphincter-like vasoconstrictions have

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FIG. 7 Microvasculature of the avian salt gland. (a) Schematic drawing of the blood supply to a secretory lobe of the duck avian salt gland after ink injection. Arterial blood from an external artery runs in a dense capillary bed from the central ductule toward the rim of the secretory lobe with subsequent venous drainage. (b) Scanning electron micrograph (SEM) of a vascular corrosion cast of a duckling head (dorsal view) showing the supraorbital salt glands (SG).Bar, 200 pm. (c) SEM of a cross section through a salt gland corrosion cast. Large veins (V), interlobular arteries, and secretory ducts (D), as well as several arterioles (arrows) dividing into capillaries are visible. Bar, 200 pm. (d) SEM of the ventromedial surface of a salt gland corrosion cast. Large veins (V),arteries (A), and the venous plexus of single secretory lobes can be distinguished. Bar, 200 pm. ( e ) SEM of

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been observed in a few arterioles close to the point of feeding into the peritubular net of capillaries (Hossler and Olson, 1990).

B. Blood Flow-Secretion Coupling Adaptation of birds kept on freshwater to a hypertonic saline regimen not only resulted in the development of highly specialized secretory tissue, but also coincided with enhanced angiogenesis as shown by morphometric analysis. Thus, angiogenesis after salt acclimation of ducklings is supported by both a marked rise in the weight of glandular corrosion castings and the incorporation of tritiated thymidine into capillary endothelial cells (Hossler, 1982; Hossler and Olson, 1990). In addition, the use of polarographic oxygen electrodes revealed a high arterial blood supply when salt gland tissue had undergone functional hypertrophy due to salt acclimation, and clearly indicated increased arteriolar perfusion with enhanced secretory activity (Fiinge el al., 1963; Peaker and Linzell, 1975). As indicated before, the carotid arteries are almost exclusively responsible for the arteriolar blood supply to the avian salt glands. Recordings of carotid artery blood flow in the Pekin duck using magnetic flow probes yielded values of about 30 ml/min for both blood vessels under thermoneutral conditions (Bech et al., 1982; R. Gerstberger, unpublished observation), indicating that the nonactivated salt glands receive some 5% of the blood perfusing the head region. During systemic osmotic stimulation (0.4 mOsm/min NaCl infusion), carotid blood flow increased to 55 ml/min at an average salt gland-specific perfusion of 15 ml/min for both glands (1.O g tissue weight) (Fig. 8), thus representing 27% of total carotid blood flow due to massive local vasodilation (Gerstberger et al., 1984a; Butler et al., 1989). Stimulated carotid blood flow values were also observed in geese maintained on hypertonic saline as drinking fluid after a systemic injection of 10 ml of a 10% saline or 20% sucrose solution, whereas arterial pressure or heart rate remained unchanged (Burford and Bond, 1968). Graded osmotic stimulation of saltwater-acclimated Pekin ducks ranging from 0.05 to 0.8 mOsm/min induced steady-state salt gland secretion matching the applied salt and water load for all steps chosen. In parallel, arterial blood flow through the glands, as measured using the radioactive microsphere technique, increased with a linear relationship between both functional parameters

the venous plexus of single secretory lobes (corrosion cast) from a duck salt gland. Bar, 200 pm. (0 SEM of a cross section through the gland showing two secretory lobes with the central canals ( m o w ) and the radiating arrangement of capillaries. Bar, 200 pm. (g) SEM of the capillary network supplying the epithelium of the main secretory duct. Bar,SO pm. [(a)From Butler ef al., 1991,copyright 0 1991; ( b g ) from Hossler and Olsen, 1990, copyright 0 1990. All by permission of WileyLiss, a division of John Wiley and Sons Inc.]

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(Kaul et al., 1983) (Fig. 8). Maximal values measured by quantitative radioactive microsphere technique were in the order of 35 ml/min/g wet salt gland tissue, thus representing a more than 20-fold rise in specific blood flow (Kaul et al., 1983; Butler et al., 1989; Gerstberger, 1991). This enormous capacity of the vascular system of the gland could also be verified in freshwater-acclimated geese, where the intravenous application of large volumes of hypertonic saline caused a marked rise in extracellular fluid volume. A subsequent increase in cardiac output at elevated heart rate but unchanged mean arterial pressure was measured. During maximally elevated cardiac output, only salt gland blood flow and coronary circulation were augmented, as determined after 25-30 min. Harderian gland blood flow was not affected by saline loading (Hanwell et al., 1971a,b). For the duck, tight coupling of blood flow and osmolal excretion at a higher time resolution could also be observed when laser-Doppler flowmetry was used to follow local superficial blood flow in the activated salt gland during systemic hypertonic saline administration (Gerstberger et al., 1988; Gerstberger, 1991). This secretion-related rise in glandular blood flow proved to be highly specific for the salt glands, as indicated by unchanged flow values for various vascular beds including brain, Harderian gland, eye, heart, lung, kidney, liver, pancreas, gut, spleen, breast muscle, and web skin (Kaul et al., 1983: Gerstberger, 1991) (Fig, 8). From the proportional increase in blood flow coupled with secretion at a known status of the extracellular fluid compartment, it could be calculated that the secretory epithelium in the duck salt gland removed a nearly constant 20% fraction of the salt delivered to it, producing an arteriovenous difference for "a+] and [Cl-] of 20 and 15 meq/liter, respectively (Kaul et al., 1983) (Fig. 8). The rate of salt gland secretion in the goose appeared to be very loosely correlated with organ blood flow. How exact extraction values for various ions from the plasma (15% for Na+, 21% for C1-, 35% for K+, and 6% for water) could

FIG. 8 Functional aspects of blood flow through the avian salt gland. (a) Schematic drawing of the position of the supraorbital salt gland in the Herring gull. (b) Diagram of the microcirculation in the salt gland of a Herring gull. Countercurrent arrangement of capillary blood flow relative to the flow of secretion within the secretory tubules. (c) Linear relation between osmolal excretion and salt gland blood flow in osmotically stimulated conscious saltwater-acclimated Pekin ducks (SW-AD) (top). and percentage of salt extracted from blood perfusing the salt glands (bottom).(d) Schematic drawing of the unequal distribution of microspheres, injected intracardially, in capillaries of various secretory lobes of the activated salt gland during osmotic stimulation in a SW-AD. (e) Pattern of local salt gland blood flow measured by laser-Doppler flowmetry (flux) during osmotic stimulation in a SW-AD. (0 Organ-specific blood flow (ml/min/g) in various organs of SW-AD during control conditions and systemic hypertonic salt loading (0.4 mOsm/min) as measured by the radioactive microsphere technique. [(a) From Schmidt-Nielsen, 1960. with permission: (b) from Fange et d . , 1958a, with permission: (c) from Kaul ef a)., 1983, with permission.]

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be calculated under these conditions remains to be clarified. The absence of a close correlation of whole organ blood flow and secretion in the goose, as demonstrated for the Pekin duck, might have been due to (a) the manner of OSmotic stimulation not allowing the animal to reach steady-state secretion and (b) the Saphirstein dilution technique, with its inherent inaccuracies such as time delay or tissue preparation (Hanwell et al., 1971b). Despite this generally tight linear relationship between whole organ blood flow and secretion rate in the duck (Kaul et al., 1983), investigations canied out under conditions of high time resolution, employing the recording of local salt gland blood perfusion or the application of various (anti)-secretagogues (see Section VII), revealed partial uncoupling of both blood flow and secretion. Locally measured lobular perfusion varied from almost total vasoconstriction to maximal vasodilation with all intermediate levels possible. The additional observation of vasomotion-like vasoconstrictions and vasodilations during the recording of local “single lobe” salt gland blood flow by laser-Doppler flowmehy, in combination with the uneven distribution of trapped microspheres in various secretory lobes at a fixed time during ongoing secretion, favors the idea of redistribution of intraglandular blood flow to active lobes at a given time with constant whole organ blood perfusion (Gerstberger, 1991) (Fig. 8). Thus the avian salt gland can (a) quickly respond to variable demands by pumping sodium and chloride against a high concentration gradient and (b) alter transcapillary fluid exchange locally due to the vasomotion of precapillary arterioles (Meyer and Intaglietta, 1986). Partial uncoupling of both parameters under various physiologic and pharmacologic conditions is also reported for other exocrine gland systems, with studies in the nonhomologous salt-secreting rectal gland yielding conflicting results. On the one hand, stimulated secretion at increased perfusion rate of an in vitro preparation and diminished excretion of chloride after reduction of the perfusion flow have been reported (Hayslett et al., 1974; Shuttleworth and Thompson, 1986). Also, using an in vivo system, augmented blood flow to the rectal gland during isotonic intravascular volume expansion at unchanged arterial pressure was demonstrated, with secretion being enhanced subsequently (Solomon et al., 1984b, 1985a). On the other hand, treatment with somatostatin or the Na-2Cl-K transport blocking agents bumetanide and furosemide suppressed stimulated secretion in the shark rectal gland, while leaving the glandular blood flow unaffected (Shuttleworth, 1983b; Solomon et al., 1984b). Exocrine secretion independent of arterial blood supply has also recently been described for gastric acid secretion in mammalian species under conditions of high blood perfusion. At low blood flow rates with possibly insufficient oxygen delivery to the stomach mucosa, however, acid secretion is linearly dependent on mucosal blood flow (Perry et d., 1983; Holm-Rutili and Berglindh, 1986; Guth and Leung, 1987). In cat salivary glands, vasoactive substances often cause marked vasodilation after intraarterial injection without concomitant secretion (Lundberg, 1981).

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IV. Salt Gland Innervation A. Nerve Supply t o the Salt Gland 1. Gross Morphology Exocrine glands such as the salivary, lacrimal, Harderian, and bronchial glands, and the pancreas, are known to be innervated by the autonomic nervous system, with the conduction pathway from the central nervous system being made up of preganglionic and ganglionic neurons. Preganglionic fibers emerging from the brain stem in cranial nerves extend through rami of the respective nerves into peripheral ganglionic collections of neurons such as the submandibular, sphenopalatine, or ciliary ganglion, often called parasympathetic ganglia, representing the synaptic sites. With regard to avian salt gland innervation and according to early anatomical observations in the goose, the ramus ophthalmicus of the fifth cranial nerve, before leaving the orbita of the skull, gives off side branches. Two of them enter the orbital ethmoidal ganglion located between the orbital wall and the Harderian gland (Cords, 1904). In addition, a nerve containing fibers of the anterior branch of the nervus facialis, the seventh cranial nerve, and the nervus glossopharyngeus as well as sympathetic fibers enters the ganglionic cell mass. Nerve branches leaving the ganglion diverge to the salt gland, the Harderian gland, or the frontal orbital edge (Cords, 1904). Serial sections of this region in the salt glands of the Pekin duck and the Herring gull did not reveal any connections between the fifth cranial nerve and the secretory nerve (Hakansson and Malcus, 1969; Cottle and Pearce, 1970) (Fig. 9). Despite lack of information due to the inaccessibility of the salt gland innervation, the anterior branch of the seventh cranial nerve must be considered the true secretory nerve, at least in the duck, goose, and gull. Gross morphologic investigations in the Adelie penguin, however, favored the hypothesis of a branch of the fifth cranial nerve innervating the salt glands, with numerous small nerve branches, possibly postganglionic in nature, passing around the frontal edge of the gland (Herbert, 1975). In lizards, the nasal salt glands opening into the anterior chamber of the nasal sac appeared to be innervated by a branch of the lateral ethmoidal nerve or the opthalmicus profundus (Oelrich, 1956; Duvdevani, 1972).

2. Nerve Stimulation-Secretion Coupling Functional indications for the efferent innervation of the salt glands playing a major role in the control of cellular Na+ and C1- transport could be derived from the suppressive action of anesthesia on salt gland blood flow and secretion in osmotically stimulated geese (Hanwell et al., 1971b). In addition, electrical stimulation of the secretory nerve (branch of the seventh cranial nerve) in the

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FIG. 9 Fiber innervation of the avian salt gland. (a) Three-dimensional schematic reconstruction of the secretory nerve (SN filaments), the secretory nerve ganglion (SNG), the ophthalmic branch of the fifth cranial nerve (Vth), and postganglionic fibers innervating the salt gland (SG) of a Pekin duck. (b) Light-microscopic cross section of the secretory nerve ganglion of a Pekin duck salt gland with the secretory nerve (SN) emerging (arrow).Bar, 100 pm.(c) Electron micrograph (EM) of a longitudinal section of a neuronal fiber branch protruding into the secretory parenchyma of a Japanese swan goose salt gland. Bar, 1 pm. (d) Nerve fiber endings on secretory nerve ganglion cells (Bielschowsky staining), revealing button-like varicosities (arrows). Bar, 20 pm. (e) EM of numerous

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anesthetized gull resulted in copious secretion with sodium concentrations as high as 900 meq/liter (Fange et a/., 1958a). To the contrary, the unilateral denervation of one salt gland led to a reduction in blood flow and secretion of the ipsilateral gland compared to the contralateral gland with intact innervation, suggesting that functional parasympathetic innervation is a necessary prerequisite for salt gland function (Hanwell et al., 1971b). The parasympathetic nature of efferent salt gland innervation was deduced from histochemical techniques (see Section IV,B). Using acetylthiocholine as substrate, cholinesterase activity was determined in salt gland tissue sections, with no variation in staining density observed between freshwater-adapted and short-term saltwater-adapted animals (Ash et a/., 1969; Fourman, 1969). Neural control of hypertonic salt secretion against a concentration gradient was also demonstrated for the shark rectal gland. Veratrum alkaloids led to a pronounced chloride secretion from isolated shark rectal glands (Erlij et al., 1981; Stoff et al., 1988). These agents are known to depolarize excitable tissues via increased sodium permeability of their plasma membranes in a way that can be blocked by the sodium channel inhibitor tetrodotoxin. In a series of experiments performed in freshwater-acclimated ducks just recovered from deep anesthesia, the complete denervation of the supraorbital salt glands in the duck including the removal of the secretory nerve ganglion resulted in the expected abolition of saline-induced secretion as well as histochemical cholinesterase staining (Ash et a/., 1969). Sectioning of the secretory nerve central to its ganglionic component, however, surprisingly did not cause glandular unresponsiveness to elevated plasma tonicity. The cholinesterase staining was not markedly reduced with choline acetylase activity lowered to less than 10% of control values. The section of the secretory nerve posterior to the ganglionic region did not appear to contain cholinesterases, indicating that the secretory nerve might be “a mixture of somatic afferent and sympathetic post-ganglionic nerves’’ (Ash et a/., 1969) and that the ganglionic cells themselves might possess osmoreceptive functions. The latter hypothesis appeared to be strengthened by the ineffectiveness of large doses of hexamethonium in influencing salt-induced salt gland secretion. The only electrophysiologic recordings of secretory nerve action potentials available indicated the presence of three fiber populations (Cottle and Pearce, 1970). The inaccessibility of these small fibers might have been the reason for

nerve terminals filled with small translucent (cholinergic) and large electron-dense (peptidergic) vesicles, running parallel at the basis of a secretory salt gland cell (Pekin duck). Bar, 0.3 pm. (f) EM of two nerve terminals in close vicinity to the basal infoldings of a salt gland secretory cell (Japanese swan goose). Bar, 0.3 pm. [(a,b,d) From Cottle and Pearce, 1970, with permission; (c) Unpublished material. courtesy of Prof. Dr. W. Kiihnel; (e) From Hootman and Emst, 1981b, reproduced from J . Cell. B i d . (1981). 91, 781-789; (0 from Kiihnel, 1972, with permission.]

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the negative cholinesterase staining reported by Ash el al. (1969) for the preganglionic nerve fibers. An intact nerve supply to the salt gland appears to be necessary not only for its function, but also for the adaptive hypertrophy during salt adaptation of the birds as demonstrated for geese and ducks with unilateral postganglionic denervation. As mentioned before (see Section KC), both glandular hypertrophy and RNA content or RNA/DNA ratio were enhanced in the intact gland compared to the denervated one (Pittard and Hally, 1973; Hanwell and Peaker, 1973, 1975).

3. The Ethmoidal Ganglion The neuronal “relay station” in avian salt gland innervation, as indicated, is represented by the orbital ethmoidal ganglion (Fig. 9), whose existence in avian species such as the duck or goose was denied in the early anatomical studies by Gaupp (1888). This structure, possibly synonymous with the sphenopalatine ganglion (Webb, 1957), was described to have connections with the nervus trigeminus (fifth cranial nerve), the nervus facialis (seventh cranial nerve), the sympathetic system, and possibly the nervus glossopharyngeus (ninth cranial nerve) (Cords, 1904; Marples, 1932; Webb, 1957). In the salt gland of the Herring gull, parasympathetic nerve fibers of the ramus palatinus of the facial nerve run parallel to the ramus ophthalmicus of the trigeminal nerve. Numerous synapses occur along the common nerve form the ethmoidal ganglion composed of two major ganglionic cell masses located at the common nerve trunk (distal mass) and the junction with fibers branching off to the salt gland and Harderian gland (proximal mass) (Hakansson and Malcus, 1969). For the duck salt gland, serial histological sections enabled exact tracing of postganglionic nerve fibers entering the parenchyma after “travelling around the opthalmic division of the Vth cranial nerve” (Cottle and Pearce, 1970) (Fig. 9). Ganglionic cells, the majority confined to the region where the secretory nerve becomes associated with the fifth cranial nerve, were also found in postganglionic fiber bundles and among fine bundles within the gland. In the Herring gull, the salt gland is evidently provided with postganglionic fibers originating from the distal ganglion portion (Hakansson and Malcus, 1969). Lightmicroscope investigations did not enable the processes and fiber endings to be followed for long distances, whereas EM revealed the presence of synaptic elements on secretory nerve ganglion cells, indicating that they indeed represent the synaptic sites (Cottle and Pearce, 1970) (Fig. 9).

4. Ultrastructure of the Nerve Endings With regard to ultrastructural aspects of salt gland innervation, larger interstitial nerves composed of several axons surrounded by a Schwann’s cell sheath have

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been reported in the duck salt gland. Nonmyelinated fibers leaving the secretory nerve ganglion with subsequent passage into the anterior portion of the gland have been described for the Herring gull salt gland (Fange et al., 1958a). Fawcett (1962) was the first to describe these nonmyelinated fibers running outside the basement membrane, often penetrating the basement membrane to ramify in the gland parenchyma (Fig. 9) with terminal endings in close spatial relationship to secretory cells as well as to collecting duct cells (Kiihnel, 1972; Hootman and Emst, 1981b). Nerve endings were often found in close association with the base of the secretory cells, separated from them by the basal lamina and a layer of connective tissue only (20 nm) (Fig. 9). Terminal axonal swellings have been described in the salt glands of the swan, goose, Pekin duck (Kiihnel, 1972), Eider duck, and Japanese swan goose (W. Kiihnel, personal communications). Pre- and postsynaptic membrane specializations typical of synaptic contacts between adjacent neuronal cells or the neuromuscular junction were not reported. Both in the perivascular space and independent from blood vessels, nerve endings could be located, with their axoplasm containing neurotubules, neurofilaments, mitochondria, and synaptic vesicles of electron-translucent and electron-dense content and varying size (Kiihnel, 1972; Hootman and Emst, 1981b; Lowy and Emst, 1987) (Fig. 9). The smaller, agranular vesicles of 50-nm diameter are characteristic of cholinergic endings, whereas aldehyde fixation in the absence of ferrocyanide revealed that the larger vesicles (100 nm diameter) appeared to have a dense core separated from the surrounding membrane by a thin electron-translucent region typical for peptidergic synaptic vesicles described in other exocrine glands (Lundberg, 1981).

6. Cholinergic Innervation 1. Histochemistry

As indicated before, the avian salt gland receives mainly parasympathetic innervation, as is typical for other exocrine glands. Histochemically, the positive staining for acetylcholinesterase activity was most often taken as a strong indication of cholinergic innervation, despite some difficulties in the interpretation of results obtained due to the presence of this enzyme in some nonneuronal tissues and the possibility that the substrates employed might also be used by butyrylcholinesterase of nonneuronal origin (Peaker and Linzell, 1975). On the one hand, using acetylthiocholine as substrate (Fig. lo), cholinesterase activity was determined in salt gland tissue sections with no variation in staining density observed between freshwater-acclimated and short-term saltwater-acclimated animals (Ash et al., 1969; Fourman, 1969). Ellis and co-workers (1963), on the other hand, reported a different pattern for both groups of animals, with

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FIG. 10 Cholinergic and catecholaminergic innervation of the avian salt gland. (a) Histochemical demonstration of acetylcholinesterase in a salt gland tissue section of a saltwater-acclimated Pekin duck (SW-AD). Bar, 200 pm. (b) Acetylcholinesterase activity near the periphery of salt gland lobes (SW-AD). Bar represents 200 pm. (c) Fluorescent adrenergic nerve fibers in the salt gland following treatment of tissue sections with formaldehyde vapor. Fibers run parallel with secretory tubules with button-like varicosities. Bar, 100 pm. (d) Receptor autoradiogram of intact salt gland sections incubated with [3H]quinuclidinyl benzilate (QNB), a muscarinic receptor antagonist. Silver grain densities are highest over the basal and lateral surfaces of secretory epithelial cells (arrows). Bar, 5 pm. [(a,b) From Ash e r a / . , 1969, with permission; (c) from Peaker and Linzell, 1975, with permission; (d) from Hootman and Ernst, 1982, with permission.]

cholinesterase-positive nerves running through the interlobular, intralobular, and peritubular connective tissue, interlacing fibers adjacent to the secretory tubules but also some endothelial cells of glandular capillaries. Comparative studies in reptilian salt-secreting glands revealed that the compound branched tubular salt-

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secreting glands of sea turtles receive a dual innervation, with one type of subcapsular nerve fibers staining positive for cholinesterase (Abel and Ellis, 1966; Dunson, 1976).

2. Pharmacologic and Physiologic Characterization To elucidate the physiologic significance of parasympathetic innervation for the directed ion transport activity of salt gland secretory cells, the cholinomimetic agent metacholine was administered to isolated salt gland slices, increasing their oxygen consumption by about 50% whereas an increase in the sodium concentration of the medium inhibited cellular metabolism (Borut and SchmidtNielsen, 1963). Treatment of dispersed salt gland cells with metacholine as a muscarinic agonist also resulted in augmented cellular oxygen consumption and enhanced binding of [3H]ouabain, suggestive of stimulated turnover of intrinsic sodium pumps (see Section V,A) (Hootman and Emst, 1981a). Using structurally polarized sheets of primary secretory cell cultures with transmural resistance values in the order of 300 !2 X cm2, the functional (para)sympathetic innervation of the secretory cells themselves could be tested by measuring alterations in short-circuit current (SCC) across the cell layer during drug application (Lowy ef al., 1985a,b) (Figs. 10,12). To mimic parasympathetic innervation, the addition of the cholinergic agonist carbachol to the abluminal side induced an increased SCC in a positive orientation from the luminal to the abluminal side. This transport-stimulating effect could be abolished by the abluminal application of the muscarinic antagonist atropine (Fig. 12). Cell layers grown in the presence of carbachol revealed a marked agonist-induced desensitization, possibly by down-regulation of the muscarinic receptor (Lowy et al., 1985b). To characterize and quantify acetylcholine receptors in the avian salt gland, the tritiated muscarinic receptor antagonist quinuclidinyl benzilate (QNB) was used as radioligand (Hootman and Emst, 1981b, 1982; Hildebrandt and Shuttleworth, 1991b). Binding to an enriched salt gland membrane fraction proved to be saturable and of high affinity with half-maximal binding values (K,) of 35-40 pM regardless of the osmoregulatory status of the animal. Concerning the regulation of receptor numbers per cell or unit of membrane protein during the process of salt acclimation, contradictory results have been obtained by various authors. Hootman and Emst (1981b), working with ducklings adapted to 1% saline for at least a month, reported an up-regulation of cholinergic receptor molecules from 8800 to 14,000 sites per dissociated cell. Hildebrandt and Shuttleworth (1991b), on the other hand, just recently described a down-regulation with values of 532 fmol/mg protein versus 165 fmol/mg protein, using freshly dissociated cells of control ducklings and animals adapted to a 1% saline regimen for only 48 hr. Competitive displacement studies demonstrated high potency of muscarinic antagonists to displace radiolabeled QNB, with lower

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affinities for muscarinic agonists. The ion transport inhibitor furosemide reduced QNB binding to the muscarinic receptor in the duck salt gland, quite different from measurements in other tissues (Hootman and Emst, 1981b). [3H]Quinuclidinyl benzilate binding sites could be localized autoradiographically in principal secretory cells, primarily along the basolateral membrane surface (Hootman and Emst, 1982) (Fig. 10). Whole animal studies yielded the final proof for a functional cholinergic innervation of the avian salt gland. Acetylcholine administered to the carotid arteries perfusing the supraorbital salt glands of conscious saltwater-acclimated ducks or gulls, thus mimicking cholinergic innervation, induced increased 0smolal excretion (Fange et al., 1958a; Gerstberger et al., 1988). Supporting the idea of Bonting and co-workers (1964) that “the cholinergic mechanism controls the gland indirectly through cholinergic vasodilator nerves” is the finding that acetylcholine also markedly enhanced glandular vasodilation with an unchanged general cardiovascular status of the animal (Gerstberger et al., 1988) (Fig. 12). In addition, ongoing steady-state salt gland secretion was totally inhibited by muscarinic antagonists such as atropine or tridihexetylchloride. Glandular blood flow as measured by laser-Doppler flowmetry or the radioactive microspheres technique was reduced to a low atropine-resistant level (Kaul et al., 1983; Gerstberger et al., 1988), as already observed by Fange and colleagues (1958a). The ineffectiveness of atropine to inhibit salt gland blood flow in the goose (Hanwell et al., 1971b), despite abolished secretion, is difficult to discuss. Only two experiments were performed with low doses of atropine. Thus, atropine may not have been able to interfere with vascular cholinergic receptors, while being effective in blocking the muscarinic receptors associated with the secretory principal cells.

C. Vasoactive Intestinal Polypeptide 1. Immunologic Characterization Dense-core vesicles of 100-120 nm in diameter, often containing peptidergic neuromodulators (Lundberg, 1981), were first shown by Kuhnel (1972) for the avian salt gland to be coexistent with small translucent cholinergic vesicles in the same nerve terminal. Analogous to studies in other exocrine glands, such as salivary glands (Bryant et al., 1976), lacrimal glands (Lundberg, 1981), or the pancreas (Sundler et al., 1978), vasoactive intestinal polypeptide (VIP) was found to be a plausible neuromodulator candidate. Immunocytochemistry using VIP-specific antisera indeed revealed the presence of VIP-positive nerve fibers throughout the salt gland parenchyma with terminals ending in the intimate vicinity of both arterioles and the basal membrane of secretory tubular cells (Lowy el al., 1987; Gerstberger, 1988) (Fig. 11). Beaded VIP-positive fiber

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structures could also be localized penetrating into the elastic medial layer of larger arteriolar blood vessels. Although preganglionic fibers did not stain for VIP-like material, ganglion cells and postganglionic fibers showed immunoreactivity, indicative of VIP synthesis in the ethmoidal ganglion. The chromatographic separation of peptidergic extracts with subsequent quantification by radioimmunoassay allowed detailed characterization of endogenous VIP in the salt gland of the saltwater-acclimated Pekin duck, whereas other well-known peptidergic neuromodulators involved in body fluid homeostasis were undetectable (Gerstberger, 1988). Electron microscopically, VIP immunoreactivity could be clearly located within the peptidergic dense-core vesicles in the synaptic nerve endings using either the peroxidase anti-peroxidase (PAP) preembedding or the immunogold postembedding techniques (R. Gerstberger and F. Niirnberger, unpublished observations) (Fig. 11). Thus, VIP-like immunoreactivity was localized in vesicles of synaptic profiles otherwise dominated by translucent cholinergic vesicles. The VIP nerves of the avian salt gland may therefore not be of the “ptype” reported by Baumgarten er al. (1980). Comparable to the findings in the duck salt gland, VIP-like immunoreactive nerve fibers could also be localized in the rectal gland of Squalus acanthias. Thick fibers exhibiting VIP-like immunoreactivity were detectable in its fibromembranous capsule, with thinner branches taking off into the glandular parenchyma and running alongside the epithelial cells surrounding secretory tubules (Chipkin et al., 1988a,b). These fibers were nonmyelinated, as demonstrated by EM. Within the nerve terminals and consistently located along the basal aspects of peritubular cells, the reaction product was confined to densecore vesicles 80-120 nm in diameter. The authors argue that VIP released in the capsular zone, which contains major circumferential branches of the rectal gland artery and plexus of smaller vessels supplying the parenchyma, might affect blood flow to the gland (Chipkin er al., 1988b).

2. Pharmacologic and Physiologic Characterization Comparable to the results obtained with cholinergic agonists, coherent sheets of principal secretory cells responded to VIP treatment with dose-dependently augmented ion transport rates, as indicated by stimulated SCC. Short-circuit current values increased transiently even above the sustained plateau induced by cholinergic agonists (Lowy et al., 1987) (Fig. 12). Vasoactive intestinal polypeptide applied close-arterially to the salt glands of conscious ducks under experimental conditions of threshold secretion (Hammel et al., 1980) caused a marked dose-dependent rise in glandular water and salt elimination from the extracellular space at augmented arterial blood flow through the gland (Gerstberger er al., 1988) (Fig. 12). Representing the morphologic and biochemical correlate for the physiologic findings, high-affinity binding sites specific for radiolabeled VIP

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were found to be concentrated at the basal aspects of the secretory tubules surrounding the central canal of a secretory lobe (Fig. 11). Ligand binding could be inhibited by unlabeled VIP and a peptidergic salt gland extract, but not various VIP analogs tested (Gerstberger, 1988). In the shark rectal gland, veratrum alkaloids induced the release of VIP-like material into the venous effluent of the preparation. The suppression of both veratridine-induced secretion and VIP release by tetrodotoxin or neural blockade suggest that a VIP-like peptide might act as functional neuromodulator in the efferent control of the shark rectal gland (Shuttleworth and Thorndyke, 1984; Stoff el al., 1988).

3. VIP-Acetylcholine Interaction Acetylcholine and VIP could be located in different populations of vesicles in the identical nerve terminal, and both agents enhanced arteriolar blood supply to, and osmolal excretion by, the avian salt gland. Putative interactions of both neurotransmitters and neuromodulators were therefore tested in vifro and in vivo. First, pretreatment of the cultured cells with the muscarinic antagonist atropine did not inhibit the V1P-mediated response (Lowy et al., 1987), nor did muscarinic antagonists suppress VIP-induced vasodilation in the duck salt gland (Gerstberger et al., 1988). Second, the coinfusion of both acetylcholine and VIP did not result in superposition of the vasodilatory actions of each neuromodulator alone, whereas stimulated osmolal excretion appeared to be additive, thus excluding interaction of both agents at the same target. The interplay between cholinergic agonists and VIP in the control of glandular functions proved to be even more complex in other exocrine glands such as the cat salivary glands (Lundberg, 1981). Acetylcholine caused both glandular vasodilation and secretion. Different from the avian salt gland, however, the local intraarterial application of exogenous VIP induced an atropine-resistant vasodilation but no salivary excretion. On the other hand, the infusion of VIP antiserum

FIG. 11 Vasoactive intestinal polypeptide (VIP) as putative neuromodulator in avian salt gland innervation. (a) Light-microscope immunocytochemic demonstration of VIP-positive fibers found throughout the secretory parenchyma of the duck salt gland. Bar, 20 pm. (b) Indirect immunofluorescence for VIP-like material in salt gland tissue sections, with positive labeling of nerve terminal aggregates at the base of secretory tubules (ST). Bar, 10 pm. (c) Electron-microscope demonstration of granular vesicles (G) contained in nerve terminals (NT) close to the basal membrane (BM). immunopositive for VIP-like material using the preembedding Sternberger-PAP technique. Bar, 0.2 pm. (d) Postembedding immunogold staining of granular vesicles (G) in salt gland nerve terminals (NT) containing VIP-like material as neuromodulator. Bar, 0.2 pm. (e) Receptor autoradiogram of an intact salt gland tissue section using radioiodinated ‘2sI-labeled VIP as ligand. Highest receptor densities can be localized at the outer portions of the secretory lobes and associated with main arteries. Bar, 500 pm. [(a,b,e) From Gerstberger. 1988, with permission.]

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FIG. 12 Functional nervous control of avian supraorbital salt gland function. (A) Response pattern of local salt gland blood flow measured by laser-Doppler flowmetq (upper panel) and osmolal excretion (lower panel) to the intracarotid short-term (10 min) application of acetylcholine (ACH), vasoactive intestinal peptide (VIP), and norepinephrine (NOR) in saltwater-acclimated Pekin ducks. (B) Alterations in SCC oriented positive from the luminal to the abluminal side of polarized primary cell cultures of the duck salt gland during bath (luminal or abluminal) application of the muscarinic agonist carbachol (CARB), the phosphodiesterase inhibitor theophylline (THEO), the muscarinic antagonist atropine (ATRO), epinephrine (EPI), the P-adrenergic antagonist propranolol (PROP), the Na-K-ATPase inhibitor ouabain (OUAB), or amphoterecin B (AMPHO) to increase membrane permeability for cations. [(B) From Lowy et al., 1985b. with permission.]

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reduced both vasodilation and salivary secretion. Vasodilatory and secretory responses were augmented during coapplication of acetylcholine and VIP. A possible explanation for this potentiation is presented by (a) a marked increase in immunoreactive VIP in the venous effluent of the gland simultaneously with salivary secretion and vasodilation and (b) the enhancement of muscarinic receptor binding in the presence of VIP (Lundberg ef al., 1982). To make matters even more complicated, muscarinic blockade partially reduced organ-specific vasodilation in the cat submandibular gland only at low parasympathetic stimulation, whereas at high parasympathetic stimulation muscarinic blockade even enhanced glandular vasodilation, possibly due to simultaneously augmented release of VIP. Salivary gland secretion was always totally abolished (Lundberg, 1981). A comparable experimental approach for the avian salt gland might yield interesting results with regard to the co-joint action of two “first messengers” at a common target.

D. Adrenergic Innervation 1. Histochemistry The existence of a sympathetic portion in avian salt gland innervation has already been proposed by early morphologists. Using the Falck-Hillarp technique to demonstrate the presence of monoamines, adrenergic fibers could finally be demonstrated, showing a distribution pattern similar to those stained for cholinesterase in the avian salt gland (Haase and Fourman, 1970) (Fig. 10). Electron microscopy revealed that adrenergic and cholinergic nerve terminals were in close proximity to each other. The synapses of the “bouton de passage” type contained either clear vesicles of 50-60 nm (cholinergic) or granular vesicles measuring 80-100 nm in diameter (monoaminergic). Colocalization of both transmitter substances in the same individual axon terminal could not be established. Fluorescent adrenergic fibers could also be detected in the secretory nerve ganglion following treatment with formaldehyde, with fluorescent nerve terminals surrounding nonfluorescent nerve cell bodies, indicative of sympathetic modulation of postganglionic signal transfer (Peaker and Linzell, 1975). Adrenergic depletion due to reserpine treatment of the animals was found to abolish both catecholamine fluorescence at the light-microscopic level and the presence of granular synaptic vesicles (Haase and Fourman, 1970).

2. Pharmacologic and Physiologic Characterization With regard to a possibly functional sympathetic innervation, the incubation of confluent secretory cell cultures with epinephrine, norepinephrine, or the padrenergic agonist isoproterenol resulted in a dose-dependent stimulation of transepithelial ion transport, with isoproterenol revealing the highest potency.

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The adrenoceptor-mediated response was fully abolished after abluminal application of the P-adrenergic antagonist propranolol, but not the a-specific antagonist phentolamine (Fig. 12). Cultures pretreated with propranolol remained refractory to subsequent incubation with epinephrine, whereas cultures desensitized for muscarinic agonists remained responsive to P-adrenergic stimulation (Lowy et al., 198513; Lowy and Emst, 1987). The authors, however, did not rule out the presence of a-adrenoceptors, which could be demonstrated throughout the salt gland parenchyma employing receptor autoradiography with the radioiodinated a,-agonist iodoclonidine as ligand (Muller, 1991). It would appear that a-adrenergic receptors are present in the avian salt gland and cause vasoconstriction and inhibition of secretion, since epinephrine, norepinephrine, and cervical sympathetic system stimulation reduced salt gland blood flow at increased arterial pressure (Fange et al., 1958a, 1963). In the Herring gull, intravenously infused epinephrine transiently blocked salt gland secretion, whereas phenoxybenzamine facilitated secretion (Fange er al., 1958a; Douglas and Neely, 1969). To simulate possible sympathetic innervation, rather than the unlikely hormonal action of catecholamines upon salt gland function, norepinephrine was administered to the blood perfusing the actively secreting salt glands of saltwater-acclimated Pekin ducks. At increased arterial pressure, steady-state salt gland secretion was only slightly inhibited at strongly reduced arteriolar perfusion of the gland (Gerstberger, 1991) (Fig. 12). A possible synopsis of the functional adrenergic innervation of the avian salt gland might be obtained by taking the simultaneous activation of both inhibitory presynaptic a,-adrenoceptors and stimulatory postsynaptic P-adrenoceptors into consideration. This hypothesis would be in agreement with (a) the marked labeling of salt gland tissue sections by a,-specific ligands (Muller, 1991). (h)the rather low potency of the nonselective a-adrenergic antagonist phenoxybenzamine to displace a,-specific ligands, ( c ) the low physiologic potency of phenoxybenzamine to diminish salt gland blood flow or secretion, but to facilitate ongoing secretion at high drug concentrations (Fange et al., 1958a; Douglas and Neely, 1969; Kaul et al., 1983; Muller, 199I), and (4 the transport-stimulating activity of P-adrenergic agonists in a salt gland tissue culture system (Lowy and Emst, 1987).

V. Secretory Mechanism The precise nature of the cellular mechanisms underlying the elaboration of a hypertonic secretion by salt gland epithelial cells is far from being completely understood, and several models have been advanced by various investigators to explain the extraction of NaCl from blood to lumen, against a large concentration gradient. In addition to the in vivo approach, a variety of in vitro techniques

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have been applied in studying the avian salt gland secretory mechanism, with special emphasis being placed upon the elucidation of intracellular ionic concentrations and fluxes. Although there are significant disparities between the postulated models, one consistent characteristic is that the enzyme Na+- and K+dependent adenosine triphosphatase (Na-K-ATPase) plays an integral role.

A. Na-K-ATPase The salt glands of several avian species have been found to be rich in Na-KATPase (Hokin, 1963; Emst et al., 1967). The enzyme molecule is thought to be a structural component of the plasma membrane, and in all cells its physiologic function is to extrude Na+ from the cell in exchange for K+ (Baker, 1972; Schwartz et al., 1975). Na-K-ATPase can be inhibited or blocked by the cardiac glycoside ouabain not only in avian salt glands but also in other salt-secreting epithelia, including the shark rectal gland (Skou, 1965; Schwartz et al., 1975; Silva et a/., 1977; Kirschner, 1980). The use of this pharmacologic tool has yielded valuable information about the molecular structure, regulation, and cellular distribution of Na-K-ATPase and its function in the salt-secreting process. Procedures for the purification of Na-K-ATPase from the duck salt gland, and also the shark rectal gland, have been determined, and the active form of solubilized salt gland Na-K-ATPase with a molecular mass of 265 kDa appeared to comprise of two a-subunits and two P-subunits with molecular masses of 96-98 kDa and 54 kDa, respectively (Stewart et al., 1976; Lingham et al., 1980; Smith, 1988; Skou and Esmann, 1988) (Fig. 13). Biochemical analysis indicated specific interactions of the protein with certain phospholipids for the Eider duck and Hemng gull (Karlsson et al., 1974). Spin-label studies performed on the Pekin duck salt gland Na-K-ATPase suggested the stoichiometric association of 66 lipids with the intramembranous surface of the Na-K-ATPase molecule (Esmann et al., 1985; Esmann, 1986, 1988; Esmann and Skou, 1988). Ion-dependent protein phosphorylation studies of detergent-purified membranes revealed the marked labeling of the 96- to 98-kDa Na-K-ATPase protein subunit in the presence of Na+, but absence of K+. Highly specific, affinity-purified polyclonal antisera against the Na-K-ATPase a-subunit recognized a 96-kDa protein exclusively (Stewart et al., 1976; Russo et al., 1987) (Fig. 13). In freeze-fracture replicas, avian salt gland Na-K-ATPase was visualized, forming aggregated intramembranous protein particles 8-10 nm in diameter, arranged into clusters and strands. Integration of the solubilized particles into artificial lipid vesicles also resulted in randomly oriented 8- to 10-nm particles (Gassner and Komnick, 1983) (Fig. 13). Binding studies using radiolabeled ouabain in the presence of cyclic AMP and phosphodiesterase inhibitors such as theophylline revealed the existence of at least two Na-K-ATPase subtypes in salt-secreting cells with cyclic AMP-induced transition of site I1 from a negative

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FIG. 13 Sodium-potassium ATPase (Na-K-ATPase) of the secretory cell. (a) Ion-dependent phosphorylation of the 96-kDa Na-K-ATPase subunit (arrowhead) purified from the duck salt gland in the presence of ( I ) Tris buffer alone, (2) Tris buffer + 120 mM NaCI, (3) Tris buffer + 120 mM NaCI + 20 mM KCI, or (4) Tris buffer + 20 mM KCI. Lanes 5-7 are derived from SDS-purified membranes in the presence of ( 5 ) Tris buffer alone, (6) Tris buffer + 120 mM NaCI, or (7) Tns buffer t 20 mM KCI. (b) lmmunoprecipitation of the 96-kDa protein (arrow) in its phosphorylated form ( I ) , and dephosphorylated in the presence of 120 mM NaCl (2) or 20 mM KCI (3). (c) Negative staining of unfixed purified Na-K-ATPase membranes from the duck salt gland. Surface particles are arranged into clusters and strands. Bar, 0.05 pm. (d) Na-K-ATPase-specific histochemical recation product is localized almost exclusively to the intracellular site of the plasma membrane in the basal area of a secretory cell. Bar, 0.3 pm. (e) Alteration in the plasma membrane surface of

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to a noncooperative binding state (Marver et a f . , 1986). When subcellular fractionation procedures and quantification of marker enzymes were employed, the preparation of a membrane fraction from salt glands enriched in Golgi membranes was achieved. This “Golgi fraction” contained high concentrations of one Na-K-ATPase subtype, suggestive of the enzyme being processed in that section of the endoplasmic reticulum (Addis et al., 1987). With regard to the cellular localization, the presence of a potassium- and magnesium-dependent phosphatase component of the Na-K-ATPase sensitive to ouabain was traced histochemically at the cytoplasmic site of the lateral and basal plasma membranes of principal secretory cells in the duck and Herring gull salt gland. Only small amounts of the enzymatic reaction product were found to be associated with the apical cell surface (Abel, 1969; Emst, 1972a,b; Ernst and Mills, 1977; Barmett et al., 1983) (Fig. 13). Using cytochemical lead techniques, adenosine triphosphate phosphorylase activities could also be located within the mitochondria1 matrix (Abel, 1969). Similar results were obtained in binding studies by coupling of a heme peptide bearing peroxidatic activity to the Na-K-ATPase inhibitor ouabain (Mazurkiewicz et al., 1978), or using radioactively labeled ouabain (Emst and Mills, 1977). Reptilian salt-secreting glands, although showing a basic morphology slightly different from that of avian salt glands with columnar cells lining the secretory tubules and mitochondria-rich tuft cells forming the terminal elements, exhibit comparable localization of Na-K-ATPase (Philpott and Templeton, 1964; Crowe et a f . , 1970; Van Lennep and Komnick, 1970; Ellis and Goertemiller, 1974). In the salt-secreting lacrimal gland of the green turtle (C. rnydas), the end product of the cytochemical reaction for Na-K-ATPase was detected primarily along the plications of the plasma membrane bordering the intercellular channels of the secretory epithelium, whereas luminal membranes and cytoplasmic inclusions were rarely observed (Ellis and Goertemiller, 1976). The same holds true for the shark rectal gland (Eveloff et al., 1979; Dubinsky and Monti, 1986) as well as the gills of the killifish (Stirling, 1976). In contrast, polyvalent antibodies raised against the catalytic a-subunit of NaK-ATPase purified from the duck salt gland reacted with the basolateral as well

cells in a duck salt gland (left panel), in the Na-K-ATPase activity, the binding capacity for tritiated ouabain and the number of mitochondria (right panel) under conditions of euhydration ( C ) ,saltwater adaptation (AD) and subsequent deadaptation (DE-AD). (0 Freeze-fracture of unfixed purified Na-K-ATPase membranes with particle-rich (open arrowhead) and particle-poor (solid arrowhead) fracture faces. Bar, 0.2 pm. (g) Freeze-fracture micrograph with a particle-rich fracture face at higher magnification. Bar, 0.1 pm. [(a,b) from Russo et al., 1987. with permission of John Wiley & Sons, Ltd.; (c,f,g) from Gassner and Komnick, 1983, with permission; (d) from Emst, 1972a. with permission; (e) from Merchant et al., 1985 (modified), with permission.]

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as apical membranes of avian principal secretory cells. Within the cells immunolabeling was partially associated with small vesicles or lysosomes, indicating biogenesis or degradative events. Using the immunogold technique, the reaction product was localized on the surface of the microvilli at the apical membrane, independent of the animal’s physiologic state (Russo et al., 1987). Similar observations were reported for the tuft cells of reptilian lacrimal glands with positive labeling of microvilli projecting into the tubular lumen. The rich endowment of the apical tuft cell membranes with Na-K-ATPase is discussed with regard to the rather high concentration of potassium in the secretory fluid of reptilian salt-secreting glands (Ellis and Goertemiller, 1974). The putative functional importance of apical Na-K-ATPase presence in the avian salt gland remains to be elucidated. It might also have been artifactual or due to the staining of cross-reactive proteins. With regard to possible regulatory influences on salt gland Na-K-ATPase activity, the cellular content of Na-K-ATPase increased when freshwateracclimated birds were given hypertonic saline as their only source of drinking fluid (Ernst et al., 1967; Fletcher et al., 1967; Fletcher and Holmes, 1968) (Fig. 13). Long-term saline treatment resulted in a fourfold increase in Na-K-ATPase activity in salt gland tissue accompanied by a comparable fivefold increase in plasma membrane densities as deduced from morphometric methods (Stewart et al., 1976; Merchant et al., 1985; Russo et al., 1987). The report by Holmes and Stewart (1968) of elevated levels in whole cell RNA and ribosomal concentration during salt acclimation of ducklings (see Section II,C) indicated de n o w synthesis of Na-K-ATPase rather than up-regulation of enzymatic turnover. This is supported by the augmented incorporation of radioactivity labeled amino acids into the catalytic subunit of the Na-K-ATPase using slices or an organotypic culture system of duck salt glands under conditions of salt acclimation. In parallel, phospholipid concentrations were significantly stimulated in isolated membranes (Stewart et al., 1976; Lingham et al., 1980; Mazurkiewicz and Barrnett, 1981).

6. Early Models of Salt Secretion Since the earliest studies of avian salt gland function some 30 years ago, a number of models have been put forward to explain the mechanism by which salt gland fluid is secreted. These models have been reviewed in detail by Holmes and Phillips (1983, and only a brief outline of these hypotheses will be given here.

1. Active Na+ Transport Across the Apical Membrane On the basis of their observation that the lumen of actively secreting salt glands was positively charged with respect to the blood, Thesleff and Schmidt-Nielsen

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( 1962) postulated that the secretory mechanism involved active transport of

Na+ by a pump located in the apical membrane. Since this transport could be blocked by ouabain, it was assumed to depend on a Na-K-ATPase system (Thesleff and Schmidt-Nielsen, 1962; Bonting et af.,1964; Van Rossum, 1966) (Fig. 14). Subsequent studies by two independent investigators produced modifications that incorporated Na+ pumps at both the basal and the luminal cell surfaces. In one model (Hokin, 1967) it was postulated that Na+ is concentrated in the secretory cells by means of a ouabain-insensitive process and that a ouabain-sensitive pump, driven by ATP, extrudes Na+ across the luminal membrane. In the other (Peaker, 1971) it was proposed that the concentration gradient for Na+ is established across the luminal membrane by an electrogenic Na+ pump, with entry of Na+ and C1- into the cell across the basal membrane being achieved by an active mechanism involving the exchange of Na+ for H+ and C1- for HC0,- (Peaker and Stockley, 1973, 1974).

2. Active Na+ lkansport into Intercellular Spaces The localization of Na-K-ATPase to the basolateral membranes using cytochemistry and autoradiography with ['Hlouabain (Emst, 1972a,b; Emst and Mills, 1977) together with the fact that intracellular "a+] in salt gland slices was generally shown to be low (Van Rossum, 1966; Peaker, 1971; SchmidtNielsen, 1976) prompted Emst and Mills (1977) to suggest a model for salt gland secretion that omitted an apical pump mechanism. These investigators postulated that the Na-K-ATPase complex was orientated in a way that made possible the transfer of Na+ from the cell to the intercellular space, followed by its paracellular movement via leaky junctions into the lumen of the secretory tubules. This type of shunt pathway maintains a concentration gradient that enables Na+ to enter the secretory cell passively from the blood, taking with it C1-, which then diffuses into the lumen along a favorable electrochemical gradient. The fallability of the idea of the passive role of C1- in salt gland secretion became apparent with the demonstration that furosemide, a C1- transporter antagonist, inhibited salt gland secretion, as did the substitution of C1- by other anions (Gilmore et al., 1977a.b; Hootman and Emst, 1981a; Ernst and Van Rossum, 1982). Accordingly, the above model of the secretory process was subsequently modified to include a secondary active C1- transport coupled with that of Na+.

3. Two-Stage Process A third type of model, advanced by Ellis and co-workers (1977), suggested that salt gland fluid was produced in two stages and required contributions by the two types of secretory tubular cells. The hypothesis proposed that the principal

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FIG. 14 Transepithelial ion transport mechanism in the avian salt gland. (a) Scanning electron micrograph of salt gland secretory cells in primary cell culture forming intercellular tight junctional complexes (arrows). These cultures can be used to study ion transport. Bar, 2 bm. (b) Trace of the voltage potential measured between the blood and the duct of a duck salt gland during secretory nerve stimulation (white bar) in the absence (A) or presence (B) of the Na-K-ATPase inhibitor Gstrophanthin. (c) Schematic presentation of transport processes involved in the excretion of sodium and chloride against an enormous concentration gradient in the avian salt gland. Basolateral location of the ouabain-sensitive Na-K-ATPase, the Na-K-2Cl cotransporter inhibitable by furosemide and a major K+ conductance sensitive to TEA and Ba2+ ions. CI- channels are located at the luminal membrane, and Na+ penetrates into the lumen via paracellular pathways. (d) Transepithelial resistance of salt gland cells for varying periods of time in primary culture after seeding of cultures with dissociated cells. [(a) From Lowy et al., 1985a, with permission; (b) from Thesleff and SchmidtNielsen, 1962, with permission; (d) from Lowy et al., 1985b (modified), with permission.]

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cells do not secrete the hypertonic effluent of the glands; instead, water is passively reabsorbed through the leaky cell junctions from an isoosmotic fluid secreted by the peripheral cells. The driving force of this water movement is the osmotic gradient that exists within the intercellular space due to the presence of Na+ extruded by pumps of the lateral membrane.

4. Post-tubular modification The three types of theoretical models described above all assume that the hyperosmotic fluid that appears at the salt gland nares is elaborated by the cells of the secretory tubules. One study that disputes this has advanced a model depicting salt gland secretion as a modification of the primary hypoosmotic or issosmotic secretion, occurring in the duct system. Marshall and co-workers (1985) used quantitative electron probe microanalysis to measure luminal and intracellular ionic concentrations in fractured, frozen samples of duck salt glands. The results of this analysis showed that the lumen of the secretory tubules contains a secretion that is either hypoosmotic or approximately isoosmotic to blood, certainly not hyperosmotic. Only as the fluid proceeds along the duct system does it become progressively more concentrated, either by the secretion of salt into the lumen or the reabsorption of water. Employing the same technique, Andrews and co-workers (1983), however, obtained results supportive of an active energy-requiring process of the principal secretory cells consistent with active chloride transport as the basis for salt secretion in this tissue.

C. Current Model Using a cell culture system with confluent sheets of isolated duck salt gland cells developing junctional complexes between cells with concomitant increases in transepithelial resistance after 3 days of culturing (see Section IV), Lowy and colleagues (1985a,b) were able to measure changes in short-circuit current and ionic fluxes produced by various agonists and antagonists of salt gland function (Fig. 14). This also allowed differentiation between the apical and the basal sites of drug action. Results of their studies produced a model for NaCl secretion that very much resembles those thought to exist in other secretory tissues, especially the salt-secreting rectal gland of the dogfish (Kirschner, 1980; Smith et al., 1982; Shorofsky et al., 1984; Greger et al., 1986; Lowy et al., 1989). This mechanism of salt secretion is based upon a secondary Cl- transport mechanism. The active process is dependent on ( a ) a ouabain-sensitive Na-KATPase, (b) a furosemide-sensitive Na-Cl symport mechanism, and (c) a barium- and tetraethylammonium (TEA)-sensitive K+ conductance, all situated at the basolateral membrane (Fig. 14). The importance of the Na-K-ATPase could be deduced from experiments where exogenous stimuli such as cholinergic

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agonists were able to increase the turnover rate of plasmalemmal Na-K- ATPase at a constant number of enzyme molecules. Ouabain binding studies revealed the existence of more than 25 million molecules of Na-K-ATPase per dispersed principal secretory cell (Hootman and Emst, 1981a). Ouabain markedly decreased basal and metacholine-induced cellular respiration, and preferentially inhibited active ion transport as measured via altered SCC (Emst and Van Rossum, 1982; Lowy et al., 1989). Evidence for the coupled basolateral carrier-mediated uptake of Na+ and C1- was obtained from experiments demonstrating profound furosemide sensitivity of the salt gland secretory activity, agonist-enhanced oxygen consumption of salt gland slices and dissociated cells (Hootman and Ernst, 1981a; Ernst and Van Rossum, 1982), binding of vitiated ouabain (Hootman and Ernst, 1981a), and active ion transport (Lowy et al., 1985b, 1989). As in other epithelia, this uptake mechanism may also involve K+ cotransport, i.e., a Na-2CIK system, with the added influx of K+ being balanced by back-diffusion through basolateral conductance channels (Silva et al., 1977; Frizzell et al., 1979; Greger and Schlatter, 1984a,b). Using cell-attached and inside-out patch clamp technology. outward-rectifying K+ channels of large conductance could be identified according to their blockade with both external barium and TEA. These K+ channels could be activated in the presence of muscarinic agonists (Richards ef al., 1989). The inward Na+ gradient established by Na-K-ATPase provides the energy for the Na+-coupled uptake of CI- and finally drives the C1- secretion. The accumulated cellular C1- (Andrews et al., 1983) probably leaves the cell across the apical membrane via regulated C1- conductance channels, which have not yet been demonstrated for avian salt glands but are known to be present in the rectal gland of the dogfish (Greger et al., 1985, 1987a,b; Gogelein et al., 1987). This is supported by recent immunolocalization for a cystic fibrosis transmembrane conductance regulator (CFTR) homolog, which is now considered to be a C1- channel, at the apical membrane of the duck salt gland (S. A. Emst, personal communication) and of the shark rectal gland (Marshall, 1991). The K+ diffusion into the blood and the CI- movement into the lumen generate a lumen-negative transepithelial voltage which then secondarily drives Na+ through the paracellular pathway into the tubular lumen. Recent studies employing the Na+ transport channel blocker amiloride ruled out the possible participation of such a channel in the mechanism of salt gland secretion. In addition it was suggested that the paracellular pathway might be similarly permeable to Na+ and K+, as judged from the fixed relationship between plasma and salt gland fluid Na+/K+ ratios under all experimental conditions (Simon and Gray, 1991). The stoichiometry, apparent from Fig. 14, indicates that for each mole of Na+ actively transported by the Na-K pump, 2 mol of NaCl is secreted. This economy of NaCl transport is also present in the rectal gland of the dogfish (Greger et al., 1986) and the thick ascending limb of the loop of Henle in the mammalian kidney. It may be characteristic of CI--transporting epithelia in general.

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VI. Receptive Systems for the Control of Salt Gland Secretion Over the years numerous investigations have been undertaken to elucidate the factors important in the afferent control of salt gland function in birds. Most of this work has involved the application of various osmotic and volume loads given via differing routes. In the majority of cases these studies have been performed in domestic avian species, in particular the duck. Although a picture has emerged from these studies, a number of inconsistencies have also been apparent, particularly with regard to the relative importance of the different regulating stimuli and the sensitivity of the receptive systems in the various avian species examined. However, it should be pointed out that many of the documented experiments were performed using birds that were not saltwater-acclimated and therefore have poor secretory ability (Holmes et al., 1961; Schmidt-Nielsen and Kim, 1964, Peaker et al., 1973). These birds are not appropriate models for examination of the afferent control of salt gland function. In addition, in view of the fact that osmotic status has such an influence on salt gland activity (see below), some of the quantitative characteristics attributed to them should be treated with caution. This is especially true for birds taken from holding pens and subjected to experimental manipulations without first being in a near threshold or steady state of salt gland secretion. Because of the variations in osmotic status known to exist in individual birds (Simon, 1982; Simon and Gray, 1989), statements such as “a two percent increase in plasma NaCl content is required to initiate salt gland secretion” do not carry sufficient unequivocal information. Notwithstanding, it is now clearly evident that ECFT and ECFV can be distinguished as parameters by which the avian salt glands are controlled, and on which salt gland activity feeds back. For the nonhomologous rectal gland of marine elasmobranchs, although increases in ECFV and ECFT have both been shown to stimulate secretion (Burger, 1962; Solomon et al., 1984a,b, 1985b), the effects of changes in tonicity appear to be indirect, acting via the induction of ECFV expansion. Since the plasma of marine elasmobranchs is slightly hypertonic to the seawater environment in which they live, even small elevations in ECFT further enhance the ongoing inward diffusion of water, resulting in ECFV expansion, which drives rectal gland activity. Isovolemic increases in serum osmolality per se, produced by simultaneous hypertonic saline infusion and hemorrhage, do not stimulate rectal gland secretion (Solomon et al., 1985a).

A. Tonicity The salt gland-stimulating effect of increases in ECFT was convincingly demonstrated in many early investigations (Schmidt-Nielsen et al., 1958;

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McFarland, 1964; McFarland et al., 1965; Lanthier and Sandor, 1967; Ash, 1969; Hajjar et al., 1970; Carpenter and Stafford, 1970), which have been thoroughly reviewed by Peaker and Linzell (1975) and Skadhauge (1981). More recent studies have tried to determine the nature and location of the monitoring receptors. Experiments have been designed specifically to evaluate whether the tonicity receptors are located centrally and/or systemically, and whether they are osmosensitive and/or sodium sensitive.

1. Central vs Systemic Tonicity Receptors The idea that increased plasma tonicity activates salt gland secretion via the stimulation of receptors located in the central nervous system (Fiinge et al., 1958a.b; Schmidt-Nielsen, 1960) has been examined by a number of investigators in analogy to the central nervous location of osmosensitive elements involved in the control of water intake, antidiuretic hormone (ADH) release, and renal antidiuresis in mammals and birds (Vemey, 1947; Andersson et al., 1967; Andersson, 1978; Bie, 1980; Simon et al., 1987). Hanwell and co-workers (1972), using geese that had never seen saltwater, found that the intracarotid (ic) administration of hypertonic NaCl did not produce greater salt gland stimulation than the intravenous (iv) route. Moreover, cross-circulation and perfusion studies showed that a raised NaCl concentration in the blood perfusing the head was ineffective in evoking secretion, suggesting that the plasma tonicity must be monitored elsewhere in the body. Since rapid injection of salt into the right atrium initiated salt gland secretion that could be abolished or inhibited by vagotomy or vagal blockade, it was concluded that the tonicity receptors are located in the heart and that the reflex runs in the vagal nerves. In contrast to the above findings, subsequent studies in ducks have demonstrated that brain tonicity receptors do exist. Hammel and colleagues (1983) compared the salt gland responses of saltwater-acclimated ducks to alterations of carotid artery and systemic venous plasma tonicity. Reduction in the osmolality of the blood going to the head, caused by an ic injection of hypotonic glucose, produced a far more pronounced inhibition of ongoing salt gland secretion than did the same dose of glucose given systematically. To further investigate the cephalic contribution to salt gland control, studies utilizing a technique of constant volume split infusion (Simon-Oppermann and Simon, 1982) were undertaken in saltwater-acclimated ducks during steady-state salt gland secretion driven by hyperosmotic NaCl (ic) and isoosmotic glucose (iv) infusions (Gerstberger el al., 1984a) (Fig. 15). Exchange of both infusion lines, with application of the isoosmotic glucose to the carotid arteries and the hypertonic saline to the peripheral circulation, resulted in a fast 13 mOsm/kg decrease in carotid blood tonicity at unchanged peripheral plasma tonicity and markedly reduced osmolal excretion via the salt glands. Additional exchange of both infusates with hypertonic saline administered to the cephalic circulation caused a

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FIG. 15 Central osmosensitivity and integrative hypothalamic control of salt gland function. (a) X-ray roentgenogram of a duck skull (sagittal section), showing the position of a bouble-barrel cannula (C) inserted into the lumen of the third cerebral ventricle (VIII) for local perfusion of the VIlI with artificial cerebrospinal fluid (aCSF). To demonstrate the localization, the ventricular system was filled with an inert contrast medium. Bar, 5 mm. (b) Sagittal section through the hypothalamus of a Pekin duck (Kliiver-Barrera staining). AC, anterior commissure; AP, anterior pituitary; CP, choroid plexus; ME, median eminence; OC, optic chiasma; PC, posterior commissure. Bar, 2mm. (c) Centrally induced inhibition and stimulation of salt gland secretion in saltwater-acclimated Pekin ducks (SW-AD). Simultaneous infusion of hypertonic NaCl (hatched bar) and isotonic o-glucose (stippled bar) into the carotid arteries (ic) and a wing vein (iv), respectively, during constant steady-state excretion (dotted line). Subsequent switching of both infusions at constant total salt and water administration. (d) Inhibition (A) and stimulation (B)of salt gland secretion rate and osmolality of the secreted fluid in SW-AD at steady-state salt gland excretion during intracerebroventricular perfusion with aCSF made slightly hypotonic (A) or hypertonic (B). [(c) From Gerstberger er al.. 1984a (modified), with permission; (d) from Gerstberger el a/.. 1984b (modified). with permission.]

13 mOsm/kg increase in carotid plasma tonicity with parallel 70-170% enhancement in glandular osmolal excretion. The constancy of whole body loading with salt and water strongly suggests the participation of cephalic osmosensitive elements in the perception of these osmotic stimuli. On the other

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hand, the existence of systemic osmoreceptors or volume receptors could be deduced from the reestablishment of steady-state secretion despite the persisting hypotonic or hypertonic infusion into the carotids, resulting in a 9 mOsm/kg increase and 11 mOsm/kg decrease in systemic plasma osmolality at continuous salt and water administration, respectively. To exclude nonosmotic effects of the glucose solution on the sensitivity of the postulated receptive elements or on transport functions of the secretory tissue itself, the isoosmotic D-glucose was replaced by equiosmolal solutions of other nonelectrolytes, all of them producing identical, temporary inhibition of salt gland activity. Considering the differences between these solutes with regard to cellular permeability and metabolic substrate suitability, it seems likely that the inhibition of salt gland secretion induced by them is solely attributable to their common reduction of carotid blood tonicity (Gerstberger et al., 1984a). The indication that tonicity-sensitive structures controlling salt gland activity and accessible via the carotid vascular system lie within the brain has been evaluated by monitoring salt gland responses in the Pekin duck to alterations of hyi pothalamic ECFT. Although a preliminary hypothalamic microinjection study failed to demonstrate any effects of hypertonic NaCl on salt gland activity (Deutsch and Simon, 1980), a subsequent investigation using an intracerebroventricular (icv) microperfusion technique (Fig. 15) in the same species showed that graded icv osmotic stimulations at the diencephalic level produced graded changes in ongoing salt gland secretory activity (Gerstberger et al., 1984b). Hypertonic icv stimulation enhanced salt gland secretion at three times the sensitivity monitored for the inhibitory responses observed during hypotonic icv stimulations (Fig. 15). Comparable results have been reported with regard to the central control of renal function and ADH release in the conscious duck, goat, monkey, and sheep using both ic and icv application of the hypertonic stimuli (Andersson, 1978; McKinley et al., 1978; Swaminathan, 1980; SimonOppermann and Simon, 1982; Gerstberger et al., 1984b). The fact that the effects of icv osmotic stimulations in the duck experiments were obtained under conditions of strong antidiuresis as well as of significant urinary excretion indicates that the alterations in salt gland function were not secondary to changes in body fluid content caused by changes in urinary output. The perfusion technique employed in this study most likely produced stimuli restricted mainly to the rostro-caudal portion of the periventricular tissue adjacent to the third ventricle (Figs. 15,16). Evidence for the important role in osmoresponsiveness which this area of the avian hypothalamus might play could also be derived from central icv stimulations with regard to ADH release and renal water elimination in the duck, and drinking behavior in the pigeon (Gerstberger et al., 1984b; Thornton, 1986; Kanosue et al., 1992). Light-microscope (LM), EM, and scanning electron-microscope (SEM) investigations of the ependymal and subependymal brain parenchyma revealed the presence of numerous neuronal elements penetrating the ependymal layer

AVIAN SALT GLANDS

FIG. 16 Scanning electron micrographs (SEM) of the third cerebral ventricle (VIII)in the duck brain. (a) SEM of a sagittal section through the brain showing the surface of the whole VIII. AC, anterior commissure; AV3V,antenoventral third ventricular region; CP,choroid plexus; ME, median eminence: OC. optic chiasm; PVO, paraventricular organ; RI, infundibular recess. Bar, 200 pm. (b) SEM of a single ependymal cell showing numerous microvilli and cilia (C)as surface membrane protrusions. Bar, 0.5 pm. (c) SEM of single hexagonal cells typical for the AV3V region, ME, or the subfomical organ, lacking a blood-brain barrier, with limited membrane specializations such as cilia (C). Bar, 1 pm. (d) SEM of neuronal dendritic processes (D)originating from liquor-contacting neurons in the PVO.Bar, 0.5 pm.

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of glial origin and protruding into the third ventricular cerebrospinal fluid (CSF) of various avian species including the duck (Vigh, 1971; Vigh-Teichmann et al., 1971; Mikami, 1975; Takei et al., 1978; Korf et al., 1983; Gerstberger et al., 1989). Whereas most of the ventricular lining is composed of specialized ependymal cells with numerous surface cilia and microvilli (Fig. 16), these so-called CSF-contacting neurons occur primarily in areas for which morphologic criteria predict receptive functions, such as the circumventricular organs (CVO) lacking a blood-brain barrier (BBB) or regions like the anteroventral third ventricular zone (AV3V) involved in autonomic control circuits (Vigh and Vigh-Teichmann, 1973; Leonhardt, 1980; Korf et al., 1982; Panzica et al., 1986; McKinley et al., 1991). Scanning electron-microscope studies of the third ventricular wall in the duck and quail revealed a hexagonal shape of cells poor in surface specializations in these regions with bulb- or knoblike dendritic endings sent into the ventricular lumen (Fig. 16). Tanycytes can be traced at the LM and EM level to contact both CSF and subependymal neuronal elements (Korf et al., 1983; Panzica et al., 1986) (Fig. 17). Although all of the afferent projections of these putative receptive neurons have not yet been identified, some of them are known to monosynaptically connect to magnocellular neurons of the paraventricular nucleus (PVN) as demonstrated by retrograde transport of horseradish peroxidase injected into the PVN (Korf et al., 1982) (Fig. 17). These periventricular neurons are therefore thought to be implicated in the release of antidiuretic hormone caused by icv hypertonic stimulation (Gerstberger et al., 1984b; Simon et al., 1992). To demonstrate directly osmosensitivity as a property of neurons located particularly in the periventricular layer, being labeled by retrogradely transported tracers applied to the PVN, single unit recordings were performed. These extracellular in vitro recordings were obtained from neurons in various hypothalamic areas of the duck brain using a duck hypothalamic slice preparation (Korf et al., 1982; Kanosue et al., 1990). Accordingly, hypertonic stimulation due to slightly elevated NaCl concentration in the artificial CSF used for brain slice incubation excited 25 and inhibited only 4 of 48 periventricular layer neurons. Hypotonic stimulation inhibited 9 of 10 neurons activated by hypertonic stimulation (Fig. 17). Osmoresponsive units in other areas of the hypothalamus such as the magno- or parvocellular PVN were rarely observed in the duck. In the rat brain, however, intrinsic osmoresponsiveness of hypothalamic cells could be demonstrated for the supraoptic nucleus (SON), organum vasculosum laminae terminalis (OVLT), or AV3V region, with the formation of an osmoreceptive complex (Honda et al., 1990). Afferent connections of this periventricular osmosensitive zone to salt gland-regulating centers cannot be excluded; on the contrary, they are very likely. Moreover, the additional, yet unidentified, location of salt gland tonicity receptors in brain structures that are accessible to both CSF and blood, e.g., CVO structures, would be compatible with the experimental data.

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FIG. 17 CSF-contacting neurons as putative central osmosensors. (a) Small neurons in the periventricular layer (PL) of the third cerebral ventricle (VIII) are retrogradely labeled by horseradish peroxidase microinjected into the magnocellular portion (MC) of the paraventricular nucleus. Bar, 100 pm. (b) Golgi impregnation of a frontal section through the duck hypothalamus showing a magnocellular neuron (MCN) of the paraventricular nucleus with its dendrites connected to a neurite (arrows) originating from a subependymal liquor-contacting neuron (CN) close the VIII. Bar, 50 pm. (c) Higher magnification of the subependymal CN. Bar, 20 pm. (d) Osmosensitivity of a neuron located in the periventricular subdivision of the hypothalamic paraventricular nucleus (PL) adjacent to the third cerebral ventricle in the duck brain. Extracellular recordings of neuronal discharge rate in a hypothalamic slice preparation during hypotonic (left) and hypertonic (right) stimulation. [(a)From Korf er al.. 1982, with permission; (b,c) from Korf er al., 1983, with permission; (d) from Kanosue er al., 1990 (modified), with permission.]

2. Osmosensitivity vs Sodium Sensitivity The first report on salt gland function reported that iv infusion of sucrose stimulated a cormorant to produce a nasal gland secretion of a concentration similar to that induced by an infusion of NaCl solution (Schmidt-Nielsen et al., 1958). The indication of this finding that any elevation of the osmotic concentration of the blood will stimulate salt gland secretion appears to have been subsequently confirmed by the observation that an increase in the plasma concentration of compounds as diverse as mannitol, LiCl, KCl, and arginine-HC1 can activate

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avian salt glands (Hajjar et al., 1970; Hanwell et al., 1972; Deutsch et al., 1979; Erbe et al., 1988). However, salt glands do not respond to hyperosmotic doses of MgCl, or urea (Carpenter and Stafford, 1970; Deutsch et al., 1979); therefore, a simple increase in the osmotic concentration of the plasma does not unequivocally drive salt gland activity. As originally proposed by Vemey (1947), a tonicity receptor is thought to be stimulated by a reduction in its cell volume, caused by an efflux of water down an osmotic gradient created by a rise in extracellular solute concentration (Hammel, 1981). Thus, if all other factors are equal, the receptor-stimulating potency of a particular blood solute should be inversely proportional to the rate at which it enters the receptor. For systemic receptors, solute specificity would be determined solely by its receptor membrane permeability. For central receptors, solute permeability of the BBB also needs to be taken into consideration. With regard to the putative systemic tonicity receptors, available information indicates that they are generally osmosensitive. Accordingly, they can be stimulated by hypertonic mannitol and sucrose, which are by and large excluded from receptive cells, but not significantly affected by hypertonic urea, which permeates cell membranes. As for central receptors, the situation is somewhat more complex, with interpretation depending on whether they are thought to be situated on the blood (e.g., CVO) or the brain side of the BBB. However, available data can be taken to indicate that receptors are present in both locations, and that they are predominantly Na+ (or cation) sensitive. Accordingly, salt gland activity driven by the ic infusion of hypertonic NaCl (3000 mOsm/kg) is markedly decreased when the saline is replaced by equiosmolal sucrose (Gerstberger et al., 1984a). The reduction of the "a+] in the cephalic compartment, at a constantly elevated blood osmolality, appears to be the reason for this response and is compatible with the idea that the relevant Na+ receptors are located on the blood side of the BBB. These or other Na+ receptors involved in salt gland control are also accessible to CSF, since a reduction in CSF "a+] by the icv application of hypertonic mannitol (Deutsch and Simon, 1980) or artificial isoosmotic CSF in which NaCl was replaced by sucrose (Gerstberger et al., 1984b) caused a reduction in ongoing secretion. Recently, evidence has been obtained extending the concept of a specific Na+sensitive receptive mechanism, to include general cations. Osmoresponsive neurons present in hypothalamic slice preparations have been shown to respond equally to stimulation by artificial CSF made hypertonic by either NaCl or LiCl, but not sucrose (Kanosue et al., 1990). Taken together with the fact that the brain receptors thought to be involved in the osmotic stimulation of ADH release in ducks are sensitive to Li+, Ca2+and Mg2+,as well as to Na+, but again not to sucrose or mannitol, the concept of nonspecific cation channels gains support (Deutsch and Simon, 1980; Gerstberger et al., 1984b; Kanosue et al., 1992).

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6. Volume Early investigations concerning the significance of volume influences on salt gland secretion produced no consensus of opinion on the matter (Holmes, 1965, 1972; Hanwell et al., 1972; Ruch and Hughes, 1975; Zucker et al., 1977; Peaker, 1978). More recently, however, clear evidence for the role of a volume component in avian salt gland function has emerged, although some degree of species variation is apparent with regard to sensitivity, particularly between ducks (the most studied avian species) and gulls (Hughes, 1987, 1989). For the analog structure of the shark rectal gland, isotonic ECFV expansion was shown to stimulate CI - secretion using an in situ preparation as well as intact anesthetized animals (Erlij et al., 1980; Solomon et al., 1984a; Erlij and Rubio, 1986). There are a number of examples in which changes in avian salt gland activity have been reported in response to alterations in ECFV, with current emphasis being placed on the relative importance of vascular and interstitial fluid volume (ISFV).

1. Vascular Volume A number of studies examining the effect of hemorrhage on salt gland function have been carried out. In ducks, removal of blood in amounts ranging from 7 to 30% total blood, before (Phillips and Harvey, 1980) or during (Deutsch et al., 1979; Hammel et al., 1980; Simon-Oppermann et al., 1984) salt gland secretion, reduced salt gland activity (Fig. 18). Reinfusion of the removed blood increased ongoing secretion. In gulls, neither the removal of 20% blood volume nor the infusion of 20% additional blood had any effect on salt gland secretion (Hughes, 1987). It has been suggested, at least in the case of ducks, that an increase in body fluid content might diminish the threshold tonicity for salt gland activation (Hammel et al., 1980; Simon, 1982). Consequently, vascular expansion by iv infusion of isoosmotic saline with or without dextran or slightly hypotonic NaCl induced salt gland secretion (Zucker et al., 1977; Hammel et al., 1980; Gray et al., 1986). Evidence for this volume effect to be acting via a change in the secretory threshold tonicity originated primarily from experiments in which the composition and volume of the body fluid in Pekin ducks were altered by iv infusions of various NaCl solutions (Hammel et al., 1980) (Fig. 18). Control experiments consisting of an iv infusion of hypertonic saline induced rates of secretion of salt and water by the salt glands that matched the rates of infusion, when the ECF "a+] exceeded some preestablished threshold concentration ("a+],,). Hydration of the ducks with an infusion of hypoosmotic saline, with the same amount of salt applied as during the hypertonic stimulation, had the effect of reducing ECF "a+] by 8 meq/liter and expanding ECFV due to the retention of 72% of the water infused. Repeating the control infusion of hyper-

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FIG. 18 Afferent volume regulatory and hypothalamic integrative control of salt gland function. (A) Inhibitory and stimulatory actions of isotonic extracellular volume alterations (bleeding, reinfusion of blood) on salt gland osmolal excretion in the saltwater-acclimated Pekin duck (SW-AD) during steady-state secretion (dotted line). (B) Nasal salt gland excretion in SW-AD (solid line) in response to hyperosmotic (0.4 ml/min of 1000 mOsm/kg NaC1) (A,C) and almost isotonic (1.4 ml/mm of 277 mOsm/kg NaCI) (B) infusions into the systemic circulation (dotted line). (C) Effect of graded hypothalamic cooling on salt gland activity in a SW-AD receiving a continuous systemic hypertonic salt load. (D) Effects of hypothalamic cooling and warming on salt gland activity in an Adelie penguin receiving a continuous systemic hypertonic salt load (dashed line). [(A) From Simon-Oppermann et al., 1984 (modified) with permission; (B) from Hammel et al., 1980, with permission; (C) from Simon-Oppermann et al., with permission; (D) modified from “The Proceedings of the Third SCAR Symposium on Artic Biology” by H. T. Hammel, J. E. Maggert, E. Simon, L. Crashaw, and R. Kaul. Copyright 0 1977 by Gulf Publishing Company, Houston, TX. Used with permission. All rights reserved.

tonic saline under these conditions indicated no significant change in the ratio of the amount infused and secreted. Since ECF “a+] had been reduced by the hypoosmotic infusion, the conclusion reached was that the increase in ECFV lowered the “a+],, for secretion. Further evidence for the putative inverse relationship between “a+], and ECFV could be obtained by comparing the ratio of the salt secreted to that infused before and after removing 15% of the total blood, showing that ECFV depletion reduced salt gland secretion by 24%.This result is in good agreement with results on ducks (Deutsch et al., 1979; Kaul

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and Hammel, 1979), which demonstrated that dehydration-induced ECFV reduction raised the “a+],, for salt gland secretion.

2. Interstitial Fluid Volume From the above experiments it is impossible to evaluate which, if any, compartment of the ECF, i.e., vascular or ISFV, is the most important in salt gland regulation, since the induced changes occurred in both. To make possible the evaluation of the contribution of both ECF compartments in afferent salt gland control, the salt gland response to iv dextran was monitored. The salt glands showed a diminished secretory response to a hyperosmotic (Hammel et al., 1980) and isoosmotic (Keil, 1990; Keil et al., 1991) NaCl load administered together with hyperoncotic dextran solutions. The substantial reductions in hematocrit values after dextran infusion clearly indicated a considerable rise in the intravascular volume, at the expense of the extravascular volume. These findings have been interpreted to indicate that salt gland activity is more dependent on the ISFV than the vascular volume (Simon, 1982; Keil et al., 1991). However, although the resultant salt gland inhibition conforms to the view that the activity of these glands is influenced by the ISFV, the fact that secretion continued (at a reduced level) with the infusion of an isotonic volume load (Keil et al., 199I), which is presumably driven by intravascular expansion, makes it impossible to determine which compartment is more important. Rather it is more accurate to say that salt gland activation by the ECFV is dependent on the algebraic sum of excitatory and/or inhibitory stimuli arising from both the intravascular and the extravascular compartments.

3. Extracellular Fluid Volume Receptors The implication that changes in the ECFV affect salt gland function raised the questions where and how these changes are monitored and transduced. Volume is an extensive property and since only intensive properties of the body may be transduced into neural signals, tension receptors somewhere in the ECF must monitor the size of that space and elicit the salt gland effects produced by changing the ECFV. In the case of intravascular volume, stretch receptors possibly located in the vicinity of the heart with afferent fiber passage in the sensory component of the nervus vagus (tenth cranial nerve) connecting the transducers with higher brain stem centers might be postulated. Vagal transsection or anesthetic blockade markedly inhibited ongoing salt gland secretion (Hanwell er al., 1972; Gilmore et al., 1977b; Simon-Oppermann et al., 1980). Concerning the extravascular volume, nothing is known about either the structure or the location of the postulated interstitial tension receptors. With regard to the afferent control of volume-induced rectal gland function in the dogfish or salt gland activity in various reptiles, only limited data are

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available about localization and characterization of putative volume-sensitive elements involved in the signal perception and transduction processes (Burger, 1962; Dunson, 1976). The stimulatory effect of localized application of veratridine into the atria or the conus arteriosus of the dogfish heart led to the questionable postulate of a receptive mechanism situated in the vicinity of the heart to monitor changes in ECFV or vascular filling in these animals (Erlij and Rubio, 1986). Preliminary studies performed in various sea turtle species of the genus Chelania indicate that secretion of the lacrimal salt glands is not initiated and controlled by increased ECFV (Hudson and Lutz, 1986; Marshall and Cooper, 1987; Nicolson and Lutz, 1989). Recently Hammel (1989) raised the interesting possibility that the neural control of avian salt gland secretion is enhanced and sustained by autofacilitation. On the basis of the fact that soon after a salt gland response has been initiated by an iv infusion of salt the rate of excretion exceeded the rate of infusion, Hammel argued that salt gland activity must be sustained by some unknown facilitation of the neurons regulating secretion, even as the initiating stimuli return to threshold. Further evidence for autofacilitation by a positive feedback loop was indicated by the finding that once the activity of the regulating neurons is sustained by their own activity, they are less easily inhibited by regulatory hormones or ISFV reduction due to iv dextran administration. However, when inhibitory hormones or dextran accompany a saline infusion driving salt gland secretion from the onset, autofacilitation is impaired.

C. Neural Integration The multiplicity of afferent inputs regulating salt gland function (cerebral and extracerebral osmoreceptors, body fluid volume receptors) indicates the need for an integrating center in the efferent control. Most of the available evidence indicates that this is accomplished by hypothalamic neuronal networks, which in addition may also integrate salt gland activity with other regulatory systems including those of kidney function, cardiovascular control, and thermoregulation. Afferent control of salt gland secretion is known to be closely coordinated with that of kidney excretion (Skadhauge, 1981; Simon, 1982; Simon-Oppermann and Gerstberger, 1989; Simon and Gray, 1991), with the receptive elements controlling both systems probably being colocalized but not identical (Gerstberger et al., 1984b). The presumably osmosensitive afferents from the wall of the third ventricle, as well as ascending information from the vagal and glossopharyngeal nerves, were shown to converge in the PVN at sites where microelectrostimulation elicited antidiuretic reactions. Vagal and glossopharyngeal afferents are relayed in sensory nuclei of the avian brainstem such as the nucleus tractus solitarii (NTS) (Korf et al., 1982; Korf, 1984). Neurons from this region of the hypothalamus apparently descend to the parasympathetic brain-

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stem nuclei efferently innervating the salt glands. This can be inferred from the observation that hypothalamic cooling inhibited ongoing salt gland activity in penguins and ducks (Hammel et al., 1977; Simon-Oppermann et al., 1979), whereas hypothalamic w m i n g transiently activated penguin or duck salt glands (Hori et al., 1986) (Fig. 18). The existence of a temperature dependence of signal transmission in hypothalamic neural integration supports an additional integration between osmo- and thermoregulation at this level. With regard to possible agents involved in the hypothalamic integration of various afferent osmoregulatory and volume-regulatory signals, there are clear indications that central neuromodulators such as norepinephrine or angiotensin I1 (ANGII) play a major role in the homeostatic control of salt gland secretion. The avian hypothalamus is strongly innervated with tyrosine hydroxylase- and dopamine P-hydroxylase-positive fibers, two key enzymes in the synthesis of brain catecholamines. The existence of 01- and P-adrenergic receptors in hypothalamic structures involved in central osmoperception and integration, such as the PVN, the anterior hypothalamus, or the periventricular zone, is consistent with the postulated hypothesis (Simon-Oppermann and Giinther, 1990; Gerstberger et al., 1992; Miiller and Gerstberger, 1992) (Fig. 19). A brain-intrinsic ANGII system has been detected in the saltwater-acclimated Pekin duck, with ANGII being regulated in a clearly ECFV-dependent manner (Gray and Simon, 1987; Ramieri, 1988). Receptive systems for the peptide could be localized in areas within the BBB such as the PVN, SON, AV3V region or medullary centers such as the NTS or olivary complex, all involved in homeostatic control circuits of salt and water balance or the cardiovascular system (Gerstberger et al., 1987a,b, 1992; Simon et al., 1987, 1992). Although physiologic data concerning the action of brain-intrinsic norepinephrine on avian salt gland function are still lacking, the central application of ANGII is well known to inhibit ongoing salt gland secretion in the duck. This is probably mediated via altered parasympathetic outflow to the glandular vasculature and secretory parenchyma (Gerstberger et al., 1984~).

VII. Hormonal Control of Salt Glands Hormones defined in the classical way as mediators “secreted by specialized endocrine cells into the circulating blood and travelling relatively long distances to targets they act on” (Norris, 1985) have also been extensively investigated with regard to their putative modulatory role of avian salt gland function (Holmes and Phillips, 1985; Butler, 1984; Butler et al., 1989). Emphasis was placed on the pituitary-adrenal axis and various peptide hormones of osmoregulatory importance, such as the antidiuretic hormone AVT, blood-borne ANGII, or the recently discovered atrial natriuretic factor (ANF).

FIG. 19 Receptor autoradiographical demonstration of high-affinity binding sites for neurotransmitters involved in the hypothalamic integration of afferent signals important for the control of avian salt gland function. (a) Distribution of hypothalamic angiotensin I1 binding sites labeled with the radioiodinated receptor antagonist '251-labeled 'sar-*ile-angiotensin 11. (b) Distribution of hypothalamic a,-adrenergic binding sites labeled with the radioiodinated receptor antagonist 251-labeled HEAT (c) Distribution of hypothalamic a,-adrenergic binding sites labeled with the radioiodinated receptor agonist 1251-labeledclonidine. AC, anterior commissure; AM, amygdala complex; AV,V, anterioventral third ventricular region; LS, lateral septum; PVN, paraventricular nucleus; SFO, subfornicnl organ. Bar,2 mm. [(a) From Gerstberger et al. 1987a. with permission; (b,c) Unpublished material, courtesy of Mr. A. Muller.]

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A. Pituitary-Adrenal Axis The idea that components of the pituitary-adrenal axis may be involved in the regulation of avian salt gland function originated from the observations that adenohypophysectomy (Wright et al., 1966; Holmes et al., 1972) or adrenalectomy (Phillips et al., 1961; Thomas and Phillips, 1975a,b) inhibited or blocked salt gland responses to hypertonic saline loading. Moreover, birds with the best developed salt glands have the largest adrenals (Holmes et al., 1961; More and Patil, 1988). Salt loading, both acute and chronic, elevated plasma corticosteroid concentrations, although not in every case (Donaldson and Holmes, 1965; Macchi et al., 1967; Allen et al., 1975; Harvey and Phillips, 1980, 1982; Klingbeil, 1985a,b). Corticosterone, as the main naturally occurring adrenocorticoid in avian species (DeRoos, 1961; Sandor et al., 1963; Donaldson et al., 1965), restored salt gland secretion in adrenalectomized birds and augmented secretion in normal animals (Holmes et al., 1961; Holmes, 1972; Harvey and Phillips, 1982). In addition, active salt glands accumulated much greater amounts of corticosterone than other tissues (Bellamy and Phillips, 1966; Takemoto et al., 1975), and two types of corticosterone receptors have been isolated from cytosolic fractions (Sandor and Fazekas, 1973, 1974; Allen et al., 1975; Sandor et al., 1977). Although the above findings seem to indicate a regulatory role for corticosterone in salt gland function, recent observations have produced reasons to doubt that this role involves a direct effect on the initiation or maintenance of salt gland secretion (Butler, 1980, 1984, 1987; Butler et al., 1989). It was shown that adrenalectomized ducks given 0.9% saline as drinking fluid to counterbalance postoperative loss of salt and water, or force-fed to overcome anorexia, had salt glands that functioned almost completely normal within 7 days of adrenal gland removal (Miller and Riddle, 1943; Thomas and Phillips, 1975a; Butler and Wilson, 1985; Butler, 1987; Butler et al., 1989) (Fig. 21). Similarly, the absence of adrenal glands does not block the adaptive hypertrophy associated with transfemng ducks from fresh water to saline (Butler, 1984). Other experimental observations which indicate that corticosterone has no direct action on salt gland activity include its lack of effect in vitro on salt gland slices and the absence in vivo of any alteration in the salt gland secretion of birds given corticosterone acutely or long term (Wilson and Butler, 1980; Butler, 1984; Harvey et al., 1985). The possibility that the effect of adrenalectomy upon salt gland function is related to cardiovascular alterations induced by steroid depletion was evaluated by Butler and Wilson (1985). Although removing the adrenal glands produced no changes in blood volume, there was a gradual reduction of both systolic and diastolic pressures during the first 3 days after adrenalectomy. This decrease was prevented by betamethasone, demonstrating that steroids have an essential role in avian blood pressure maintenance. Accordingly, it appeared that the

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reduction in plasma corticosterone levels caused by hypophysectomy or adrenalectomy produced cardiovascular failure and resultant blood flow insufficiency, which prevented an increase in blood supply essential for salt gland activity (Hanwell and Peaker, 1973; Kaul et al., 1983; Butler et al., 1989). Corticosterone may also have another indirect stimulatory effect on avian salt glands by elevating plasma glucose concentration, which itself has been shown to enhance salt gland secretion by increasing the availability of metabolic substrate for the sodium-transporting mechanism (Peaker et al., 1971; Holmes, 1972; Allen et al., 1975). In addition, it has been suggested that corticosterone is physiologically relevant for the homeostatic adaptations of salt glands by regulating transcriptional events leading to protein induction required by hypertrophying glands (Sandor and Mehdi, 1981; Harvey and Phillips, 1982; Sandor et al., 1983; Harvey et al., 1985; Holmes and Phillips, 1985). The observation that adrenalectomized ducks, without corticosterone, progressively secreted fluid at a higher rate and electrolyte concentration, however, indicates that any role of corticosterone in developing glands is not essential (Butler, 1980). The fact that adenohypophysectomy completely inhibited salt gland secretion, which could be restored by adrenocorticotrophic hormone (ACTH) supplement, is likely to be dependent on the role of ACTH in maintaining plasma corticosterone concentrations rather than any direct effect it may have on salt glands (Wright et af.,1966; Bradley and Holmes, 1971; Holmes et af.,1972). Harvey and co-workers (1985) found that ACTH injection had no effect on salt gland function in ducks, and although in another investigation ACTH did stimulate salt gland secretion, its effect was thought to be due to an elevation of plasma glucose and potassium, both of which can stimulate salt glands (Peaker et al., 1971; Holmes, 1972). In the same study, however, ACTH did reduce nasal fluid “a+] and [K+], an effect not seen with hyperglycemia or hyperkalemia. It may represent a direct influence of ACTH on water movements in the salt gland. For aldosterone, there is no evidence that a direct effect on salt glands is of physiologic significance. Although this steroid has been shown to delay salt gland secretion and reduce the sodium content of the fluid, the duration of secretion is prolonged. Thus, total osmotic excretion is unaffected (Gill and Burford, 1968). Other studies have failed to demonstrate any type of effect of aldosterone on salt gland activity (Phillips and Bellamy, 1962; Holmes, 1972), and it is generally believed that this mineralocorticoid plays no role in salt gland regulation (Thomas and Phillips, 1975a; Harvey and Phillips, 1982; Simon and Gray, 1989).

B. Angiotensin II The octapeptide ANGII, the foremost product of the renin-angiotensin system, effects avian salt gland function in a way that is consistent with its generally

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accepted role of salt and fluid conservation (Peach, 1977), namely inhibition of salt gland secretion. This effect, first demonstrated in ducks by Hammel and Maggert (1983) has been repeatedly confirmed in this species (Butler, 1984; Wilson et al., 1985; Gray et al., 1986) as well as in gulls (Gray and Erasmus, 1989a) (Fig. 20). In each of these studies it was shown that elevation of the circulating level of ANGII reduces the salt and fluid output of active salt glands. In addition, reduction of the systemic concentration of ANGII by blocking its production using captopril, a converting enzyme inhibitor, enhanced extrarenal excretion in saline-loaded ducks (Wilson et al., 1985), although this effect may have involved a reflex compensation for the reduced renal output induced by captopril. The threshold plasma concentration of ANGII for its inhibitory action seems to be about 150 pg/ml (Gray and Erasmus, 1989a), although this is likely to be very much dependent on the drive to stimulate salt gland secretion (Hammel, 1989). This concentration of ANGII is well within the range measured in the blood from various bird species with salt glands (Gray and Simon, 1985; Gray and Erasmus, 1988); therefore the effect of ANGII is clearly physiologic. The inhibitory action of peripheral ANGII on salt gland secretion could be abolished by coapplication of the specific ANGII receptor antagonist 1sar-8ile-ANGII.but not 'sar-8ala-ANGII commonly applied to mammalian species (Hammel and Maggert, 1983). Specific blood flow measurements using the radioactive microspheres technique or laser-Doppler flowmetry in conscious animals clearly demonstrated that ANGII markedly inhibited both salt gland secretion and arterial blood perfusion of the gland at almost unchanged arterial pressure, heart rate, and cardiac output (Butler, 1984; Gerstberger, 1991). The seemingly obvious direct action of ANGII on both the vasculature and the secretory parenchyma of the avian salt gland, however, can be ruled out due to (a) the higher efficacy of intracarotid ANGII application in inhibiting ongoing secretion compared to that of systemic peptide administration, (b) the lack of ANGII-specific binding sites in the salt gland tissue (Gerstberger et al., 1987a,b; Gerstberger, 1992), and (c) the abolition of ANGII-induced salt gland inhibition after ganglionic blockade (Butler et al., 1989). Figure 20 shows that duck salt glands stimulated to secrete by an intravenous continuous infusion of hypertonic saline were switched off transiently by a single injection of ANGII. When steady-state salt gland secretion had been reestablished, blockade of ganglionic transmission with subsequent total reduction of secretory activity could be reversed by iv infusion of metacholine, a cholinomimetic drug. With the glands secreting in the absence of any neural input, ANGII then failed to have any inhibitory effect on their fluid or salt excretion. Blood-borne ANGII did not directly regulate NaCl transport by secretory cells in salt glands, nor did it cause direct vasoconstriction of blood vessels supplying them. The inhibitory action of ANGII was mediated via modulation of the neural control to the salt gland, presumably within the central nervous system. Whether this is limited to a

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reduction of the cholinergic input or also involves a stimulation of sympathetic fibers innervating the glands is unclear. Neuronal entities without a well-established BBB, such as CVOs, represent the prime targets for circulating ANGII to inhibit salt gland function through its central action. Receptor binding studies revealed the presence of high-affinity ANGII binding sites in the SFO and the median eminence of the duck hypothalamus being accessible to blood-borne ANGII, comparable to data available for mammalian species with regard to centrally induced drinking (Gerstberger et al., 1987a,b, 1992; McKinley et al., 1991) (Fig. 19). In addition, brain nuclei localized within the BBB, such as the PVN, SON, amygdala, or NTS, showed positive labeling for ANGII receptors (Gerstberger et al., 1987a,b). Neuronal activity recorded extracellularly in vitro from hypothalamic slice preparations of the duck brain has revealed large fractions of ANGII-responsive neurons in the SFO with activation as the prevailing ANGII effect (Matsumura and Simon, 1990a). In analogy to possible inhibitory effects on salt gland function and competitive actions at the ANGII receptive sites in the SFO, angiotensin I and angiotensin I11 proved to be uneffective, with lsar-sile-ANGII fully inhibiting the ANGII-induced stimulation of neuronal discharge (Fig. 20). Adaptation of birds reared on freshwater to hypertonic saline caused hypertrophy of the supraorbital salt glands, elevated plasma and cerebrospinal fluid concentrations of ANGII (Gray and Simon, 1985, 1987; Brummermann and Simon, 1990), and an up-regulation of ANGII receptor density in the SFO exclusively at unchanged binding affinity. This might indicate that elevated endogenous ANGII reduced salt gland output of water and electrolytes via central nervous action under conditions of reduced ECFV (Gerstberger et al., 1987a). Moreover, the threshold concentration of ANGII excitation for SFO neurons was found to decrease from M ANGII in freshwater-adapted birds to M ANGII in those adapted to saline, suggesting that the salt-adapted ducks are more responsive to ANGII than freshwater birds (Simon et al., 1989; Matsumura and Simon, 1990b).

FIG. 20 Hormonal control of avian salt gland function (angiotensin 11). (A) Dose-dependent (A-C) inhibitory action of peripherally administered angiotensin I1 (ANGII) on salt gland function in the Kelp gull under conditions of steady-state osmolal excretion (dashed line). (B) Effect of ANGII on constant sodium excretion via the supraorbital salt glands of a duck in response to hypertonic saline loading (SALT) before (left) and after ganglionic blockade of secretion by mecamylamine (MECA) application (right). To reestablish steady-state secretion despite effective ganglionic blockade, the muscarinic agonist metacholine (MCH) was infused systemically. (C) Firing rate recorded from a subfomical organ (SFO) neuron in a duck hypothalamic tissue slice that was superfused with medium containing ANGII (upper left), angiotensin 111 (ANGIII) (upper right), the highly specific receptor antagonist 'sar-*ile-ANGII with ANGII (lower left), and again ANGII (lower right). [(A) From Gray and Erasmus, 1989a (modified), with permission; (B) from Butler e t a / . . 1989 (modified), with permission; (C) from Matsumura and Simon, 1990a (modified), with permission.]

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C. Atrial Natriuretic Factor There is now abundant evidence to show that the heart functions as an endocrine organ to process and secrete a peptide that plays a vital role in osmoregulation. This hormone, ANF, has been most extensively characterized by studies in mammals (DeBold et al., 1981; Genest and Cantin, 1985; Bie et al., 1988). Atrial natriuretic factor is also present in birds (Miyata et al., 1988; Toshimori et al., 1990), and its actions appear to be designed to counteract saltand fluid-conserving systems (Schiitz and Gerstberger, 1990; Gray et a[., 1991a,b; Schutz et af.,1992a). Early studies with mammalian forms of ANF failed to demonstrate any effect of this peptide on avian salt gland function (Wilson, 1987a; Langford and Holder, 1988). The successful isolation and identification of ANF from the chicken atria, however, enabled this peptide to become commercially available (Miyata et al., 1988). One study with this avian form of ANF has provided compelling evidence that ANF has a role in the regulation of salt glands. Schutz and Gerstberger (1990) showed that elevation of the circulating levels of ANF within the physiologic range by ic and/or iv infusion in ducks with actively secreting salt glands resulted in a marked enhancement of both the rate of secretion and the osmotic concentration of the fluid (Fig. 21). Preliminary data also indicated a vasodilatory effect of bird-specific ANF on the salt gland vasculature, and enhanced secretion even in the presence of ganglionic blockade, suggestive of a direct glandular ANF action in general. Moreover, receptor autoradiography of the salt glands revealed a uniform distribution of specific ANF binding sites, with a marked density throughout the parenchyma of the glands, but not in the arterioles supplying them (Fig. 21). Further receptor studies with enriched plasma membrane fractions of salt gland tissue showed that the binding sites were of a single class and high affinity. The direct action of ANF on salt gland secretion is short-lasting, and it has been suggested that this may be due to receptor-mediated internalization of the hormone (Schutz and Gerstberger, 1990). This would be consistent with the observations of Lange et al. (1989) using antibodies directed against mammalian ANF. The authors demonstrated the presence of ANF-like immunoreactivity in duck salt glands, although this could not be repeated by Schiitz and Gerstberger (1990). The transiency of the salt gland response to ANF may also indicate that systemic ANF acts as an emergency hormone, e.g., in cases of rapid vascular volume expansion resulting from postprandial salt and fluid absorption. The nonhomologous salt-secreting rectal gland of the elasmobranchs was also shown to be stimulated by ANF of mammalian sequence as well as peptidergic extracts of the shark heart (Solomon et al., 1985b). Blood flow through the gland and glandular chloride secretion were enhanced to a degree comparable to that resulting from ECFV expansion known to induce atrial ANF release in these animals. Experimental treatment of monolayer cultures of rectal gland ep-

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FIG. 21 Hormonal control of avian salt gland function (adrenal steroids and ANF). (a) Comparable time-dependent sodium excretion via the salt glands in sham-operated and adrenalectomized Pekin ducks during osmotic stimulation. (b) Stimulatory action of atrial natriuretic factor (ANF) infused into the carotid arteries of a saltwater-acclimated Pekin duck at threshold conditions of secretion on salt gland osmolal excretion. (c) Receptor autoradiogram of a duck salt gland tissue section incubated with radioiodinated bird-specific ANF ( '251-BH-labeled BH-chANF). Positive staining can be localized throughout the secretory tissue of single glandular lobes (GL). Bar, 800 pm. [(a) From Butler e t a / . , 1989 (modified), with permission; (b,c) from H. Schiitz and R. Gerstberger, Endocrinologv, 127, 1718-1726, 1990; 0The Endocrine Society.]

ithelial cells with ANF led to a pronounced rise in Cl--dependent, bumetanidesensitive SCC irrespective of the site of ANF application (luminal, abluminal) (Karnaky et al., 1991). Silva and co-workers (1987), however, reported that ANF per se proved to be ineffective in eliciting rectal glandular ion transport. These authors claim an indirect ANFergic action via release of VIP from neural stores within the gland with subsequent VIP-induced CI - secretion. In analogy to the postulated action of ANGII on brain structures outside the BBB, ANF might also modulate osmoregulatory effector systems such as the supraorbital salt gland via interaction with neurons in the SFO or organum vasculosum of the laminae terminalis (OVLT), two CVOs heavily endowed with ANF-specific binding sites (Schutz et al., 1992b). The existence of an avian brain-intrinsic ANF system in addition to that of the periphery can be derived from immunocytochemic and radioimmunologic data, and might be of importance with respect to the long-term control of salt gland function. Experiments

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using hypothalamic application of ANF by icv perfusion or local microdialysis techniques may provide valuable information concerning the role of central ANF not only in salt gland regulation but also in avian osmoregulation in general.

D. Prolactin There is conflicting evidence concerning the possible role of prolactin in salt gland control. Prolactin has been shown to partially restore salt gland function depressed by adenohypophysectomy (Ensor and Phillips, 1972; Ensor et al., 1972), and to induce marginal salt gland secretion (Peaker and Phillips, 1969; Peaker et al., 1970). The onset of salt gland secretion was also found to be much earlier in prolactin-treated birds than in those given ACTH, indicating that prolactin may have a direct effect on salt glands (Peaker et al., 1970). In contrast to these findings, further studies have shown that prolactin has no effect on salt gland secretion (Harvey and Phillips, 1982) and, moreover, blockage of prolactin secretion by bromocryptine did not alter the salt gland response to hypertonic saline loading. It has been suggested (Harvey and Phillips, 1982; Holmes and Phillips, 1985) that any effect prolactin may have upon salt gland function is indirect and probably related to its stimulation of food and water intake (Ensor, 1975, 1978). Since food or water deprivation reduced salt gland activity (Ensor and Phillips, 1972; Phillips and Harvey, 1980), the restoration of these parameters to normal in hypophysectomized birds or the maintenance of feeding in birds adapted to hypertonic saline may be the means by which prolactin indirectly influences avian salt gland function.

E. Arginine Vasotocin The role of arginine vasotocin (AVT), the avian antidiuretic hormone, in the control of renal function has been firmly established (Ames et al., 1971; Braun and Dantzler, 1972; Stallone and Braun, 1985, 1986; Gerstberger et al., 1985; Gray and Erasmus, 1988). Strong evidence indicates, however, that AVT has no, or only limited, effect on extrarenal salt gland secretion. First, neurohypophysectomized ducks, which lack AVT and demonstrate all the symptoms of diabetes insipidus, have salt glands that respond normally to hypertonic saline loading (Wright et al., 1967; Bradley and Holmes, 1971). Second, there is no consistent relationship between circulating AVT levels and salt gland activity, so that high plasma AVT concentrations can exist during conditions of both salt gland stimulation and inhibition (Gerstberger et al., 1984b,c). The effects of AVT on salt glands reported so far have not been consistent, with facilitation (Holmes and Adams, 1963; Lanthier and Sandor, 1967) and inhibition (Gill and Burford, 1969; Peaker, 1971) being observed. Intracarotide-

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ally administered AVT (20 ng/min/kg bw) did not alter ongoing steady-state salt gland secretion in the saltwater-acclimated Pekin duck, but did induce mild vasoconstriction in the salt glands at unchanged mean arterial pressure and heart rate (Gerstberger, 1991). In all cases, however, the doses of AVT required to elicit these effects resulted in supraphysiologic plasma concentrations of the hormone, thus questioning the physiologic significance of its actions (Simon, 1982; Arad and Skadhauge, 1984; Stallone and Braun, 1986; Gray and Erasmus, 1989a,b). Receptor binding studies using tritiated arginine vasopressin as radioligand did not result in positive labeling of secretory or vascular salt gland structures, but did reveal high densities of specific binding sites in both glomeruli and tubules of the collecting ducts in the avian kidney (Keil, 1990; Keil et al., 1990). Therefore AVT does not appear to play a major role in hormonal control of avian salt gland function.

F. Other Hormones There are a number of hormones that have been incidentally considered possible candidates for a role in salt gland regulation. In each case, however, it is difficult to ascribe a clear physiologic action to the hormones simply because there are insufficient data available. Three of these hormones are discussed below. 1. Thyroid Hormones

Ensor et al. (1969) demonstrated that the response of salt glands to an oral salt load is delayed by thyroidectomy. If the same load is administered iv, the salt gland functions normally. This suggests that thyroid hormone depletion affects intestinal transport rather than directly influencing salt glands. Additionally, salt glands may depend on thyroid hormones indirectly via their regulation of cell metabolism and growth (Harvey and Phillips, 1982).

2. Substance P

In one study in ducks, large doses of substance P stimulated the rate of fluid output at marginal salt gland activity, without changing its electrolyte concentration (Cheeseman et al., 1975). Conversely, Wilson (1987b) found that in the same species substance P reduced the electrolyte content of nasal fluid without affecting the rate of its secretion. To add even more confusion, in the same study substance P increased the secretion rate in glands inhibited by ANGII, without reversing the inhibitory effect of ANGII on salt excretion. The apparent ability of substance P to dissociate changes in solute composition from changes in flow rate of salt gland fluid needs to be reevaluated and could give important insights into mechanisms underlying salt gland fluid elaboration.

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3. Catecholamines The comparison of circulating epinephrine and norepinephrine concentrations between ducks maintained on freshwater and those acclimatized to hypertonic saltwater yielded similar plasma levels of epinephrine in both groups, with norepinephrine plasma levels lower in saltwater-adapted animals (Brummermann, 1988). Indications for a catecholamine-induced modulation of salt gland function should, however, not be derived from these data. Although bilateral adrenalectomy inactivated salt gland function, injections of cortisol were able to completely restore their activity without any catecholamine supplement (Phillips et al., 1961; Butler, 1980). In addition, partial depletion of catecholamines by reserpine had no effect on the salt gland response to a hypertonic saline load (Wilson and Van Pham, 1985), and the systemic application of the a-adrenergic antagonist phenoxybenzamine did not alter either resting blood flow or the proportionality between blood flow and secretion (Kaul et d., 1983). The recent demonstration that ic injections of norepinephrine markedly reduced salt gland blood flow at only moderate reduction in osmolal excretion (Gerstberger, 1991) is probably explained by a mimicking of enhanced sympathetic innervation rather than a hormonal role for catecholamine (see Section IV,D).

VIII. Stimuludecretion Coupling To elucidate the molecular events involved in the regulation of epithelial salt secretion in the avian salt gland, the intracellular pathways transferring the extracellular signals to the cellular machinery with the final induction of Na+ and CI - secretion against an enormous concentration gradient have been studied. The contributions of the major “second and third messenger” systems, namely cyclic adenosine 3‘3‘-monophosphate (CAMP),cyclic guanosine 3 ’ ,5’monophosphate (cGMP), the inositol phosphate cycle with inositol-l,4,5trisphosphate [Ins( 1,4,5)P,] and diacylglycerol (DAG), and the intracellular calcium concentration ([Ca2+],) have been investigated primarily in isolated duck salt gland cells.

A. Cyclic Adenosine 3’,5’-Monophosphate Although some early studies tended to exclude the adenylate cyclase product cAMP from being physiologically relevant in avian salt gland secretion (Peaker and Linzell, 1975; Stewart et af., 1979), a more recent investigation (Shuttleworth and Thompson, 1987) provided evidence to the contrary. Accordingly, the application of muscarinic agonists, exogenous cAMP or forskolin, an activator

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of adenylate cyclase, to duck salt gland tissue slices stimulated ouabain-sensitive oxygen consumption. The nature of the extracellular signal responsible for activation of the adenylate cyclase-cAMP system is as yet unclear with muscarinic innervation being excluded as the natural stimulus. As mentioned earlier, duck salt glands were shown to possess VIPergic innervation in addition to the postganglionic cholinergic one, and VIP stimulated glandular secretion via membrane-bound receptors (Gerstberger, 1988). Cyclic AMP has been identified as a common second messenger of VIP in various systems (Gespach et al., 1983). This coupled system also exists in the shark rectal gland (Stoff et a/., 1979; Solomon et a/., 1984a,b). Using primary cultures of avian salt gland principal cells, the cAMP analog 8-Br cyclic AMP as well as forskolin elicited a SCC that could be potentiated by theophylline, and inhibited by both furosemide and ouabain, suggestive of an intracellular pathway involving CAMP. That VIP also stimulates ion transport in these cultures in a furosemide- and theophylline-sensitive way strongly indicates that VIP represents the prime candidate using cAMP as appropriate second messenger system in the avian salt gland (Lowy and Emst, 1987; Lowy et al., 1987) (Figs. 12,22). The role of cAMP as a second messenger in avian salt gland function might be derived from data concerning the importance of cAMP in shark rectal gland function. Using an intact animal system, ECFV expansion in the dogfish S . acanthias resulted in elevated rectal gland tissue concentrations of cAMP (Erlij and Rubio, 1986). Experiments with either the isolated gland, isolated tubules, or tissue slice preparations then showed stimulated glandular vasodilation. Hyperpolarization of tubular membranes consistent with electrogenic chloride transport, enhanced ouabain binding, and oxygen consumption due to the application of cAMP could also be demonstrated. All of these effects were abolished in the presence of furosemide, indicative of a direct stimulatory action of cAMP on the furosemide-sensitive entry of Na+ into the cell (Shuttleworth and Thompson, 1980; Forrest et a/., 1983; Shuttleworth, 1983a,b). In accordance, forskolin was found to increase basolateral membrane K+ conductance in cultured rectal gland cells, thus maintaining the driving force for apical C1- extrusion. (Valentich and Forrest, 1991; Moran and Valentich, 1991). Direct interaction of cAMP with the apical chloride conductance has been discussed as an additional mode of action of this second messenger in regulating cellular Na+ and CI- secretion in the rectal gland (Greger et al., 1987a,b; Sullivan et al., 1991).

B. Cyclic Guanosine 3’,5’-Monophosphate Another cyclic nucleotide, namely cGMP, formed by guanylate cyclase activity of membrane-associated or cytosolic origine, is also able to activate the secretory process of salt glands. Cyclic GMP has been considered by some authors to be part of the mechanisms by which neural or hormonal control of avian salt

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FIG. 22 Intracellular signal transduction in freshly dissociated avian salt gland cells. (A) Salt secretion via the principal secretory cell appears to be efferently controlled by parasympathetic innervation (muscarinic) using inositol 1,4,5-trisphosphate (IP,) and intracellular Ca2+ ([Ca2+Ii) as second messengers (SM), VIP using cyclic AMP (CAMP) as SM, and ANF using cyclic GMP (cGMP) as SM. (B) Model of agonist (Ach)-induced increase in the release of Ca2+ from intracellular stores after phosphoinositol hydrolysis with generation of diacylglycerol (DG) and IP,, and extrdcellular Ca2+ influx via specific channels. The intracellular Ca2+ pool is refilled via extracellular Ca2+ influx and energy-dependent CaZ+ uptake from the cytosol. (C) Enhanced [Ca2+], due to cholinergic stimulation (CCh) is dependent on refilling of the intracellular Ca2+ pools via extracelMar Ca2+ influx. Replacement of extracellular Ca2+ after agonist-induced depletion of internal Ca stores in Ca2+-freemedium results in a transient rise in [Ca2+],itself (asterisk). (D) Oscillations of [Caz+],increase in frequency depending on the dose of the muscarinic agonist carbachol (CCh) employed. Graded elevations in baseline [Ca2+],also occur as agonist dose increases, reaching maximally elevated sustained levels of [Ca2+Ii.(E) Oscillations in [Ca2+Iielicited by the application of CCh to the cell culture system persist in the presence of the calcium channel blocker Ni2+ at slightly reduced frequency and diminished baseline [Ca2+Ii.C,D, and E represent results from single cell analysis. (F) Time-dependent phosphorylation of a 170-kDa protein by salt gland secretory cells stimulated by the muscarinic agonist CCh. [(B.C) From Stuenkel and Emst, 1990, with permission; (D,E) from Crawford e r a / . , 1991, with permission; (F) from Torchia et al., 1991, with permission.]

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glands is transduced (Stewart et al., 1979; Holmes and Phillips, 1985; Butler et al.. 1989). In salt gland slices, guanylate cyclase stimulators, such as hydroxylamine and sodium azide, stimulated ouabain-sensitive respiration as well as Na-K-ATPase activity, as did the application of cGMP itself. This action proved to be independent of Ca2+ and was not inhibited by atropine, suggestive of cGMP-induced cellular processes not related to the parasympathetic innervation of the gland (Stewart et al., 1979; Stewart and Sen, 1981). Cyclic GMP represents the second messenger that has so far been identified exclusively for ANF in mammals (Laragh and Atlas, 1988). The fact that activation of salt gland secretion by saline loading in ducks not only increased glandular tissue cGMP concentrations but also elevated the circulating level of ANF (Stewart et al., 1979; Gray et al., 1991b) makes cGMP a more than plausible candidate involved in the secretagogue effect of ANF (Fig. 22). Comparative studies showed that the ANF-induced activation of the shark rectal gland is also mediated, at least in part, by this nucleotide, although enhanced C1- secretion in the isolated rectal gland during 8-bromo-cGMP application could not be observed (Silva et al., 1987; Karnaky et al., 1991).

C. Phosphoinositols and lntracellular Calcium The idea that activation of muscarinic acetylcholine receptors in salt gland tissue increases the turnover of cellular phosphoinositides was already indicated by the early studies of Hokin and Hokin (1967). During cholinergic stimulation of salt gland slices in vitro, the incorporation of radioactive 32P into phosphatidic acid and phosphoinositides was markedly enhanced, whereas incorporation into phosphatidyl choline and ethanolamine, major phospholipids of the plasma membrane, was negligible. These observations could then be verified using dissociated duck salt gland cells, which were either prelabeled with 32P or tritiated inositol (Fisher et al., 1983; Snider et al., 1986). Accordingly, by analogy with a number of other tissues (Agranoff et al., 1984; Berridge, 1984), receptormediated breakdown of phosphatidyl-inositol 4,5-diphosphate (PIP,) led to the formation of Ins( 1,4,5,)P, and DAG. Ins( 1,4,5,)P, generation induced via muscarinic agonists proved to be sensitive to atropine (Shuttleworth, 1990; Hildebrandt and Shuttleworth, 1991b). To identify possible effects of differentiation in avian salt gland cells on inositol phosphates, Hildebrandt and Shuttleworth (199 1b) reported that upon muscarinic receptor activation both Ins( 1,4,5)P, and Ins( 1,3,4,5)P, increased to higher levels in unstressed isolated salt gland cells compared to fully differentiated ones. This suggests a possible significance of this second messenger system in cell proliferation or differentiation during the process of salt acclimation. Ins( 1,4,5)P, has been shown to induce subsequently the release of intracellularly stored Ca’+, particularly from the endoplasmic reticulum (Berridge,

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1981; Streb et al., 1983; Burgess et al., 1984; Putney, 1986; Benidge and Galione, 1988). As indicated in Fig. 22, in avian salt glands as well phosphoinositols appear to mediate Ca2+ release from intracellular stores and may also stimulate extracellular Ca2+ entry, the latter representing the essential signal for secretory activity (Shuttleworth and Thompson, 1989). The importance of a rise in [Ca2+Iifor the secretory process could be derived from tissue slice experiments, where cholinergic stimulation of the secretory process, as indicated by changes in ouabain-sensitive respiration, proved to be fully dependent on the presence of Ca2+ in the medium (Stewart et al., 1979). Using confluent cultures of salt gland cells, cellular ion transport could be induced with the Ca2+ ionophore A23187, which also elicited a SCC blockable by ouabain and furosemide (Lowy et al., 1985a,b). Agonist-induced changes in [Ca++], have been measured in a number of studies using dissociated secretory cells from duck salt glands. In the first (Snider et al., 1986) it was found that the increase in cytoplasmic Ca2+ was entirely related to extracellular Ca2+ entry. Use of a more refined method to measure Ca2+, however, demonstrated that the initial rise in [Ca2+Iioriginated from intracellular stores, followed by an increased extracellular Ca2+ influx to maintain the elevated [Ca2+Iiconcentration and to replenish depleted intracellular stores (Shuttleworth and Thompson, 1989; Stuenkel and Emst, 1990) (Fig. 22). The mobilization of intracellularly stored Ca2+ was extremely rapid and transient, reaching a peak within 2 sec and declining to basal values after 2 min in the absence of an extracellular source of the cation. In the presence of Ca2+-containing medium, however, the fall in [Ca2+], was offset by extracellular Ca2+ entry across the plasma membrane, with [Ca2+],being sustained at or near peak values while maximally stimulating concentrations of agonist are present (Shuttleworth and Thompson, 1989; Stuenkel and Emst, 1990) (Fig. 22). This pattern of Ca2+ mobilization can be demonstrated in a large variety of nonexcitable cells (Hallam and Rink, 1985; Hallam and Pearson, 1986; Merrit and Rink, 1987; Negulescu and Machen, 1988) and in each case appears dependent on stimulation of inositol lipid breakdown. Single-cell microfluorometry of changes in [Ca2+Ii revealed [Ca2+Ii oscillations to be inducible by extracellular application of the acetylcholine analog carbachol in a dose-dependent manner. Oscillation frequencies reached extraordinarily high values compared to those of other cell types (Berridge and Galione, 1988; Cobbold, 1989; Rink and Hallam, 1989), whereas the spike amplitude remained unchanged (Crawford et al., 1991) (Fig. 22). In other exocrine glands such as rat pancreatic or parotid acinar cells, [Ca2+Iioscillation frequency proved to be independent of agonist concentration (Gray 1988; Tsunoda et al., 1990). Blockade of extracellular Ca2+ entry or reduction in extracellular Ca2+ concentration reduced frequency, but not amplitude of [Ca2+Iioscillations. It could not fully inhibit the refilling of depleted intracellular calcium stores necessary for the propagation of [Ca2+Iioscillations, in-

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dicative of both Ins( 1,4,5)P,]sensitive and -insensitive intracellular calcium pools (Hildebrandt and Shuttleworth, 1991a; Crawford et al., 1991). Analysis of inositol phosphates in principal secretory cells revealed also that raising [Ca2+Iidid not cause an increase in cellular Ins(2,3,4,5,)P4 concentration, suggestive of physiologically insignificant activation of Ins( 1.4.5)P3 3-kinase (Shuttleworth and Hildebrandt, 1991).

D. Protein Phosphorylation In salt glands, the cellular events following [Ca2+Iielevation are essentially unknown, although it is to be assumed that the Ca2+-activated K+ channels of the basolateral membrane, which are thought to play a role in the secretory process, represent an important aspect of the Ca2+-regulated stimulus-secretion coupled mechanism (Richards et al., 1989). In addition, alterations in the phosphorylation status of membrane-intrinsic proteins appeared to play a major role in the regulation of cellular ion transport (Nishizuka, 1986). Accordingly protein kinase A or C (PKA, PKC) activity might also be stimulated in the avian salt gland by extracellular primary messengers resulting in the phosphorylation of various proteins, first denied in a study by Fisher and colleagues (1983). Thus, muscarinic stimulation of suspended single secretory cells of the duckling salt gland resulted in both time- and concentration-dependent increases in the phosphorylation of a 170-kDa protein blockable by the antagonist atropine (Torchia et a/., 1991) (Fig. 22). Pharmacologic experiments using phorbolesters known to activate PKC as well as PKC inhibitors indicated that muscarinic receptor activation leads to stimulation of PKC with subsequent phosphorylation of the 170-kDa membrane-intrinsic protein. The putative physiologic significance of this protein for the secretory process is supported by phosphorylation experiments with microsomal membranes revealing the ion-dependent phosphorylation of an equal-sized protein parallel with the labeling of the catalytic subunit of the Na-K-ATPase (Russo et al., 1987).

IX. Concluding Remarks Descriptive morphologic and experimental works surveyed in this article substantiate the current concept of avian salt gland function. These supraorbitally located glands represent one of the most effective organs in the vertebrate kingdom involved in the epithelial transport of ions (sodium and chloride) against a marked concentration gradient. In an orchestrated system together with the kidneys they help to maintain avian body fluid homeostasis, and marine and estuarine birds would not survive without them.

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Acknowledgments The authors highly appreciate the support, critical suggestions, and careful revision of the manuscript by Prof. Dr. S. A. Ernst, Ann Arbor, Michigan, and hof. Dr. W. Kiihnel, Liibeck, Germany. Dr. Kiihnel supplied the authors with numerous unpublished electron micrographs of avian salt gland structure, some of them being incorporated into this review. The authors are very much indebted to Prof. Dr. E. Simon and Dr. H. Schiitz for valuable discussions and support, and to Mrs. H. Holzinger for excellent photographic work and artwork.

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Mitosis: Dissociability of Its Events Sibdas Ghosh*vt and Neidhard Paweletzt ‘Centre of Advanced Study in Botany, University of Calcutta, Calcutta 700019, India tResearch Program IV, German Cancer Research Center, D-6900 Heidelberg, Germany

1. Introduction Although the regulation of cell division is one of the most important basic questions in the context of development and differentiation, it still remains one of the least understood processes in cell biology. An understanding of the control mechanism of mitosis is essential not only to understand the basic control of the cell cycle but also to obtain a better insight in the basis for malignant growth. One approach to understanding this process is to analyze events associated with this process and to see how far these are interlinked. At its simplest, mitosis can be conceived as a linear sequence of events, each of which is required to take place before the next can occur. All these events may be induced by a common signal and they proceed in a cascade. On the other hand, the events may be only casually but not causally linked. In such case, omission or blocking of one or two events may not inhibit the occurrence of other events. Moreover, in such cases, the induction of these events would demand separate initiation factors or activators. In this review, we analyze some events associated with mitosis, such as condensation of chromosomes, breakdown of the nuclear envelope, microtubule rearrangement, development of trilaminar kinetochore, centrosome-kinetochore interaction, chromatid separation, chromosome movement, and nuclear reformation. Our main intention is to see how far these events are dissociable, independent, and inducible. As such, we have concentrated mainly on this particular aspect. A large number of reviews on mitosis have appeared in recent years. Some of them are referred to in this discussion. Apart from them, the readers’ attention is drawn to two special issues of journals (Science 1989, 246, 537-724; J . Cell Sci. 1989, Suppl. 12) in which a number of fine articles on the cell cycle and mitosis appeared. Experimental induction of mitotic events was first achieved by fusing mitotic cells with cells in interphase by Rao and Johnson (1970) using the cell-fusion 217

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technique. Components responsible for the induction of some of the mitotic events such as chromosome condensation and nuclear envelope breakdown were first identified in Xenopus oocytes and were termed maturation promoting factor (MPF) (Masui and Markert, 1971; Wu and Gerhart, 1980). A similar activity has been subsequently reported from Xenopus embryos, yeast, sea cucumber, and mammalian cells (Sunkara et d., 1976; Weintraub et d . , 1982; Kishimoto et al., 1982; Gerhart et al., 1984). It has been suggested that MPF triggers entry of cells into mitosis by initiating a cascade of protein phosphorylation reactions leading to the expression of a series of mitotic events (MiakeLye and Kirschner, 1985; Burke and Gerace, 1986). If true, this hypothesis would mean that a blockage of one early event should result in the disruption of later events. In that case, these events would not be dissociable. The last few years have witnessed a spurt of activities in understanding the mechanism of mitosis through concerted approaches by geneticists, cell biologists, and biochemists. At this juncture, we consider that a critical analysis of the events associated with this process would help toward a better understanding of the mechanism of governing the fundamental process of cell divisionmitosis.

II. Mitotic Events A. Historical Background According to Mazia (1961), Schneider described nearly all the stages of mitosis without giving exact interpretations in 1873. By 1878-1879, several workers including Strassburger and Flemming had arrived at the definitive picture of mitosis (Paweletz, 1974). The early studies were mainly descriptive (Wilson, 1925). Experimental investigations on cell reproduction began around 1950. In his classical review on mitosis (1961) Mazia presented a list of events that were regarded at that time to be associated with the process of mitosis. Even at that time, he noted that these events could run parallel, as well as sequentially. He also described continued condensation of chromosomes even when the breakdown of the nuclear envelope was inhibited by treating sea urchin eggs exposed to 0.75 M mercaptoethanol 25 min before prophase. Evidently, it was assumed that changes of the nuclear membrane were not directly geared to the condensation of the chromosomes. In the same year, Lettr6 (1961) proposed two extreme possibilities for the regulation of cell division. These are (a) that the different mitotic events are all causally connected with each other, so that one step can take place only when the previous one has been successful (cascade hypothesis) and (b) that mitotic events are only chronologically associated with each other so that the failure of one step would not necessarily prevent the next one from

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occurring. From a host of experimental data and earlier observations, he concluded that (a) several mitotic steps are independent of each other, (h) some events are causally linked to each other, and (c) others are only chronologically connected. Unfortunately, these studies did not receive much attention from later workers. Only recently has the regulation of mitotic events again become a matter of great interest.

6. Standard Type of Mitosis To examine the dissociability of mitotic events, we make a survey of the variations that can be observed in the mitotic processes in different organisms, particularly in lower eukaryotes. While talking of variations, one should expect a standard type of cell division to which all deviations can be compared. Although it will be difficult to define a standard type, since no standard type naturally exists, the general mitotic process that is encountered in the higher eukaryotes including mammalian system may be taken as a standard. A standard mitosis (as seen in mammalian cells; Paweletz, 1987) may be described as follows. The first visible sign of prophase is the condensation of chromatin. The fine or coarse network of chromatin strands develop into chromosomes. This process starts within the nuclear boundaries. In mammalian cells, the nucleus enlarges and becomes light. The centrosomes that have replicated in the preceding S phase develop an aster of microtubules and begin to separate. The nuclear envelope forms a funnel-shaped depression that transforms into a zone of deep folds and indentations, and the nucleolus disintegrates. In the cytoplasm, the membranes of the Golgi apparatus and of the endoplasmic reticulum rearrange and the Golgi apparatus disintegrates into vesicles. Prophase turns into prometaphase; the condensation of chromatin continues. In tissue culture, some type of cells lose contact with the neighbors and round up. Cytoskeletal microtubules disappear and the mitotic spindle is formed. The nuclear envelope opens at the base of the crypts and microtubules penetrate into the nuclear area; the nuclear envelope then breaks down. The trilaminar structure of kinetochores becomes identifiable, and kinetochore and microtubule attachment occur. The two centrosomes continue their migration to the prospective poles, thereby creating the two half-spindles to form the bipolar mitotic apparatus. At the end of prometaphase, the chromosomes become arranged in the equatorial plate of the spherical cell. In metaphase, the bipolar mitotic apparatus exhibits its typical spindle-shaped form and condensation of chromosomes reaches its maximum. The chromosomes are found oscillating around the equatorial plate. The membranous vesicles of the former Golgi apparatus are distributed all over the spindle area while the majority of the cisternae of the endoplasmic reticulum-nuclear envelope complex encase the mitotic apparatus.

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Anaphase starts with the separation of sister chromatids and the chromatids are translocated toward the poles. Chromatin begins to decondense. A new part of the spindle, the midbody, is formed during the separation of the chromosome groups; the osmiophilic streak that is the zone of overlapping of the half-spindle becomes visible. The spherical shape of the cell transforms into an ellipsoid, which continues to elongate. Cytokinesis begins with a ring-shaped shallow cleavage furrow. The cytoplasmic membranous system undergoes further rearrangements. Telophase is characterized by the continuation of cytokinesis. The chromosomes have reached their destinations. The nuclear envelope reforms around the chromosomes and surrounds the chromosome masses to form the daughter nuclei. The midbody regresses, and the Flemming body is formed in the middle of the cytoplasmic bridge between the two daughter cells. Nucleologenesis begins, the chromatin decondenses, and endoplasmic reticulum and Golgi apparatus reappear. The cell flattens, the cytoplasmic bridge breaks between the two daughter cells, and the Flemming body is pinched off. The mitotic cycle comes to an end (Paweletz, 1987). Unlike animal cells, cleavage does not take place in plant cells. Cytokinesis in plant cells involves the construction of new plasma membranes and a new cell wall that bisects the cell. The process depends on vesicle transport and fusion directed by a microtubule complex termed the phragmoplast. The phragmoplast appears at the equator of the mitotic apparatus at late anaphase or telophase. A cell plate is formed; the cell plate and the phragmoplast grow toward the surface to make the cytokinesis complete (see Bajer and MoE-Bajer, 1972; Inout, 1981). All these events may not be directly associated with the process of mitosis; for example, nucleolar dispersion, which was earlier considered to be an essential mitotic event (Das, 1962; Gimenez-Martin et al., 1971, 1977), takes place as a consequence of mitosis (Ghosh, 1987). Even in 1961, Mazia regarded the breakdown of the nucleolus not to be an obligatory event of mitosis. For the sake of simplicity in this review, we concentrate mainly on some events that are directly associated with the process of mitosis, e.g., chromosome condensation, nuclear envelope breakdown, centrosome activation, formation of kinetochoremicrotubule attachment, splitting of centromeres, translocation of chromatids toward poles, chromosome decondensation, nuclear envelope reformation, and cytokinesis. These events can be classified in three different series: (a) the processes that take place in the cytoplasm, (b) the events linked with the formation of the mitotic apparatus, and (c) the events taking place within the nucleus and associated with the chromosomes.

C. Variations in Lower Eukaryotes Mitotic division is a multistep process by which the genetic material is equally distributed to two daughter cells, which, in general, do not differ from their

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mother. This can be realized in a number of different ways. If during this process one or the other step is lacking or an additional step is included, but the result of the process remains almost the same, this can be regarded as a variation of the standard type. Such variations point out that the sequence of events is not strictly obligatory for the successful completion of this process. Although the variation of mitotic events is limited in higher plant and animal cells, there is much variation in lower plants and animals. An extensive and comprehensive review of variant mitoses in lower eukaryotes is presented by Heath (1980), who describes the differences of the mitotic process in algae, fungi, and protists in detail and presents them in tables. Interested readers are also referred to Kubai’s review (1975) in which the differences are discussed in relation to the evolution of the mitotic spindle. It is not the purpose of this brief survey to enumerate all major and minor variations of mitotic events in all cell types investigated so far. However, we will point to some important alterations that indicate the dissociability of mitotic events. Let us first consider the behavior of the nuclear envelope. Whereas in higher plants and animals nuclear envelope breakdown is a major event, in many lower eukaryotes the envelope persists during mitosis. Two major forms of persistent nuclear envelopes are known: In some species, particularly in Zoomastigina and Ciliophora, the nuclear envelope remains completely intact during the entire course of mitosis (Franke, 1974; Kubai, 1975; Heath, 1980). During separation of chromatids in anaphase of these cells the nucleus greatly elongates and is cleaved to give rise to two daughter nuclei; cytokinesis then follows. This type of mitosis is termed “closed cell division” or “closed mitosis.” There is a gradual transition from closed to open mitosis (our standard mitosis-as described above). In some fungal species, belonging to Heterobasidiomycetes (McCully and Robinow, 1971a,b), only a few holes are formed, which reseal around the microtubules. In some species of Rhodophyceae and fungi (see Heath, 1980), gaps in the nuclear envelope forming “polar fenestrae” develop through which the spindle is formed. A few further special features of the closed mitosis will be mentioned here. In micronuclei of some ciliates, the nuclear envelope of the daughter nuclei develops within the sheath of the mother envelope, indicating that reformation of the new envelope is independent of the existence of an “old” envelope (Franke, 1974; Heath, 1980). A very rare case occurs in Stylocephalus, in which the nuclear envelope disperses but each chromosome becomes enwrapped within a layer of double membranes (nuclear envelope) and these micronuclei are then incorporated into the spindle. In some species, the nuclear envelope remains completely intact at the beginning of mitosis, but opens in postmetaphase stage (see Heath, 1980). A very interesting case can be observed in Physarum. Aldrich (1969) described the persistence of the nuclear envelope in cenocytic plasmodia1 mitoses,

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whereas the nuclear envelope breaks down at prophase in myxamebae of the same species. In this context, the existence of cytoplasmic control (factor) for either persistence or breakdown is discussed (Ross, 1968). We can assume that this factor is quite independent of other mitotic factors and neither the occurrence nor the lack of nuclear envelope breakdown is essential for the process of cell division, and the other mitotic events in this organism are likely to be regulated independently. In cell types with open mitosis, the complete disintegration of the nuclear envelope into smaller cisternae and patches seems to be compensated by a layer of cisternae of the endoplasmic reticulum-nuclear envelope complex around the major parts of the mitotic apparatus (Paweletz, 1981). In some algae and a few other species, a system of the endoplasmic reticulum is formed around the mitotic nuclei, resembling the formation of two concentric nuclear envelopes around the mitotic chromosomes as in Stylocephalus (Heath, 1980). A large number of vesicles are found within the closed or around the open mitosis in many species of fungi or algae, as in higher organisms. One of the most conspicuous events of mitosis is the condensationdecondensation cycle of chromatin. In higher eukaryotes, the condensation of chromatin starts at the end of the S phase and culminates at metaphase; decondensation starts in anaphase, continues in telophase, and culminates in the G , phase. However, there are some differences between the condensation-decondensation cycle of the hetero- and euchromatin of the same nucleus (Frenster, 1974). In lower eukaryotes, we can find two extremes. One is that there is no condensation of chromosomes, which are very small and show no differentiation into kinetochores, centromeres, or nucleoli-organizing regions, as in Saprolegnia (Heath, 1978) and Saccharornyces (Peterson and Ris, 1976). It is not only the size of the chromosomes that determines the condensation process, since cells with an equally small amount of DNA per chromosome as in Coprinus clearly show chromatin condensation. The other extreme is no decondensation at the completion of mitosis and the chromosomes also remain in their condensed state during interphase (e.g., in some dinoflagellates such as Euglena) (Grell, 1964; Ris and Kubai, 1974). If we assume the existence of a factor controlling the process of condensation and decondensation of chromatin, it then follows that this factor is quite independent of the signals controlling other events of mitosis. Another interesting feature can be observed in Cryptophytes and Diatoms. The condensation process leads to an apparently unorganized accumulation of chromatin around the central spindle (Pickett-Heaps and Tippit, 1978), but the chromatin does not condense into individual chromosomes. There is also a wide variation in the spindle structure and function in lower eukaryotes compared to higher organisms. In higher eukaryotes, the structure and function of the mitotic apparatus are in principle the same and the spindle is always involved in the arrangement and distribution of chromosomes. In general, two basic components can be observed: a framework of microtubules

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(MTs) that either continuously or by overlapping runs from pole to pole (PMTs) and microtubules that connect the chromosomes (kinetochores) to the poles (KMTs). The latter are mainly involved in the movement of the genophores. In many lower eukaryotes, however, this is not the case. Excellent reviews on this subject have been presented by Kubai (1975), Heath (1980), and Fuge (1982). Functionally, the microtubules in some lower eukaryotes are quite different, which may indicate that microtubule-oriented chromosome movement in higher eukaryotes originated quite independently of other mitotic events. In some dinoflagellates, as in Crypfhecodinium cohnii, it can be seen that microtubules are present in the cytoplasm during mitosis but do not participate in the movement of chromosomes inside the nucleus (Soyer, 1969; Kubai and Ris, 1969; Ris and Kubai, 1974; Kubai, 1975). The chromosomes are transported along the inner nuclear membrane to polar regions in the elongated nucleus; a cleavage then follows, developing two daughter nuclei. There the microtubules act as cytoskeletal elements. In some other dinoflagellates, as in Trichonympha agilis and Hypermastigina, microtubules attach to the nuclear envelope to which the chromosomes are fixed at the inner side (Kubai, 1973). Here, cooperation between the microtubules and the nuclear envelope is necessary to move chromosomes. In this case, special parts of chromosomes are attached to the nuclear envelope for contact with the microtubules outside the nucleus. In fact, special chromosomal regions (kinetochores) can be found at the inner nuclear membrane (as in C . cohnii) in some pockets of the nuclear envelope, which is attached to the microtubules (as in T. agilis), or in nuclear pores or holes in direct contact with the microtubules (as in Syndinium sp.; Kubai, 1973). Kinetochores have been considered part of the nuclear envelope (Franke, 1974; Pickett-Heaps, 1974), but perhaps they are distinct chromosomal regions that developed contacts with the nuclear envelope in some lower eukaryotes as a means of transportation to the poles. It is now well established that in higher eukaryotes the kinetochores are intranuclear without any attachment to the nuclear envelope (Ghosh and Paweletz, 1987a; Paweletz and Lang, 1988). In lower eukaryotes, the morphology of the kinetochores ranges from very small funnel- or disc-like structures, as in C . cohnii, Haplozoon axiothellae (Siebert and West, 1974), and Amphidinium sp. (Oakley and Dodge, 1974), which are almost indistinguishable from the nucleoplasm, to large multilayered complexes, as in Oedogonium (Coss and Pickett-Heaps, 1973, 1974). In most lower eukaryotes, the kinetochores are without typical trilaminar differentiation (in higher plants the kinetochores are ill-defined, less-electron-dense ball-like structures), but are quite able to fulfill their role in chromosome distribution in mitosis either by attachment to the nuclear envelope or the microtubules directly or by means of the nuclear envelope. In species in which mitosis is closed, the presence of intranuclear kinetochores is doubtful (see Heath, 1980). In the micronucleus of Paracineta limbata intranuclear MTs are formed at mitosis. These MTs are parallel and no definite attachments to either the nuclear envelope or the poorly defined chromosomes is

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apparent (Hauser, 1972). In Trypanosoma rhodiense the MTs radiate from an intranuclear spindle pole body and elongate until the pole bodies reach the nuclear envelope. Microtubules lengthen and the polar bodies are pushed. Chromatins become distributed along the inner nuclear membrane, well-separated from the central microtubular mass. Here also, there are no definite connections between the MT and the chromosome (Vickerman and Preston, 1970). The nuclear envelope possibly plays a role in genophore distribution. In general, cell division comprises karyokinesis and cytokinesis. Under normal conditions, karyokinesis is followed by cytokinesis. In many lower eukaryotes, e.g., in Myxomycetes Physurum, the plasmodium is a syncytium in which intranuclear divisions take place synchronously without subsequent cytokinesis. Upon a special signal, however, cell walls are formed and cytokinesis proceeds to develop myxamebae (Lloyd et al., 1982). A large number of fungal, algal, sporozoal, and cilophora species present cenocytic conditions, in which karyokinesis is not followed by cytokinesis (Heath, 1980). There is a wide range of variation in the structure of the spindle pole in lower eukaryotes. As Heath (1980) states, “there is perhaps no other feature of mitosis that exhibits such a range of variation as the structures that lie at the poles of the mitotic spindle.” On the basis of the variation in the structure, they have different names such as spindle plaque, centrosomal plaque, nucleus-associated body, nucleus-associated organelle, spindle pole body, and centrosome (Heath, 1980), but the function of these polar structures (whether intra- or extranuclear) remains the same: to embody the mitotic pole. The presence of centriole is not always essential in the polar body (to be discussed later). It seems to be an accessory structure. The presence or absence of centrioles can be demonstrated in the same organism depending on its physiological state: in Myxomycetes (Nuegleria) and in few other lower organisms (Heath, 1980), centrioles are not always present during vegetative mitosis, but instead they synthesize centrioles de novo when flagellum production is necessary. Kubai (1975) and Heath (1980) have presented excellent accounts of mitotic variation in lower eukaryotes. They have tried to establish evolutionary sequences from very primitive types of cell division to a standard type such as that found in higher eukaryotes. This review does not focus on evolutionary sequence of mitosis. However, it is clear that many of the major mitotic events did not evolve simultaneously, as evidenced in lower eukaryotes. The events evolving independently are also likely to have independent control.

D. Variations in Higher Eukaryotes There have been consistent reports of different types of variations of the mitotic process in a few forms of higher eukaryotes, some of which later proved to be misinterpretations. There has been some consistent discussion on the possible

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persistence of the nuclear envelope in plant cells as a boundary around the mitotic apparatus (Wada, 1955, 1957; cited in Mazia, 1961). However, these were light-microscope observations. It is now known that layers of cisternae of the endoplasmic reticulum-nuclear envelope complex can be present around the major parts of the mitotic apparatus (Paweletz, 1981). In light microscopy, this may give an impression of a persistent nuclear envelope. In many plant cell types, a DNA-replication cycle can be observed within the nuclear envelope and without spindle formation. These are endomitotic and endoreduplication cycles (Nagl, 1981). Both cycles lead to endopolyploidy. Endomitosis (Geitler, 1939) and endoreduplication (Levan and Hauschka, 1953) differ from each other in that structural changes comparable to those seen in mitosis occur in the former event, whereas no mitosis-like process can be seen during an endoreduplication cycle. Endomitosis as such can be regarded as an intraenvelope variant of mitosis, leading to a high degree of polyploidy in different plant tissues. Endomitosis leading to polyploidization can also be occasionally met in animal cells. Mammalian bone marrow megakaryocytes reach an octaploid state (Paulus, 1968). Such endopolyploidy is also noted in silk gland cells of Bombyx mori, trophocytes of insect ovaries, etc. (Nagl, 1981). Another interesting deviation from endomitosis is the nonseparation of sister chromatids in endoreduplication so that in the next mitosis each chromosome exhibits four (two pairs) chromatids (Takanari and Izutsu, 1981; Goyanes and Svartzman, 1981). In repetitive endoreduplication (Levan and Hauschka, 1953; Rizzoni and Palitti, 1973), chromosomes show four pairs of chromatids (quadruplochromosomes). The chromatids fail to separate, since the G, cells are believed to escape mitosis (to be discussed later). Apart from polyploidization due to endoreduplication and endomitosis, other types of polyploid cells, as caused by the action of antimitotic drugs are often formed in differentiated regions of plants (Nagl, 1981) and different animal tissues, as in liver and other glands, megakaryocytes, and vegetative ganglia (Brodsky and Uryvaeva, 1977). In specific tissues of mammals and also of some other animals, polyploid cells are formed as a result of aberration in the mitotic process during the later phases after the separation of chromosomes. Such a mode of formation of polyploid nuclei is termed “mitotic,” in contrast to endomitotic polyploidization. In Drosophila, fragmentation of the nuclear envelope occurs at the spindle pole only (Strafstrom and Staehelin, 1984), resembling the semiopen mitosis found in many lower eukaryotes. The most prominent type of “abnormal” mitosis found in higher eukaryotes (especially in plants) is the delayed cytokinesis and often cytokinesis not following karyokinesis. The most common occurrence can be found in nuclear endosperms. Free nuclear conditions may persist throughout as in Floerkea, Limanthes, and Oxyspora or the wall formation may take place later as in Helianthus, Triticum, and Haemanthus (Bhatnagar and Sawhney, 1981).

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Such phenomena can also be encountered in animal cells. During the embryonic development of Drosophila, only nuclear divisions take place at the beginning and a syncytium is formed from the fertilized egg. The nuclei move to the cortex. From the fourteenth division on, however, the embryo becomes cellularized and all nuclear divisions are now always followed by division of the cytoplasm (Zalokar and Erk, 1976; Foe and Alberts, 1983). In following sections, experimental evidence will be put forward to show that karyokinesis is a process independent of cytokinesis, but that karyokinesis and cytokinesis have been interlinked to ensure smooth cellular division.

111. Dissociation of Mitotic Events A. Genetic Evidence The problem of the dissociability of mitotic events has been successfully approached by the study of cell cycle mutants and temperature-sensitive (ts) mutants. Three main cell types or organisms have been used for this purpose. Lower prokaryotes (especially fission and budding yeasts) have been studied extensively and they have contributed much to the understanding of cell division. Drosophila, another main object investigated by geneticists, has been shown to develop mutants that are defective in the regulation of individual mitotic steps. Mammalian cells cultivated in vitro represent the third group of cell types in which a number of mutants blocking or altering mitotic events have been observed. 1. Lower Eukaryotes In an excellent review, Hartwell (1978) has enumerated lower organisms in which cell or division cycle mutants have been found, such as S. cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Tetrahymena pyriformis, Ustilago maydis, Physarum polycephalum, and Chlamydomonas reinhardtii. However we will mainly concentrate on the results obtained from yeast. A cautious estimation of the number of genes involved in regulating the cell division cycle assumes as many as 400 genes; at present about 50 cell division cycle (cdc) genes have been isolated and identified (Meeks-Wagner et al., 1986). Since mitosis in yeast takes place within the nucleus, and cells and nuclei of yeast are very small, it is difficult to distinguish individual mitotic events. According to Hartwell (1978), the cell cycle in S. pombe can be subdivided into DNA synthesis, nuclear division, early plate formation, late cell plate formation, and cell separation stages. In S. cerevisiae spindle pole body (SPB) duplication, SPB separation, initiation of DNA synthesis, bud emergence, nuclear migration,

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early nuclear division, medial nuclear division, late nuclear division, cytokinesis, and cell separation can be recognized. There are cell cycle mutants affecting all cell cycle events, but we discuss here only those mutants concerned with the mitotic events. All mutants detected so far are temperature-sensitive cells that traverse the cell cycle undisturbed at their permissive temperature. However, when they are transferred to the nonpermissive (restrictive) temperature they become arrested at a respective “landmark.” Seven mutated genes can be found in cells that show defects in nuclear divisions (Culotti and Hartwell, 1971). It is likely that defects could be correlated with normal mitotic events, as seen in mammalian cells; the authors have, however, used different terminology suited to yeast mitosis such as mitotic arrest at an early stage or a medial stage or a late stage of nuclear division. Four other genes are responsible for defects in cytokinesis (Hartwell, 1971). At the restrictive temperature cells of these mutants undergo several rounds of DNA synthesis and nuclear division, but cytokinesis is blocked, which leads to the development of multinucleate cells. Methylbenzimidazole-2-yl carbamate (MBC) inhibits the division cycle between DNA synthesis and the completion of nuclear divisions (Quinlan et al., 1980). Using MBC as a tool, Wood and Hartwell (1982) tried to analyze the MBC-sensitive step by means of cdc mutants. The completion of DNA replication was found to be independent of the execution of the MBC-sensitive steps. Uemura and Yanagida (1986) found mutations of the topoisomerase I1 locus (top 2). At the nonpermissive temperature, such mutants show an uncoordinated mitosis. Normally the spindle is formed and starts to pull, but because the chromosomes are not condensed, they behave abnormally. The spindles show normal kinetochore function and spindle elongation. Here the independence of the process of chromosome condensation from all other mitotic events is evident. The authors furthermore show that topoisomerase I1 is intimately involved in the condensation-decondensation cycle of chromatin. Hiraoka ef al. ( 1984) have isolated a ts mutant that is defective in the production of P-tubulin. At the restrictive temperature, this mutant lacks a spindle and cytokinesis does not take place, even though the chromosomes condense normally. Toda et al. (1984) also report a mutant defective in a-tubulin. Two genes [nim 1, (new inducer of mitosis) and cdc 251 are responsible for the initiation of mitosis, which may be induced at a reduced cell size (compared to the wild type) when cdc 25 is lacking and is compensated by an increased expression of nim 1 (Russel and Nurse, 1987a). Mutants of the Wee 1 locus start cell division at a smaller size than the wild type. If the mitotic inducer of cdc 25 genes is overproduced, the activity of the Wee 1 locus is necessary to prevent a lethal premature mitosis. Cell division is delayed until the cells have grown to a larger size as soon as the Wee 1 expression is increased (Russel and Nurse, 1987b). The product of Wee 1 is obviously an inhibitor of mitosis. These products of the mitosis-regulating genes have been shown to be protein kinase

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homologs. Although these data have no bearing on the dissociability of mitotic events, they clearly show that the initiation of mitosis is regulated independently of the control of cell size, supporting the cell bi-cycle theory of Mazia (1974). Hartwell et al. (1974) proposed a scheme of the cell cycle of S . cerevisae derived from mutant phenotypes. According to this scheme, cdc mutants 9, 13, 16, 17, 20, and 23 block medial nuclear division; cdc 5, 14, and 15 block late nuclear divisions; and cdc 3, 10, l l , and 13 inhibit cytokinesis. Nurse et al. (1976) have proposed a scheme of the cell cycle of S . pomhe, showing diagnostic landmarks of the mutant types. According to this scheme, mutants 1, 2, 5 , and 6 block nuclear division; cdc 7, l l , 14, and 15 block early cell plate formation, and cdc 3, 4, 8, and 12 inhibit late cell plate formation. Thus, a large number of genes that control different events are involved in mitosis in both budding and fission yeasts. Naturally, these gene products may be considered independent factors. Frankel and colleagues have analyzed temperature-sensitive mutants in T. thermophila. In this organism, division is accompanied by the formation of a second oral apparatus and the development of a fission zone that precedes the cleavage furrow. In this ciliate, a macro- and a micronucleus are present, both of which under normal conditions go into mitosis before cleavage occurs. Frankel et al. (1980) isolated mutants in which micronuclear division takes place normally, whereas macronuclear division is totally suppressed. The formation of the fission zone is also prevented. In some other mutants, the fission zone is fully developed, but complete constitution is inhibited. Another type of mutant shows a somewhat altered development of the oral apparatus but cannot enter normal cell division (Frankel et al., 1980). Cleffmann and Frankel (1978) obtained mutants in which macronuclear division and cell divisions are blocked at the restrictive temperature, whereas the first stages of micronuclear division can take place undisturbed. DNA replication is also not affected. Frankel er al. (1980) described a series of cell division arrest (cda) mutants that shows different types of blocks in macro- and micronuclear division, development of the oral apparatus, and formation of the fission zone or cleavage at the restrictive temperature. Although the results obtained from Tetrahymena are not as conclusive as those obtained from yeasts, these mutants show that the typical stages of cell division in this species appear to be rather independent of each other. Similar ts mutants have also been isolated from Paramaecium tetraurelia (Jones and Berger, 1982). As in Tetrahymena the formation of a fission zone always precedes the cleavage furrow in normal division. In one mutant, only defective fission zones (dfz mutant) are formed, whereas karyokinesis and cytokinesis occur normally. In the defective constriction (dc) mutant nuclear division takes place undisturbed and the fission zone is formed, but constriction is only attempted and not completed. Thus, some of the main events of cell division can also be dissociated in Paramaecium. In A. nidulans, Morris (1976a) described a temperature-sensitive mutant that is blocked in nuclear division. This UV ts 706 mutant accumulates mitotic spindles

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and condensed chromosomes at an elevated temperature. A normal course of mitosis is resumed as soon as the temperature is downshifted from 40 to 32°C. Moms therefore concludes that the block occurs at anaphase. In this stage, the chromosomes remain condensed, the spindle does not regress, and the nucleus cannot divide. This shows that despite the normal beginning of mitosis, the block can occur at anaphase. The effects of the UV ts 706 mutation can be compared to the action of a number of antimitotic drugs, but the block is likely to be explained here by the assumption of a factor(s) controlling the postmetaphase mitotic stage. Thus, the old idea that once mitosis has started normally it must continue can no longer be maintained. The role of an anti-MPF factor (Adlakha et al., 1983; Gerhart et al., 1984) and the dilution of MPF activity (Miake-Lye and Kirschner, 1985) in the progression of postmetaphase mitotic events will be discussed later. A heat-sensitive P-tubulin mutation, benA33 of A. nidulans, blocks nuclear division and nuclear movements at the restrictive temperature (Oakley and Morris, 1981). Shifting of benA33 to the nonpermissive temperature results in the inhibition of chromosome movement to the poles (anaphase movement). However, the formation of the spindle is not blocked and the mitotic apparatus appears normal. The product of benA33 hyperstabilizes the spindle microtubules, preventing disassembly and thus causing arrest of chromosome movement. These effects can be compared to the action of D,O or taxol on spindle movement (Burgess and Northcote, 1969; Schiff et al., 1979). Although products of benA33 increase the stability of mitotic microtubules, thereby blocking nuclear division at anaphase, tubA1 and tubA4 mutations (Gambino et al., 1984) destabilize spindle and cytoplasmic microtubules and thus also block mitosis. Weil et al. (1986) isolated mutants of A. nidulans (microtubule-interacting protein, mip) that can suppress the block of benA33 at restrictive temperature and allow normal nuclear division at that temperature. The mip mutations are cold-sensitive. In a detailed comprehensive study on mitotic mutants in A. nidulans, Morris (1976b) reported 45 temperature-sensitive mutants that are defective in nuclear division, septation, and distribution of nuclei within the mycelium. The ts blocked in mitosis (bim) E7 mutant overrides normal control systems that prevent mitosis from prematurely occurring during S or G, (Osmani et al., 1988). In the never in mitosis (nim) group, the cells are blocked just before mitosis and cell division cannot take place. The bim mutants enter mitosis but cannot complete it. In some of these mutants, chromosome condensation does take place normally and the intranuclear spindle is formed, but the spindle is much smaller than that in the wild type. Because of its small size, it cannot move the chromosomes. Moreover, normal chromosome condensation and spindle regression do not take place. Here again the regulation of mitosis does not follow a cascade, but is actually a coordination of several events. The dissociability of cytokinesis can be documented in the septation (sep) mutations in which nuclear division is completely normal, but septation is

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inhibited. In the bimB strain (Boothroyd and Moms, 1986), giant nuclei are formed with a large number of small nucleoli. Here, chromosomes condense after reduplication, a spindle is not formed, and nuclear cleavage is prevented. After a definite time at the restrictive temperature, some of the giant nuclei cleave into smaller nuclei, generally with only one nucleolus, demonstrating that nuclear cleavage can be greatly delayed. Again, nuclear cleavage is independent of spindle formation. This situation is somewhat similar to the delayed cytokinesis in endosperm of certain plant species (Bhatnagar and Swahney, 1981). 2. Drosophila

The dissociability of the mitotic events has been extensively studied in mutants of Drosophila. Gelbart (1974) found a mutant (mitotic loss inducer, mit) in which definite chromosomes were lost in the course of mitosis. In these mutants, which appear to be very similar to some ts mutants of S. cerevisiae (Meeks-Wagner et al., 1986), neither karyokinesis nor cytokinesis is disturbed or inhibited. The failure of some chromosomes to be incorporated into the spindle may be genetically controlled and perhaps its regulation is independent of the system of regulations of all other mitotic events. A number of mutations in Drosophila were found by Smith et a/. (1985) and Gatti er al. (1983). They isolated mutants defective in condensation of chromatin. The functions of some mutant genes were necessary for the condensation of heterochromatin (mus 101) but not euchromatin. Other mutants affect the condensation of both types of chromatin (Gatti et al., 1983). Still other mutants, such as 1( 1) ZW. - 10, produce a large number of mitotic nondisjunctions, presumably due to premature centromere (kinetochore) separation (Smith er a/., 1985). The progeny of such cells exhibit an aneuploid genome. It has been shown (Smith et al., 1985) that mitosis is not inhibited or severely disturbed in these mutants, although some of the mitotic events are disturbed. Ripoll et al. (1985) describe a cell division mutant of Drosophila with an abnormally functioning spindle (asp). In this mutant, the spindle is altered. Cells are arrested in metaphase for a definite period of time and then the nuclear envelope is reformed, resulting in highly polyploid cells. Not only the mitotic but also the meiotic spindle is affected, resulting in e.g., diplo or nullo gametes. The light-microscopical images greatly resemble those of cells that are arrested in metaphase by colchicine-like spindle poisons. However, the authors demonstrated that a spindle was present in both mitotic and meiotic cells. In this respect, they resemble the benA33 mutant of A. nidulans. Thus, chromosome decondensation and nuclear envelope reformation are events that are independent of the presence and/or the function of the spindle. Freeman et al. (1986) describe a recessive maternal effect lethal mutant that they term giant nucleus (gnu). This mutant makes it evident that nuclear events of mitosis are uncoupled from many of the cytoplasmic ones. In gnu embryos,

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the centrosomes replicate and separate independently of all other mitotic events. These centrosomes are responsible for the nucleation of the mitotic asters that can be found in the periphery of the embryo. As already described, in the wild-type embryo the first 13 nuclear divisions take place without cytokinesis and the nuclei are found in a syncytium. During the fourteenth nuclear generation, the syncytium forms cellular membranes and about 5000 cells can be found. In the gnu mutant, DNA synthesis continues despite the lack of nuclear divisions. This finally results in a few giant nuclei, from which the name for this mutant is derived. Although nuclear divisions do not take place at the beginning, the centrosomes continue to replicate and separate. They then migrate into the cortex of the syncytium and nucleate a few giant asters. The nuclei break down and some spindle-like structures are formed. From this mutant, it is evident that the centrosome cycles are independent of the nuclear cycle. A large number of cell cycle mutants of Drosophilu have been reported so far. An excellent review of these studies has been presented by Glover (1989). Some of these mutants, such as mit(3)R2, mit(3)R72, and mit(3)R135, mimick the effect of colchicine. Mutants l(l)d.deg3 and l(l)d.deglO display overcondensed chromosomes and split chromatids, with no anaphase, which thus leads to polyploid cells (Gatti and Baker, 1988; cited in Glover, 1989). Endoreduplication has also been noted by these authors in mutant 1(3)13m281. A mutant polo shows abnormal spindle poles (Sunkel and Glover, 1988). Another mutant, merry-go-round (mgr), shows functional monopolar spindles (Gonzalez et al., 1988), which possibly arise due to a failure of centrosome division or due to the failure of centrosome pairs to separate. Another mutant, string (stg), blocks G, of interphase 14 of Drosophila embryo, which is the first zygotically controlled mitosis (Edgar and O’Farrell, 1989). String protein has been shown to be homologous to cdc 25+ from S . pomhe, an activator of cdc 2+, a constituent of MPF (Edgar and O’Farrel, 1990; Jimenez et ul., 1990). There are more mutants in Drosophila (Freeman et al., 1986; Glover, 1989). Many of these mutants support the assumption that the process of cell division is not a cascade but rather a sequence of events at least some of which may be regulated independently in a temporal order. 3. Mammalian Cell Lines Mammalian cells are of particular interest for the study of the regulation of mitosis, including cytokinesis. The elaboration of new techniques in tissue culture of mammalian cells enabled scientists to look for mutants that could provide some information about the genetic control of cell division. In 1969, Naha was successful in isolating ts mutants from a monkey cell line that had been treated with a mutagen. Since then, a large number of ts mutants showing blocks and deficiencies in the course of the cell cycle have been selected (Wissinger and

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Wang, 1983). Smith and Wigglesworth (1972) obtained a ts mutant that grew and divided normally at 31"C, but that developed binucleate cells when transferred to 39°C. Obviously, cytokinesis was inhibited, whereas karyokinesis could proceed undisturbed. Hatzfeld and Buttin (1975) isolated a ts mutant of a Chinese hamster cell line that was defective in cytokinesis at the restrictive temperature. However, along with rnultinucleate cells, cells with one giant nucleus with up to 100 chromosomes were also formed. Here, some unknown disturbance to karyokinesis also occurred in association with blockage of cytokinesis. Another ts mutant of Chinese hamster ovary (CHO) cells was isolated by Thompson and Lindl (1976). At the restrictive temperature, cytokinesis was inhibited. Both polyploid and multinucleate cells were produced. Ultrastructural studies revealed the failure of the midbody to develop, the part of the spindle between separating groups of chromosomes during anaphase and telophase. This is unique and has not been found elsewhere. There is no drug known that can mimick this effect. On the other hand, it is well known that the different parts of the microtubular spindle (aster, half-spindle, midbody, Flemming body) exhibit different sensitivity to cold or microtubular poisons, resulting in deficiency of only one part of the spindle at a threshold dose. This CHO ts mutant seems to be defective in production of compounds determining the differential sensitivity of the microtubules. Wang (1974) reported a ts mutant, ts-655, that grew and divided normally at the permissive temperature of 33°C. A shift to 39°C blocked many of the mitotic cells in metaphase. The light microscope showed that chromosome condensation and nuclear envelope breakdown took place normally but in metaphase the chromosomes accumulated in the central part of the cell. In this ts mutant, Wang et al. (1974) reported normal spindle formation at metaphase, which indicates that the blockage is due to some other reasons but not due to abnormal spindle function, unlike the asp mutant of Drosophila or BenA33 mutant of A. nidulans. It rather resembles the ts bim G mutant of Aspergillus (Doonan and Morris, 1989) and cold-sensitive disjoining-defective (cs-dis) mutants of fission yeast (Ohkura et al., 1988). Another interesting mutant was isolated by Wang (1976), which when transferred to 39°C showed defects in prophase progression. Chromatin condensed into dense clumps and no typical chromosomes were formed. Although the presence of the nuclear envelope was clearly evident at the beginning, the nuclear boundary could no longer be identified in later stages, indicating that the nuclear envelope had broken down in the normal sequence, the clumping of chromatin continued, and nuclear reformation could not be recognized. At the restrictive temperature, cells were arrested in prophase. The data show nuclear envelope breakdown without being followed by other mitotic events. In another mutant, ts 546 (Wang and Yin, 1976), the cells continued their cell cycle and proceeded until metaphase when switched to the nonpermissive temperature. Prophase and prometaphase took place undisturbed but then the chromosomes clumped and co-

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alesced into aggregates. The nuclear envelope reformed around these clumps of chromatin and then the chromatin decondensed, but cytokinesis did not follow. A ts mutant of murine leukemic cells described by Shiomi and Sat0 (1976) was found to be blocked in karyokinesis (probably during metaphase) and cytokinesis did not take place. Chromatin condensation, breakdown of the nuclear envelope, arrangement of chromosomes into metaphase-like configuration at the beginning of the shift to the nonpermissive temperature, and the typical rounding-up process occurred undisturbed, indicating not only that these events can be dissociated from the late mitotic events, but also that the late mitotic events are independent of spindle formation and its function. Another interesting ts mutant of Syrian hamster cells has been reported by Wang el al. (1983). It shows some disturbances in mitosis at the nonpermissive temperature. Cells that have reached prometaphase or metaphase at the shift from 33 to 39°C pass through undisturbed and result in two normal daughter cells. After 15 min of exposure to the nonpermissive temperature, the spindle formation is altered. A bipolar spindle cannot be formed. Instead, the chromosomes are arranged in a shell at the cell periphery; microtubules are present and connect the chromosomes to the four closely associated centrioles, which obviously form a monopolar mitotic apparatus near the center of the cell. This spherical monopolar mitotic apparatus soon transforms into a conical half-spindle in which the chromosomes become arranged in a metaphase-like configuration. Chromosomes move within this half-spindle in an ordered way, but chromatids do not separate. The chromosomes decondense, the nuclear envelope is reformed, and all chromosomes remain in one nucleus. Cytokinesis is attempted, but fails. Such cells can go into several subsequent rounds for up to 5 days, resulting in cells with hundreds of chromosomes. Here the types of microtubules responsible for the separation of the centrosomes and the erection of a bipolar spindle are, perhaps, lacking. It is obvious that only one type of microtubule is defective, whereas the rest are functional. We now have enough evidence from mutants of lower eukaryotes, Drosophila, and ts mutants of animal cell lines that cell division is not a cascade event, but at least some of these events can be dissociated from others without endangering the whole process or affecting the later events. Other experimental evidences have also confirmed these observations.

6 . Other Experimental Evidence

1. Cytokinesis Is Independent of Other Mitotic Events In general, mitosis comprises karyokinesis followed by cytokinesis. It has already been mentioned that in a large number of lower eukaryotes karyokinesis

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is not followed by cytokinesis resulting in the formation of syncytia or cenocytic cells. Such abnormality is also encountered in endosperm of higher plants and in the embryonic development of Drosophila. In yeast, a number of mutants, cdc 3, 10, l l , and 13, inhibit only cytokinesis, allowing other nuclear events to proceed. Cytokinesis can also be experimentally inhibited or delayed from the events of karyokinesis. In a classical experiment, Kihlman and Levan (1949) showed that caffeine could inhibit cytokinesis in plant cells, resulting in the formation of binucleate cells. Cytokinesis in plants itself comprises overlapping phases such as ( a ) production of Golgi vesicles, (h) vesicle accumulation, (c) vesicle arrangement, and (d)vesicle coalescence (Lopez-Saez et al., 1982). Caffeine inhibits vesicle fusion, thus inhibiting cytokinesis (Paul and Gaff, 1973). Other chemicals such as methylxanthine and 2,6-dichlorobenzonitrile are also known to cause inhibition of cytokinesis in plants (Gonzales-Reyes et al., 1986). In animal cells, cytochalasin B, a mold metabolite from Helminthosporium demafioideum, causes multinucleate cell formation by suppressing daughter cell separation following an otherwise normal nuclear division (Carter, 1967;Smith et al., 1967). In time lapse cinematographic observations, the treated mitotic cells round up, chromosomes segregate, and a normal cleavage furrow is seen to develop. The resulting daughter cells move away from each other, but remain connected by a bridge showing the midbody. This connecting bridge fails to break and the daughter cells reunite and form a large binucleate cell (Krishan and RayChaudhuri, 1969). Binucleate cells can also be induced by metabolic inhibitors such as sodium vanadate (Navas et al., 1986). This is possibly due to the inhibition of ATPase by this metabolic inhibitor (Cande and Wolniak, 1978).When the drug is withdrawn from the medium, binucleate cells are found to undergo cytokinesis and convert to the mononucleate state (own observations). This phenomenon may indicate that the factor for cytokinesis was present but could not act due to energy depletion. We have seen that karyokinesis may occur without being followed by cytokinesis. However, no cell type is known in which cytokinesis takes place under normal conditions without prior karyokinesis. Under definite experimental conditions (Lettrk, 1961),cleavage can be observed after which one “cell” is anucleate, whereas the second daughter cell contains all the genetic material. In erythroblasts, the nucleus is extruded under normal conditions, giving rise to anucleate erythrocytes. This process may be compared to the one referred to above. Hiramoto (1956, 1965) removed the mitotic apparatus in fertilized sea urchin eggs by micromanipulation or by injection of sucrose solution. He could also displace the mitotic apparatus by injecting paraffin, oil, or seawater. In all cases, cleavage took place almost normally despite the absence of the mitotic apparatus or its displacement. In the myxomycete Physarum, the plasmodium is a syncytium in which karyokinesis is not followed by cytokinesis. However, upon a special signal cell wall formation is initiated and karyoki-

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nesis is followed by cytokinesis to develop myxamebae. Similarly, cytokinesis in Drosophila embryonic development follows karyokinesis only from the fourteenth division onward. Such late wall formation is also reported in the free nuclear endosperm of certain higher plants. In the developing endosperm of Citrullus fistulosus (Chopra, 1955; cited in Bhatnagar and Swahney, 198l), the haustorium becomes cellular by segmentation into multinucleate chambers. These chambers finally subdivide to give rise to uninucleate cells. Here, the process of cytokinesis in delayed cellularization is not immediately preceded by karyokinesis. These observations indicate that cytokinesis requires a special signal (factor), but in normal mitosis it is being coordinated with karyokinesis.

2. Dissociability of Mitotic Apparatus Associated Events from Other Mitotic Events About half a century back it was noted by Blackeslee (1937) that in plant cells the chromosome number could be doubled by treatment with colchicine. Later colchicine was found to be the most specific poison acting on nearly all types of cells of animal and plant kingdom, combining effectively with tubulin (Eigsti and Dustin, 1955; Deysson, 1975). Colchicine depolymerizes the microtubules and inhibits polymerization of tubulin, but allows the other mitotic events to proceed. The chromosomes condense and the nuclear envelope breaks down, but the events associated with the mitotic apparatus are inhibited. The chromosomes remain arrested in metaphase for a considerable length of time and fail to segregate, but the chromatids separate, indicating splitting of centromeres. Then, the decondensation of chromatin starts and a new nuclear envelope is formed (Fig. 1). Cytokinesis is inhibited, resulting in the formation of a polyploid cell or a cell with a number of nuclei formed from single or a group of chromosomes (Ghosh and Paweletz, 1984a). The effect of this chemical clearly shows that the events associated with the mitotic apparatus are quite independent of other mitotic events. Moreover, cytokinesis is also a dispensable event of mitosis. It also indicates that separation of sister chromatids is not a direct function of the microtubules (Lambert, 1980). After the discovery of colchicine several other substances were found to have an action similar to that of colchicine, the best known of which are podophyllotoxins, the vinca alkaloids such as vinblastine and vincristine (Kelly and Hartwell, 1954; LettrC, 1965). Hexachlorocyclohexane or gammexane was found to have a similar effect in plant cells (D’Amato, 1969). A large number of other substances have been reported to be microtubular poisons in specific cases (see Table 5.3, Dustin, 1978). It is of interest to note that an antibiotic, griseofulvin, obtained from Penicillum griseofulvum induces polyploidy in the myxomycete P. polycephalum, which has the closed type of mitosis (Gull and Trinci, 1974). A recent addition to the list of these inhibitors is nocodazole, which shows an effect almost identical to that of colchicine (DeBrabander et a/., 1986).

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FIG. 1 Electron micrograph of a colcemid-treated rat kangaroo cell showing chromosome decondensation and nuclear envelope reformation around metaphase chromosomes, which failed to show anaphase movement. Bar, 1 pm. (From Ghosh and Paweletz, 1984a. with permission.)

Heavy water and taxol, a diterpenoid isolated from Taxus brevifolia, have effects opposite to those of colchicine and promote microtubule assembly (Burgess and Northcote, 1979; Schiff et al., 1979). Taxol also stabilizes microtubules against cold and microtubule inhibitors (DeBrabander et al., 1986). But the net effect of taxol on mitosis seems to be similar to that of microtubule inhibitors. Chromosome condensation proceeds, the nuclear envelope breaks down, and cells round up, but the chromosomes remain dispersed and immotile in an abortive metaphase stage followed by formation of restitution nuclei and readhesion of the cell. The inactivation of the mitotic chromosome movement is caused by the additional and abnormal assembly of microtubules (DeBrabander et a / . , 1986). Here again, we find that mitotic apparatus (MA)-associated events are quite dissociable from the other mitotic events. Spontaneously arising polyploid cells are occasionally met in differentiating plant tissues (Nagl, 1981) and in some animals, including mammalian cells (Brodsky and Uryvaeva, 1977). These polyploid cells are believed to be formed as a result of aberrations in the mitotic process itself. The mode of formation of such polyploid nuclei is termed mitotic (Brodsky and Uryvaeva, 1977). It seems

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that antimitotic drugs mimick these naturally occurring polyploids and they result from a mitotic cycle in which some of the events are missing. That the events associated with the mitotic apparatus are independent of the chromosome cycle in mitosis is evident from some other experiments using metabolic inhibitors, such as 2,4-dinitrophenol and sodium azide, which cause inhibition of anaphase chromosome movement (Hepler and Palevitz, 1985; Spurck et al., 1986; Ghosh el al., 1989). These arrested chromosome groups are able to form restitution nuclei. These results are somewhat comparable to the effect of taxol. Even the anaphase type of chromosome movement is not sequential to other mitotic events. McNeill and Bems (198 1) irradiated one of a pair of kinetochores of chromosomes arranged in the equatorial plane with a laser microbeam. Immediately, all the chromosomes moved to the pole to which the other chromatid was attached. Thus anaphase-type movement including the shortening of KMT fibers may occur even during metaphase. With respect to that particular chromosome, the MA is monopolar. Monopolar mitotic apparatus has been reported in a ts mutant of Syrian hamster cells (Wang et al., 1983), as already discussed. The Drosophila cell line mutant mgr is a!so functionally monopolar (Gonzalez et al., 1988). Such monopolar MA was experimentally induced in fertilized sea urchin eggs after treatment with mercaptoethanol (Mazia et al., 1981). In such cells chromosomes condense, the nuclear envelope breaks down, and chromosomes are aligned in a metaphase-like configuration and are then transported to one pole in an anaphase-like movement. The second halfspindle does not develop. A nucleus is reconstructed, complete cleavage fails, but attempts are made. Similar results are described in newt lung cells (Bajer et al., 1980), where a monopolar mitotic apparatus is spontaneously formed but as a rare event. In sea urchin, Harris (1983) noted a caffeine-induced monoaster cycling in fertilized eggs. In cold-sensitive lethal mutant ndc-1-1 of yeast, chromosomes remain attached to one pole and thus delivered to one daughter cell only (Thomas and Bottstein, 1986). It is a mutant of a cell cycle gene required for attachment of chromosomes to the spindle pole. However, normally a monopolar mitotic apparatus is formed due to the failure of the centrosomes to migrate to opposite poles and the chromosome-to-pole connections are made only by kinetochores that face centrosomes (Mazia et al., 1981). The centrosome embodies the spindle pole and its presence is universal in eukaryotic cells (Mazia, 1987), which has been fully established after the demonstration of the presence of centrosomes in higher plants using anticentrosomal antibodies (Wick, 1985). There can be accessory structures, which can be temporarily or permanently associated with centrosomes. Centrioles represent such structures and often serve as indicators of centrosomes in different cell types. However, centrioles are absent in typical barrel-shaped spindles in plant cells (Bajer and MoE-Bajer, 1972). As such, centrioles are not regarded necessary for mitosis. In multipolar spindles of higher animal cells, some poles can lack centrioles (Keryer et al., 1984). Mitosis proceeds undisturbed after selective laser

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microbeam destruction of the centriolar region in PtK2 cells in prophase (Berns and Richardson, 1977). In cells of higher animals, centrosomes undergo a cyclic development (Paweletz et al., 1984; Mazia, 1984, 1987). Although in general the centrosome cycle is coordinated to the mitotic cycle, it can be uncoupled by experimental means. In cells with arrested chromosome cycle with DNA synthesis inhibited by arabinosyl cytosine, the centrioles divided independently (Rattner and Phillips, 1973). Paweletz et al. (1984) demonstrated that the centrosomal cycle of fertilized sea urchin eggs can proceed whereas the chromosomal cycle is arrested by mercaptoethanol. In some insects (e.g., the gall midge; Wolf, 1980) asters can undergo up to four divisions, although nuclear divisions do not take place. When PtK1 cells are arrested in metaphase by means of colcemid for a long period, the number of centrosomes increases steadily by duplication, although nuclear division does not take place (Dey et al., 1989). The independence of the chromosome cycle from the centrosome cycle has been precisely recorded by Sluder et al. (1986). They showed that centrosomes in an enucleated cytoplasm in sea urchin eggs replicated with precise periodicity, indicating cytoplasmic control of the centrosome cycle. Naturally, it is expected that the mitotic events associated with the chromosomes and nuclei would be independent of events associated with the centrosomes. This has also been demonstrated in multinucleate cells obtained by cell fusion. Using a peroxidase-antiperoxidase method for the detection of polymerized tubulin in fused multinucleate cells, we (Armas-Portela et al., 1988) demonstrated that the transition of the microtubular cytoskeleton from interphase to mitosis is independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. Mitotic asters can be induced to form even around interphase nuclei (Fig. 2). In an earlier publication, we (Ghosh and Paweletz, 1987b) showed that S phase prematurely condensed chromosomes (PCCs) fail to interact with microtubules, although they contained fully formed kinetochores (Fig. 3). Consequently, they very often fail to segregate. Okadaic acid-induced PCCs also fail to organize metaphase spindle. It is likely that the premature centrosomes fail to respond to the factor( s) that induces nuclear envelope breakdown and premature chromosome condensation in the corresponding nuclei. Recently we have shown (Ghosh et al., 1992) that okadaic acid (0A)-induced PCCs in HeLa cells not only fail to organize a metaphase spindle, but also fail to develop trilaminar kinetochores.

3. Dissociability of Chromosomal and Nuclear Events in Mitosis The most conspicuous events in mitosis are the changes associated with chromosomes. The chromosomal events include condensation of chromatin in distinct chromosomes containing two chromatids, appearance of trilaminar

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FIG. 2 Asynchrony in the rearrangement of microtubules in a fused multinucleate HeLa cell. (a) Bright-field microscopy reveals the presence of mitotic asters near the prometaphase chromosome groups, but interphase microtubule in the other part. Note one of the mitotic asters in front of an interphase nucleus (arrow). (b) Fluorescence microscopy with Hoechst 222358. Bar, 20 pm. (From h a s - P o n e l a ef al., 1988, with permission.)

kinetochores, and separation of centromeres along with the chromatids followed by decondensation of chromatin, whereas the nuclear events consist of breakdown of the nuclear envelope and disintegration of the nuclear lamina and their reorganization in telophase. Like the disintegration of the nucleolus, the disorganization of the nuclear matrix network (Ghosh el al., 1978; Ghosh and Dey, 1986) may also be regarded as a nuclear event. But these are only consequences and are not directly involved in the process of mitosis per se. The first indication of the independence of the chromosome-related events came from observations on endomitotic cycles (Nagl, 1981). Endomitosis is regarded as an intraenvelope variant of mitosis, leading to high degrees of polyploidy in different plant tissues and mammalian cells, already described. Endoploidy has also been induced by various chemicals such as azaguanine (Nuti-Ronchi et al., 1965), hydroxylamine sulfate (Lin and Walden, 1974), and 3-deoxyadenosine (Gimenez-Martin et a!., 197I).

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FIG. 3 (a) S-phase PCC with very low degree of chromosome condensation but with fully developed kinetochore plates (arrow). (b) exhibits PCC kinetochores without microtubule attachment. Bar, 1 pm. (From Ghosh and Paweletz, 1987b. with permission.)

In this type of mitosis, the chromosomes condense, the kinetochores split, and the chromatids separate all within the nuclear envelope and without the formation of a mitotic spindle. It indicates that the chromosome cycle is independent of other mitotic events, although in normal cells they are highly coordinated. Moreover, it can be inferred that centromere splitting is a chromosomal event, which is also apparent from colchicine-treated cells (Lambert, 1980). Endoreduplication also leads to endopolyploidy (Levan and Hauschka, 1953), but here the chromosomes do not show structural change and the centromeres fail to separate. Endoreduplication has been observed to arise spontaneously in a number of cell types, as already mentioned. A larger incidence of this phenomenon can be induced by colcemid (Herreros and Gianelli, 1967; Rizzoni and Palitti, 1973), mitomycin C (Takanari and Izutsu, 1983), hydrazine (Speit ef al., 1984), and a number of other chemicals and also in plant cells by 8azaguanine (Nuti Ronchi et al., 1965) and hexyl mercury bromide (Levan, 1971). However, data on endoreduplication are scarce. In this case, we must presume that the G , chromatin in the absence of mitotic division must enter the second cell cycle to undergo another round of DNA replication to give rise to diplochromosomes in the following mitosis (Schwarzacher and Schnedl, 1965). However, it is known that G, nuclei are unable to synthesize DNA in fused multinucleate cells even in the presence of S-phase nuclei (Rao and Johnson,

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1970). The chromosomes have a continuous coiling cycle and at the end of G, they are maximally decondensed when S phase and DNA replication can begin (Mazia, 1987; Pederson, 1972). The G, chromosomes, although not so condensed as the mitotic chromosomes, must undergo decondensation before the initiation of the next DNA replication phase. As such, in endoreduplication the G2 cells are presumed to enter the next G , phase without entering the M phase. The trilaminar kinetochores appear in prometaphase (Rieder, 1982). Their development could be observed even on prematurely condensed chromosomes (Ghosh and Paweletz, 1987b). It can be assumed that in the endoreduplicated cells, the kinetochores do not develop in the absence of the mitotic factors, and centromeres fail to split and the chromatids fail to separate in absence of mitosis. Endoreduplication represents a cell cycle that omits the mitotic phase, whereas in endopolyploidy mitosis takes place without involving the nuclear and the centrosome-associated mitotic events (intraenvelope). Other evidence indicating the dissociability of chromosomal and nuclear events of mitosis can be considered in two broad categories: (a) the early mitotic events, including chromosome condensation, nuclear envelope and nuclear lamina breakdown, development of trilaminar kinetochores, and splitting of chromatids; (b) postmetaphase mitotic events, such as reformation of the nuclear envelope and nuclear lamina and decondensation of chromatin.

a. Early Mitotic Events Mitotic chromosome condensation is part of the chromosome-coiling cycle (Mazia, 1987) and the chromosomes are already well condensed when the nuclear envelope breaks down at the onset of prometaphase. As such, these two events are unlikely to be controlled by a common factor. Mazia (1961) noted continued condensation of chromosomes even when the nuclear envelope breakdown was inhibited in mercaptoethanol-treated sea urchin eggs. In fused multinucleate cells, we (Ghosh and Paweletz, 1984b) observed that the nuclear envelope breakdown was often delayed in certain nuclei but the chromosomes could reach a fully condensed state (Fig. 4). Obviously, these events are not likely controlled by a single factor and should be regarded as dissociable. This is also supported by the observations of Wagenaar (1983a). He noted that sea urchin embryos did not show chromosome condensation and mitosis when the protein synthesis was inhibited 25 min after fertilization. When the protein synthesis was inhibited 30 min after fertilization, the chromosomes condensed but the nuclear envelope failed to break down and the cells were arrested at prophase. The results indicate that chromosome condensation and nuclear envelope breakdown are controlled by two separate factors. When mitotic cells are fused with interphase cells, the chromatin of interphase cells shows premature chromosome condensation (Rao et al., 1977). The PCCs lack the nuclear envelope and show different degrees of chromosome condensation. In PCCs chromosome condensation initiates only after breakdown of the nuclear envelope (Peterson and Berns, 1979; Ghosh and Paweletz, 1984b). This

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FIG. 4 Electron micrograph of a fused multinucleate HeLa cell, showing one well-advanced nucleus in close proximity to two less-advanced nuclei. The chromosomes are highly condensed but are still enclosed with the nuclear envelope. Bar, 1 fim. (From Ghosh and Paweletz, 1&7a, with permission.)

sequence of events is quite different from that found in normal mitosis. This phenomenon actually represents premature nuclear envelope breakdown with incomplete chromosome condensation. Premature chromosome condensation is induced by the MPF synthesized or activated by another nucleus residing in the same cytoplasm. As such, premature chromosome condensation cannot be observed in mononucleate cells. A temperature-sensitive mutant, BN 2 of the BHK cell line (Syrian hamster fibroblast), may undergo premature chromosome condensation and other early mitotic events at the restrictive temperature (Nishimoto et al., 1978, 1985).

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The block to M-phase initiation can be overcome by treatment with caffeine, which induces premature chromosome condensation, nuclear envelope breakdown, and mitosis-specific phosphoprotein synthesis in synchronized BHK cells arrested in early S phase (Schlegel and Pardee, 1986). The dependence of mitosis on completion of DNA synthesis is lost in Wee mutants in fission yeast (Enoch and Nurse, 1990) (discussed later in relation to the checkpoint hypothesis). The ts bim E7 in Aspergillus also overrides the normal control system that prevents mitosis from occumng prematurely during S or G, (Osmani et al., 1988). Okadaic acid at a concentration specifically inhibiting phosphatase 1 can also induce PCCs (Yamashita et al., 1990). A possible molecular mechanism of this phenomenon has been discussed by Enoch and Nurse (1991). Mitosis in the absence of chromosome replication is disastrous, so cells had to develop a control maintaining dependence of mitosis on chromosome replication. However, even the initiation of mitosis is not nondissociably coupled with the completion of S phase. From their biochemical experiments using an in vitro system, Newport and Spann (1987) conclude that chromosome condensation occurs independently of nuclear envelope breakdown and lamina depolymerization. Chromosome condensation can be specifically inhibited by competition for a putative binding protein, whereas lamina depolymerization remains unaffected. Chromosome condensation is also blocked by inhibitors of topoisomerase I1 (Wright and Schatten, 1990). Topoisomerase I1 has been identified as a major component of protein fractions derived from mitotic chromosomes (Eamshaw et al., 1985; Eamshaw and Heck, 1985). The development of the trilaminar structure of kinetochores can be seen only after nuclear envelope breakdown (Rieder, 1982). This could indicate that kinetochore plate formation might depend on nuclear envelope breakdown, being triggered by cytoplasmic factors diffusing into the rupturing nucleus (Roos, 1973). However, we (Ghosh and Paweletz, 1987a) were able to demonstrate the presence of fully developed kinetochores on chromosomes still enclosed within the nuclear envelope (Fig. 5). This could indicate that the same factor is not responsible for the breakdown of the nuclear envelope and the development of the kinetochore plates. Fully developed kinetochores have been observed on PCCs belonging to G I , S, and G, phases (Szollosi et al., 1986; Ghosh and Paweletz, 1987b). It is possible that the same factor that controls chromosome condensation also controls the development of kinetochores. However, the top 2 mutant of yeast shows formation of normal kinetochore with normal function on abnormal chromosomes that fail to condense (Uemura and Yanagida, 1986). This shows that kinetochore plate formation is an event independent of even chromosome condensation. The PCCs belonging to the G I or S phase often fail to be connected to the spindle fibers (Ghosh and Paweletz, 1987b), due to unsynchronized chromosomal and centrosomal cycles, as has already been discussed. However, kinetochore development itself seems to be a chromosomal event.

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FIG. 5 Electron micrograph of a fused multinucleate HeLa cell (part), showing condensed chromosomes still enclosed within the nuclear envelope. Fully formed trilaminar kinetochores are visible on two chromosomes (arrow). Bar, 1 pm. (From Ghosh et al., 1987a. with permission.)

Balczon and Brinkley (1987) reported the presence of a specific protein complex on metaphase chromosomes that is contiguous with kinetochore-bound tubulin and may be involved in microtubule-kinetochore interactions during mitosis. In that case, kinetochore-microtubule interaction may depend on the synthesis of a special type of protein. Cells entering into mitosis when treated with protein synthesis inhibitors often show irregularities in microtubule kinetochore attachment (Wagenaar, 1983b), which may indicate that the factor controlling the microtubule-kinetochore interaction may be synthesized during G,, before the cells enter mitosis. Recently the role of a group of proteins (inner centromeric proteins, INCENPs) isolated from the mitotic chromosome scaffold of MSB 1 cells (chicken) in sister chromatid pairing and separation has been claimed (Cooke et al., 1987, 1990). In metaphase chromosomes, these proteins have been located all along between two chromatids. In colcemid-blocked diplochromatids they appear restricted to the centromere. Another type of protein, chromatid linkage protein (CLiP) has been isolated from sera from a CREST (calcinosis, Raynauds phenomenon, esophageal dismotility, sclerodactyly, and telangiecta-

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sia) patient, which has been shown to have identical chromosomal locations (Rattner et a!., 1988). Although these two groups of proteins seem to have identical functions, they differ structurally. It has long been claimed that chromatid separation is a chromosomal function and that chromatids can separate even in the absence of microtubule attachment to the kinetochores, as in colchicinetreated cells (Mol&-Bajer, 1958; Lambert, 1980). Even in acentric chromosomal fragments, sister chromatids are seen to separate simultaneously with their centric partners (Carlson, 1938). The assumed role of INCENPs or CLiPs in chromatid separation could explain this behavior (Earnshaw and Rattner, 1989). It is possible that some modifications of INCENPs or CLiPs occur at metaphase stage, which change their chromatid linking property, and the chromatids fall apart. This modification would then be governed by some definite mitotic factor. Alterations of such factors by mutations may also explain the behavior of ts bimG mutants of Aspergillus and cs-dis mutants of fission yeasts. It is interesting to note that both of these mutants fail to encode phosphoprotein phosphatase 1 (Doonan and Moms, 1989; Ohkura et al., 1989). On the contrary, the high frequency of aneuploid nuclei in Drosophila (1) ZW-I0 mutants appears to be the consequence of premature separation of sister chromatids at prophase-metaphase and their subsequent independent regulation at anaphase (Smith et al., 1985). This mutant may lack the CLiP or INCENP necessary for the cohesion of sister chromatids. Recently, however, it was suggested that the INCENPs represent a new class of proteins, chromosomal passenger proteins, that are canied to the spindle equator by the chromosomes and subsequently perform a cytoskeletal role following their release from the chromosomes at the metaphase-anaphase transition (Earnshaw and Cooke, 1991). Recently we demonstrated (Ghosh and Paweletz, 1992) that phosphatase 1 inhibition at metaphase by okadaic acid induces failure of sister chromatid separation even in mammalian cells. The visible nuclear event during initiation of mitosis is the breakdown of the nuclear envelope. Disassembly of the nuclear envelope begins at prophase when the pore complexes disappear and the nuclear membranes are fragmented, forming small vesicles that disperse throughout the cytoplasm and become indistinguishable from membranes of the endoplasmic reticulum (Roos, 1973). The nuclear lamina which is a supramolecular protein assembly associated with the nucleoplasmic surface of the inner nuclear membrane is depolymerized in coincidence with the disassembly of the nuclear envelope (Gerace and Blobel, 1980). Phosphorylation of lamin may lead to the disassembly of the nuclear lamina which may in turn trigger nuclear envelope breakdown. However, there is ample evidence to indicate that the nuclear envelope breakdown and lamina disassembly are dissociable events. The lamina polypeptides appear in the cytoplasm long before the nuclear envelope has disappeared (Jost and Johnson, 1981). Using a cell-free system, it has been observed that structural proteins of the nuclear lamina are hyperphosphorylated within 15 min after addition of MPF, followed by gradual depolymerization of the nuclear lamina

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until the nuclear envelope breaks down 30 min later (Miake-Lye and Kirschner, 1985). Even the disassembly of the nuclear lamina itself may not be responsible for the breakdown of the nuclear envelope. In oocytes, the nuclear lamina disappears during zygotene, but then reappears in diplotene (Stick, 1987) within an intact nuclear envelope.

b. Postmetaphase Mitotic Events Apart from anaphase A and anaphase B movements, other postmetaphase mitotic events are chromosome decondensation, nuclear envelope reformation, and reassembly of lamina. These are chromosomal and nuclear events. That these events are not dependent on successful completion of anaphase movement is evident from the effect of a large number of drugs that lead to the arrest of anaphase movement. Examples of these drugs are colchicine, nocodazole, taxol, and dinitrophenol, whose effect on microtubules has already been discussed. In fused multinucleate cells, we (Ghosh and Paweletz, 1987c; Ghosh et al., 1988) observed that neither decondensation of the chromatin nor dissolution of the spindle was associated with the induction of nuclear envelope reformation (Fig. 6 ) . Similarly, in colcemid-induced multinucleate cells nuclear reformation was found to be induced around metaphase chromosomes by diffusible factors from nearby telophase groups. In 2,4-dinitrophenol-treatedcells, nuclear envelope reformation could be observed (Ghosh et al., 1988, 1989) around condensed anaphase chromosomes with distinct trilaminar kinetochores and microtubular attachment (Fig. 7). It seems that ATP depletion does not inhibit nuclear envelope reformation per se. On the other hand, a striking chromatin condensation results from ATP depletion (Newmayer et al., 1986). It is very likely that these two events are dissociable. The assembly of the nuclear lamina is concurrent with the reformation of the nuclear envelope (Gerace and Blobel, 1980). Burke and Gerace (1986) noted a telophase-like reconstruction of the nuclear envelope around endogenous mitotic chromosomes in a cell-free system involving total homogenates from CHO metaphase cells along with dephosphorylation and assembly of the lamina around metaphase chromosomes in that array. Neither of these processes require free ATP. However, nuclear envelope reformation can take place without the formation of a lamina, as has been demonstrated by Benavente and Krohne (1986) after microinjection of lamina antibodies. Although these events are dissociable, they may be triggered by a common factor and both processes are regulated by protein dephosphorylation (Burke and Gerace, 1986). Fusion between mitotic and interphase cells demonstrates that cells in mitosis contain cytoplasmically transmissable factors that are able to induce both breakdown of the nuclear envelope and condensation of chromatin in interphase cells (Rao and Johnson, 1970). Subsequently, this cytoplasmic factor has been isolated and has been purified to a great extent (Sunkara et al., 1976; Wu and Gerhart, 1980; Adlakha et al., 1985; Lokha et al., 1988).

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FIG. 6 Electron micrograph of part of a fused multinucleate HeLa cell exhibiting meta- and/or anaphase chromosomes with microtubule attachment to kinetochores (arrow).Nuclear envelope formation around these chromosomes is almost complete. Bar, 2 pm. (From Ghosh and Paweletz, 1 9 8 7 ~with . permission.)

Originally, the activity of the MPF-inducing germinal vesicle breakdown (GVBD) and chromosome condensation was demonstrated in amphibian oocytes leading to meiotic maturation. Mitotic maturation has also been reported to be induced by MPF (Kishimoto et al., 1982; Halleck et al., 1984). Maturation promoting factor activity has been observed even in starfish, in sea cucumber (Kishimoto et al., 1982), and in yeast cells (Weintraub et al., 1982); MPF has been isolated from metaphase chromosomes, also (Adlakha er al., 1982). Even in a cell-free system MPF has been found to induce early mitotic events such as chromosome condensation, nuclear envelope breakdown, lamina disassembly including hyperphosphorylation of the lamina, and formation of the spindle (Lokha and Maller, 1985; Miake-Lye and Kirschner, 1985; Suprynowicz and Gerace, 1986). Spindle formation obviously indicates the development of trilaminar kinetochores on condensed chromatin through induction by MPF. It has been suggested that MPF triggers entry of cells into prophase by initiating a cascade of protein phosphorylation reactions leading to chromosome condensation, nuclear envelope breakdown, and other early mitotic events (Miake-Lye and Kirschner, 1985; Burke and Gerace, 1986). The activity of the MPF can be preserved by a cytostatic factor (CSF), which is thought to maintain metaphase arrest by stabilization of MPF (Masui er al., 1980; Newport and Kirschner, 1984).

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FIG. 7 Electron micrograph of part of an “anaphase” cell treated with 2.4-dinitrophenol showing reconstruction of nuclear envelope around chromosomes with distint trilaminar kinetochores and microtubule attachment. Bar, 1 pm. (From Ghosh et al., 1988, with permission.)

As MPF can induce mitotic events that appear one after another, it is believed that MPF induces a cascade mechanism that ultimately induces all the nuclear events (Miake-Lye and Kirschner, 1985; Burke and Gerace, 1986). In the cascade mechanism there is a substrate-product relationship. As such, the completion of earlier events is necessary for the initiation of later events. in this survey work we have presented many examples where later mitotic events run almost unhampered even though earlier events failed to occur. As an alternative to the cascade hypothesis Hartwell and Weinert ( 1989) have proposed a checkpoint hypothesis. According to this hypothesis the completion of an earlier event acts as a checkpoint for the later events. This hypothesis is mainly based on observations of some yeast ts mutants defective in DNA replication functions. Apart from the budding yeast mutants, several other mutants of fission yeast (Enoch and Nurse, 1990) and A. niduluns (Osmani et ul., 1988) and BN, mutants of a BHK cell line (Nishimoto et al. 1978) are known in which M phase does not depend on the completion of the S phase. This dependency is also abolished in

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hamster cells treated with caffeine (Schlegel and Pardee, 1986) or mammalian cells treated with okadaic acid (Yamashita er al., 1990; Ghosh er al., 1992). In fission yeast activation of the cdc 2 kinase at M phase requires the product of the cdc 25 gene. Again, the cdc 25 activity is countered by two inhibitors, Wee 1 and mik 1, which together maintain the cdc 2 in an inactive state (Enoch and Nurse, 1991). At the molecular level the entry into M phase requires tyrosine/phosphorylation of p34 and dephosphorylation is blocked when replication is inhibited. Uncoupling of M phase from S phase may be induced by premature dephosphorylation of p34, through loss of inhibitors or by overexpression of activators or by chemical treatments. Thus the dependency is not a block in the cascade (substrate-product relationship), but is due to an intrinsic control, which is a coordination of several molecular events. Hartwell and Weinert (1989) have also cited a dependence of anaphase on metaphase, and the delay in the transition when metaphase is hampered, as evidence of a checkpoint. The best example of such delay can be observed in colchicine-treated cells. Here, the arrest in metaphase is accompanied by delayed degradation of cyclin B (Minshull et al., 1989; Lewin, 1990). Anaphase transition is also delayed in mutants deficient in phosphatase 1 activity (Doonan and Moms, 1989; Ohkura et al., 1989). These are, as such, intrinsic molecular controls. Actually, the induction of mitosis by MPF alone does not preclude the possibility of the dissociability and independence of different mitotic events. As chromosomes condense at mitosis, histone H, and H, become highly phosphorylated (Gurley ef al., 1978). Similarly the nuclear lamina becomes phosphorylated before disassembly (Gerace and Blobel, 1980). Sahasrabuddhe er al. ( 1984) noted phosphorylation of eight major nonhistone proteins (NHPs) before the initiation of mitosis. These NHPs were rapidly dephosphorylated during M-G, transition. Naturally, it may be surmised that MPF in turn activates a number of protein kinases to induce mitotic maturation. Actually, Murray and Kirschner (1989) have proposed a model in which MPF has been suggested to induce nuclear envelope breakdown, chromosome condensation, spindle assembly, etc., independently from each other (possibly acting on different substrates). Indeed, it has been shown by Newport and Spann (1987) that MPF is not the immediate effector of mitosis. The MPF can be depleted of activities required to promote nuclear envelope breakdown by preadsorption to DNase-treated nuclei, which implies that the primary interactions between MPF and nuclei involve proteins rather than DNA. Again, preincubation of MPF is not the immediate effector of mitotic breakdown. In another elegant experiment, Lokha and Maller (1985) have shown that when sperm chromatin or somatic cell nuclei were incubated with isolated MPF, they did not show mitotic changes. However, the same supernatant containing the MPF could induce nuclear envelope breakdown, chromosome condensation, and spindle assembly when added to extracts in which particulate components were abundant. These results suggest that particulate components are also required for the nuclear changes and that the MPF

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cannot act directly on the chromatin. Obviously, the particulate material in this experiment contained inter alia nonchromosomal bodies such as centrosomes. Moreover, there is a possibility that MPF itself may contain more than a single factor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the partially purified mitotic factor indicated the presence of several polypeptides with a major band of 50 kDa (Adlakha et al., 1985). Even in a highly purified MPF preparation from Xenopus, Lokha et al. (1988) noted the presence of several proteins in minor amounts but two proteins of 45 and 32 kDa (34 kDa) were consistently present. The last few years have witnessed a confluence of genetical and biochemical approaches to the understanding of the cell cycle control. It has been established that S. pombe cdc 2+ and its homolog in S. cerevisiae cdc 28+ encode a protein kinase of 34 kDa, p34 (Hindley and Phear, 1984; Reed et al., 1985; Simanis and Nurse, 1986; Russel and Nurse, 1987a). Further, the Xenopus homolog of p34 cdc 2/28 has been shown to be a component of MPF (Dunphy et al., 1988; Gautier et al., 1988). This kinase probably acts in a complex with several other polypeptides to phosphorylate specific proteins to bring about changes associated with initiation of mitosis. The p34 level in the cells is remarkably stable throughout the cell cycle (Wittenberg and Reed, 1988; Draetta et al., 1989). However, in HeLa cells its enzymatic activity increases at least 70-fold as the cells move from G, to mitosis. It becomes inactivated during metaphase. This inactivation is associated with loss of a 62-kDa subunit from the protein kinase complex (Draetta and Beach, 1988). In G, cells, p34 becomes associated with p62 and is phosphorylated and maximally active as a protein kinase. On the contrary, another group of cell cycle proteins, the cyclins, accumulate during interphase but undergo rapid degradation at the end of each mitosis. The oscillation of cyclin levels is regulated by selective proteolysis which occurs at the metaphase/anaphase transition (Evans et al., 1983; Swenson et al., 1986; Standart et al., 1987). Earlier, it was proposed that cyclin A may have a role as an activator of MPF (Swenson et al., 1986). However, recently obtained evidence indicates that sea urchin and clam cyclins, which are a S. pombe pl3+ homolog, may be the second MPF (p45) component (Pines and Hunt, 1987; Booher and Beach, 1988; Solomon et al., 1988). It has been further shown that clam p34 is found in association with both cyclin A and cyclin B, probably not in a lrimolecular association, but as separate p34/cyclin A, p34/cyclin B complexes (Draetta et al., 1989). These authors have proposed a model depicting the relationship between p34 and cyclins in the activation-inactivation cycle of MPF. They have proposed that active MPF is created by post-translation modification of cyclin A and B/cdc 2+ complexes and is responsible for driving the cells into mitosis. It is possible that divergent regions of cyclin A and B sequences might differentially regulate the properties of the protein kinases. Indeed, the complex containing cyclin B has much more histone 1 kinase activity than that containing cyclin A (Draetta et al., 1989), although both cyclin A

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and B are equally capable of causing nuclear envelope breakdown (Lewin, 1990). The association of cyclin A with p34 begins at S phase, but that of cyclin B during G, (Pines; cited in Lewin, 1990). Some potential functional differences between the forms of the kinase assembled with either of these cyclins are suggested by the differences in the timing of their maximum activity. A new class of cyclin, PRAD 1 (Motokura et al., 1991), has been recently reported. It can also bind and activate p34. At present at least four G,-specific cyclins are known (Surana et al., 1991). All these facts point to the possibility that different types of MPF activities may be needed for the initiation of mitosis. Sloboda (cited in Lewin, 1990) reported that phosphorylation of a 62-kDa protein of the mitotic apparatus correlates with the solubilization of microtubules of the mitotic apparatus. The kinase that undertakes this activity appears to be a calcium/ calmodulin-dependent enzyme present in the mitotic apparatus. Okadaic acid, which induces a rapid activation of MPF in S-phase cells, fails to organize metaphase spindles. In S. pombe, cdc 13 may interact with microtubules, as in cdc 13-1 17 mutants cytoplasmic interphase microtubules appear cytologically normal but the mitotic spindles fail to form (Hagan and Hayams, 1988). In S. cerevisiae cdc 28-1N cells arrested with fully formed mitotic spindle indicate that they are defective in executing certain other aspects of mitosis (Surana et al., 1991). These authors have further shown that clb 2 and cdc 28-1N mutants have high levels of kinase activity and yet are delayed or completely defective, respectively, in executing mitosis. These observations strongly suggest that cdc 28 kinase (cdc 2 kinase = p34 kinase) activity per se is not sufficient for mitosis. Similarly, Osmani reports (cited in North, 1991) the requirement of a second protein kinase, encoded by the nim A gene along with the cdc gene products for the initiation of mitosis in A. nidulans. Similarly, Obara et al. (1975) noted a requirement for continuous protein synthesis in the interphase cells before fusion to induce nuclear reformation (antiMPF). It is now known that MPF inactivation requires cyclin degradation (Woodgett, 1991), which is likely mediated by phosphatases. In yeast, a number of mutants are reported which show blockage of late nuclear events. Likewise, UV ts 706 mutant of A. nidulans (Morris, 1976a) cannot proceed to anaphase at elevated temperature. Another mutant of this species, ts bim G, fails to complete anaphase (Doonan and Moms, 1989). Schizosaccharomyces pombe cs-dis mutants are defective in sister chromatid disjoining (Ohkura et al., 1988). All of them indicate that gene products control these mitotic events. Since protein phosphorylation is important for the G2/M transition, it is likely that dephosphorylation could be required for the transition from mitosis to G1 (Lewin, 1990). Both A . nidiilans bim G+ and S. pombe dis 2+ encode phosphoprotein phosphatase 1, which is highly homologous to mammalian protein phosphatase 1 (Doonan and Morris, 1989; Ohkura et d., 1989). However, only four types of serinetthreonine-specific protein phosphatases have been found in mammalian cells (Ingebritsen and Cohen, 1983). There may be more

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awaiting discovery or the restricted number of protein phosphatases may have a wide variety of substrate specificities to control different late mitotic events.

IV. Conclusion A critical survey of mitotic events in lower and higher eukaryotes shows that all the events that are associated with this process are not causally coupled. The evolutionary sequence of mitosis in lower eukaryotes also indicates the dissociability of these events. Genetic studies in cell cycle mutants in lower eukaryotes, especially in yeasts and Aspergillus, in Drosophilu, and in mammalian cell lines showing blockage at particular mitotic stages at restrictive temperatures demonstrate that particular gene functions may be required at those stages for completion of the process. In other words, different mitotic events may be governed by different gene products. Other evidence provided by experiments using the cell fusion technique, chemicals affecting different events of mitosis, antibodies and inhibitors, and cell-free systems strongly supports the dissociability of many of the mitotic events. Finally, recent biochemical studies including those on the cell cycle mutants indicate the possibility of the presence of multiple forms of MPF and protein kinases that are likely to be responsible for mitotic induction and initiation of different mitotic events. The roles of protein kinases for the transition of G,/M and phosphatases for M/G, also indicate that separate activities initiate early and late mitotic events. We conclude that several mitotic events are dissociable and run parallel and are perhaps governed by independent factors. However, we do not exclude the possibility that some mitotic events may be interdependent and may be controlled by common causal factors and that all these events are strongly coupled in normal mitotic division in a temporal order.

Acknowledgments This work is part of an Indo-German Science Collaboration Project between the Indian Council of Medical Research, New Delhi, and the Gesellschaft fur Strahlenforschung, Munich. S.G. is grateful to the German Cancer Research Center for financial assistance. We also thank Dr. D. Schroeter for help in preparing this manuscript and Ms. C. Kamp, Ms. E. Gundel, and Mrs. A. Wohlfahrt for careful secretarial work.

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The Endosymbiotic Origin of Chloroplasts Jean M. Whatley Department of Plant Sciences, Oxford University, South Parks Road, Oxford OX1 3RB, United Kingdom

1. Introduction The concept of chloroplasts as organelles that have evolved from endosymbiotic cyanobacteria was first proposed by Schimper (1883). The concept was based on his observations that chloroplasts and cyanobacteria had an essentially similar structure and function and that chloroplasts never arose de n o w but, like cyanobacteria, always reproduced by fission. The idea was pursued further by Mereschowsky (1905, 1920), who extended the hypothesis to include the possible origin of mitochondria from endosymbiotic bacteria. At the time, lack of suitable investigative techniques precluded the search for evidence to support or oppose these proposals and interest in the idea soon lapsed. The hypothesis was only resurrected in the 1960s when Ris and Plaut (1962) discovered that chloroplasts and mitochondria contained their own DNA. It was about this time also that the fundamental differences between prokaryotic and eukaryotic organisms became widely recognized and this, too, had an important influence on ideas about the endosymbiotic hypothesis. The increasing interest in the subject of chloroplast origin was marked by numerous publications, most notably “The Origin of Eukaryotic Cells” (Margulis, 1970) and by the organization of several symposia devoted to the subject. In 1970 Raven proposed a modification of the original hypothesis, which incorporated the idea of a polyphyletic rather than a monophyletic origin for chloroplasts. He suggested that those chloroplasts with the photosynthetic pigments chlorophyll a and phycobilins had indeed evolved from cyanobacteria, but that those chloroplasts with chlorophylls a and b and with chlorophylls a and c as the photosynthetic pigments had evolved from two other types of prokaryotic algal symbiont. The Raven modification was criticized because no prokaryotic algae that had chlorophyll b or chlorophyll c as accessory photosynthetic pigments were then known. However, in the mid-1970s a prokaryotic alga previously thought to be Inrrmorioaol Review, of C.vm1og.v. Vol. 144

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a cyanobacterium was found to contain chlorophylls a and b and to lack phycobilins (Lewin and Withers, 1975; Newcomb and Pugh, 1975). The discovery of this alga, renamed Prochloron, was greeted with considerable enthusiasm, for it seemed that a modem descendant of the ancestral green symbiont might have been found. A second major discovery of the 1970s was the finding within the cryptomonad chloroplast of the nucleomorph, a structure since identified as a much reduced nucleus (Greenwood, 1974; Gillott and Gibbs, 1980). This, together with other ultrastructural observations, led to further modifications of the original hypothesis, namely that some chloroplasts have evolved from eukaryotic rather than from prokaryotic algal symbionts (Gibbs, 1978; 1981; Whatley et al., 1979) and that the cryptomonad chloroplast may have been derived from a symbiotic red alga (Whatley and Whatley, 1981). By the early 1980s there was general agreement that chloroplasts had indeed evolved from photosynthetic endosymbionts and that these symbionts included both prokaryotes and eukaryotes. However, there was disagreement about the identity of the symbionts and the number of separate acts of endosymbiosis involved (Gibbs, 1981; Whatley and Whatley, 1981; Cavalier-Smith, 1982). As research was extended to additional species, and as more modem symbiotic partnerships were investigated, several anomalies appeared that made it necessary to reconsider some of the earlier interpretations. In addition, information derived from new research techniques, such as the use of antibodies, nucleotide sequencing, and analysis of the chloroplast genome, provided evidence in support of some of the earlier conclusions but cast doubt on others. On the one hand, there is new evidence supporting the red algal ancestry of the cryptomonad chloroplast (Douglas et al., 1991). On the other hand, green chloroplasts may have evolved from a cyanobacterial and not a prochlorophytic ancestor (Seewaldt and Stackerbrandt, 1982; Lockhart et al., 1991; Palenik and Haselkom, 1992; Urbach et al., 1992). Although the chloroplasts of both the red and the green algae now appear to have evolved from a cyanobacterium, it is still not known whether both resulted from the same or from separate endosymbiotic events or, in the case of the separate events, whether they evolved from the same or from different cyanobacterial ancestors. It is also now widely accepted that the ancestor of the chloroplast in cryptomonads and heterokont algae was eukaryotic, but again the number of separate acts of symbiosis is in doubt as well as the nature of the symbiont (Cavalier-Smith, 1986; Whatley, 1989; Gibbs, 1990). Indeed it is this aspect of the hypothesis, the possible monophyletic or polyphyletic origin of the chloroplasts of different algal phyla, that is currently receiving most attention. It is hoped that, as new techniques provide additional information about more species, we may look forward to a time when the ancestry of the chloroplasts in the various phyla becomes clearer.

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II. Cyanobacteria, Red Algae, and Cyanelles The chloroplasts that cyanobacteria most closely resemble, and whose ancestry is in consequence not in doubt, are those of the red algae. Cyanobacteria are free-living photosynthetic organisms enclosed in a characteristic cell wall and maintaining within their cytoplasm all the metabolic functions required for an autonomous existence. These functions are under the control of a prokaryotic type of genome. The chloroplasts are only semiautonomous. They lack a cell wall and have a reduced prokaryotic type of genome, which is present in multiple copies. The chloroplast stroma with its thylakoids is the homolog of the cyanobacterial cytoplasm but, although photosynthesis is maintained as the principal metabolic function, many other functions have been lost.

A. Pigments Cyanobacteria and red algal chloroplasts (Figs. 1 and 2) both have chlorophyll a as the primary photosynthetic pigment and phycobilins (allophycocyanin, phycocyanin, and phycoerythrin) as the main accessory pigments. In cyanobacteria and in chloroplasts of the simpler red algae the ratio of phycocyanin to phycoerythrin is higher than that in chloroplasts of the more complex species. The carotenoids in cyanobacteria and red algal chloroplasts are simpler than those in the chloroplasts of other phyla (Liaaen-Jensen, 1978). In red algae the carotenoids a-and p-carotene may also participate in photosynthesis.

6. The Structure of Chloroplasts The chloroplasts have no obvious cell wall and are instead enclosed within an envelope of two membranes. The inner membrane of the chloroplast envelope is believed to have evolved from the plasmamembrane of the symbiotic cyanobacterium. Two alternative proposals have been put forward for the origin of the outer membrane: ( a )from the membrane of the vacuole into which the ancestral cyanobacterium was sequestered (MorrC and Mollenhauer, 1974; Whatley et al., 1979; Whatley and Whatley, 1981) and (b)from the external membrane layer of the cyanobacterial cell wall (Douce and Joyard, 1981; Cavalier-Smith, 1982) (but see Section 111,C). The ultrastructure of the thylakoids is similar in both cyanobacteria and red algal chloroplasts (Figs. 1 and 2 ) . They are arranged singly in a spiral configuration with few interconnections between adjacent strands. In the more complex

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red algae the thylakoids are encircled by at least one peripheral lamella. Protruding from the thylakoids are phycobilisomes, the sites of concentration of the phycobilins (Hara and Chihara, 1974; Duckett and Peel, 1978; Gantt et al., 1986; Ueda and Chida, 1987). The core of the phycobilisome comprises allophycocyanin attached to the chlorophyll a-protein complex embedded in the thylakoid membrane. Diverging from this core are radially arranged stacks of discs, the lower ones being of phycocyanin and the upper of phycoerythrin. In the simpler red algae, as in cyanobacteria, the phycobilisomes are small and fan-shaped, whereas those in the chloroplasts of the more complex red algae are larger and hemispherical (Gantt, 1986).

C. Biochemical Features The chloroplast stroma contains strands of prokaryotic-type DNA and ribosomes that are of similar size (70s) to those in cyanobacteria but smaller than those found in the cytoplasm of eukaryotes (80s).The reserve carbohydrate derived from the photosynthetic activity of cyanobacteria is glycogen and this is deposited as granules within the cytoplasm. In red algae the photosynthetic reserves are a glycogen derivative, Floridean starch, but the starch grains in which this reserve is accumulated are laid down in the cell cytoplasm, not in the chloroplast stroma (Fig. 2). Also present in the cyanobacterial cytoplasm are carboxysomes, the sites of concentration of Rubisco (ribulose 1,5-bisphosphate carboxylase oxygenase). The homologous structures in the chloroplasts are pyrenoids. Studies of Rubisco are still at an early stage. Nevertheless they are proving to be of considerable interest with respect to chloroplast evolution. Rubisco is made up of large and small subunits. In cyanobacteria both subunits are obviously encoded in the cyanobacterial genome. It has been known for some time that in green algae and land plants the gene encoding the large subunit is still located in the chloroplast but that the gene encoding the small subunit resides in the cell nucleus. By contrast the red algae appear to show a greater resemblance to cyanobacteria, for the Rubisco genes for both the large and the small subunits may be located within the prokaryotic genome of the chloroplasts. This has been claimed for both GrifSlthsia pacijica and Porphyra yezoensis (Newman et al., 1989) and there is also circumstantial evidence that

FIG. 1 Anahaena arollae, a cyanobacterium that is an extracellular symbiont of the fern A d l a . Bar = 1 pm. FIG. 2 Porphyridium purpureum, a red alga. The chloroplast has an envelope of two membranes, spirally arranged single thylakoids and a central pyrenoid. Starch is deposited in the cytoplasm. Bar = I pm.

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both subunits are chloroplast-encoded in Cyanidium caldarium and Porphyridium aeruginosum (Douglas and Dumford, 1989). The many biochemical and ultrastructural similarities between cyanobacteria and the chloroplasts of red algae clearly point to their common ancestry. However, there is disagreement whether the endosymbiotic uptake of cyanobacteria (a) took place only once and gave rise, first, to the chloroplasts of red algae and later, by divergence, to those of green algae and perhaps also to those of other phyla or (b) took place on more than one occasion and hence gave rise independently to chloroplasts in the red and in other algal phyla.

D. Glaucophytes During the 1970s, when the endosymbiotic origin of chloroplasts was still a matter of dispute, there was considerable interest in several anomalous algal or protist species that contained photosynthetic organelles called cyanelles, which appeared to be intermediate in status between cyanobacteria and chloroplasts. The phylogenetic position of the species with cyanelles is uncertain. Species belonging to four genera are usually classified together, the best known being Cyanophora paradoxa. At various times the four genera, now usually placed in a single class, the Glaucocystophyta or Glaucophyta, have been classified with red algae, or with the cryptomonads or with the green algae (Kies and Kremer, 1990). The most recent affiliation has been with the green algae and euglenoids, and for the cyanelles to be regarded as true chloroplasts rather than as modified cyanobacteria. However, these assignments are still controversial and the general systematic position of the glaucophytes and their cyanelles remains uncertain. The biochemical information at present available is still too limited to solve the problem. Analyses of cytoplasmic 5 s rRNA have indicated an affinity between Cyanophora and Euglena gracilis (Wolters and Erdmann, 1986; Maxwell et al., 1987). Analyses of small and large subunit cytoplasmic rRNAs do not include Cyanophora but tend to show the euglenoids or their relatives, the kinetoplastids, as diverging early from the main line of evolution and green algae and land plants as diverging much later, positionings that contradict the idea of a close and direct relationship between euglenoids (and possibly Cyanophora) on the one hand and green algae on the other (Sogin and Gunderson, 1987; Perasso ef al., 1989, 1990; Douglas et al., 1991).

E. Cyanelles Cyanelles have some features that are characteristic of free-living cyanobacteria, e.g., remnants of a cell wall, and other features that are characteristic of

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chloroplasts, e.g., multiple copies of a reduced genome. The particular features of each category that are present in the cyanelles vary in the different “host” species, e.g., the extent to which the cyanobacterial cell wall is retained.

Pigments and Ultrastructure From the point of view of pigments and ultrastructure, cyanelles closely resemble both cyanobacteria and red chloroplasts, but a possible close relationship with green chloroplasts has also been proposed (see Section 111,E).Like cyanobacteria and red algal chloroplasts, the photosynthetic pigments of cyanelles are chlorophyll a and phycobilins and the carotenoids are simple (LiaaenJensen, 1978); the single thylakoids have a spiral configuration and the phycobilins are assembled in phycobilisomes. As in red algae, starch is accumulated in the cytoplasm (Trench, 1982; Kies and Kremer, 1990). The genome of Cyanophora has been mapped and this map has revealed that both the large and the small subunits of Rubisco are encoded within the cyanelle (Lambert el al., 1985). Further investigation of the cyanelles from different species may well provide information about some of the modifications that accompany the transformation of a cyanobacterium into a chloroplast.

111. Prochlorophytes, Green Algae, and Land Plants There is more information available about the chloroplasts of green algae and land plants than there is about those of any other phylum. Much of this information about “green” chloroplasts has tended to be viewed as the standard for all plastids. However, as more is learned about other phyla, the more distinctive the chlorophyte plastids appear to be.

A. Pigments Chlorophyll a is the primary photosynthetic pigment in green algae as it is in cyanobacteria and all chloroplasts. However, the main accessory pigment in green chloroplasts is chlorophyll b. Unexpectedly, a pigment chromatographically similar to chlorophyll c j and another related pigment, magnesium 2,4divinyl pheoporphyrin a5 monomethyl ester (Mg 2,4 D), have both been identified as minor accessory pigments in some prasinophytes, the subgroup generally regarded as being among the simplest chlorophytes (Jeffrey, 1989). Most green algae have a-and p-carotene as their main carotenoids and lutein, zeaxanthin, and neoxanthin as their main xanthophylls.

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6. The Structure of Chloroplasts In green (Figs. 3-6) as well as in red algae (Fig. 2) the chloroplast envelope comprises two membranes. In most respects the stromal matrix is also similar. However, the starch of green algae differs from Floridean starch, first in its chemistry, for it contains amylose as well as amylopectin, and second in its siting, for, uniquely, the starch is laid down inside the chloroplast, adjacent to the photosynthetic lamellae (Dodge, 1973; Pickett-Heaps, 1975). Although the thylakoids in green chloroplasts show a basically spiral arrangement, their structure differs from that in red chloroplasts in other ways. The absence of phycobilin pigments means that the thylakoids have no protruding phycobilisomes. It is thus possible for the membranes of adjacent thylakoids to become closely apposed or appressed. In some green algae, mainly prasinophytes, the thylakoids tend to be arranged in regular and extensive bands of three which have few interconnecting thylakoids (Fig. 4). In most other green chloroplasts the thylakoids are stacked irregularly, sometimes in broad bands and sometimes over shorter distances; single thylakoids frequently link adjacent bands (Fig. 5). Only in members of the Charophyceae, the group from which land plants are thought to have evolved, are the thylakoids assembled in the pattern of precisely organized and limited stacking known as granal (Fig. 6).

C. The Derivation of Envelope Membranes The traditional interpretation of the derivation of the two membranes of the chloroplast envelope is that the inner membrane evolved from the plasmamembane of the prokaryotic algal endosymbiont and that the outer membrane evolved from the membrane of the vacuole within which the symbiont was sequestered by the host cell, i.e., that it was part of the host cell’s endomembrane system (Morrt5 and Mollenhauer, 1974; Whatley et al., 1979). This meant that the two membranes were considered to be derived from two different organisms, one prokaryotic and one eukaryotic.

FIG. 3 Ulva lacruca, a green alga. Starch is deposited inside the large cup-shaped chloroplast. Some starch grains form a sheath around the central pyrenoid. Bar = I pm. FIG. 4 Part of a chloroplast of the green alga, Stigeoclonium sp.. showing the thylakoids arranged in bands of three, with no interconnecting lamellae. Bar = 0.5 pm. FIG. 5 Part of a chloroplast of the green alga Pseudendoclonium sp., showing the irregularly stacked thylakoids linked by single lamellae. Note the two membranes of the chloroplast envelope. Bar = 0.5 pm. FIG. 6 Part of a chloroplast of Chura glohrtlaris, showing the granal stacking of the thylakoids. Bar = 0.5 pm.

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A more recent hypothesis agrees with the concept of the plasma membrane as the source of the inner envelope membrane, but proposes that the outer membrane evolved from the outermost lipid layer of the symbiont’s cell wall; i. e, both membranes are considered to be of prokaryotic origin. This proposal was based on the observations (a) that in land plant chloroplasts the two envelope membranes are chemically similar to each other and to the cell membranes of the prokaryotic cyanobacteria and (b) that the outer envelope membrane is chemically very different from the endoplasmic reticulum (ER) and other components of the eukaryotic host cell’s endomembrane system (Douce and Joyard, 1981; Joyard et al., 1991). In addition, it was pointed out that despite many reports of continuity between the outer envelope membrane and the ER, there was no convincing evidence to support these claims. The electron micrographs that purported to show this continuity seldom did so clearly and the occasional examples that did show clear continuity (Cran and Dyer, 1973; Crotty and Ledbetter, 1973) were regarded as artifacts produced during the lengthy process of chemical fixation of the tissue. Using tissues subjected to ultrarapid freezing and fracturing, we have recently demonstrated frequent, clear, and unambiguous continuity between the ER and the outer membrane of the chloroplast envelope in the green alga Chara globularis and in the land plants Equisetum telmateia (McLean et al., 1988), Phaseolus vulgaris (Whatley et al., 1991), and Narcissus pseudonarcissus (unpublished). We believe the apparent absence of membrane continuity in thin sections of chemically fixed tissue may indeed be partly due to a fixation artifact, an artifact that causes some breakage of connections during the lengthy process of tissue stabilization (Figs. 7-9). However, more importantly, we believe that the apparent absence of clear membrane continuity is essentially a statistical matter resulting from the very low frequency with which random thin sections will be cut within the small segment of the plastid envelope occupied by the connecting membrane strand and precisely orientated in a plane perpendicular to both the plastid envelope and the long axis of that strand. When chemically fixed tissue has been subjected to careful serial sectioning, clear membrane continuity has been demonstrated (Brangeon and Forchioni, 1984).

FIG. 7 A thin section of a young chloroplast of the land plant Equisetum relmateia showing the associated sheath of ER. Scale bar = 1 pm. (Photograph courtesy of The New Phytologist.) FIG. 8 A young chloroplast of Equiserum telmareia from tissue subjected to ultrarapid freezing and fracturing. Note the continuity (arrows) between the outer membrane of the chloroplast envelope and the associated sheath of ER. Bar = I pm. (Photo courtesy of Dr. Barbara McLean, Oxford University, and The New Phytologist.) FIG. 9 Two species of endosymbiotic bacteria in the multinucleate amoeba Pelomyxa palustris. The vacuolar membranes are continuous with the ER; these membranes are well preserved and electron dense, whereas the outer membrane of the bacterial cell wall is broken intermittently and only weakly stained. Bar = 1 jm.

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The consistency and frequency with which we have observed clear, if intermittent, continuity between the ER and the outer membrane of the plastid envelope in rapidly frozen and fractured tissue support the hypothesis that the outer envelope membrane is indeed a part of the eukaryotic cell’s endomembrane system, and thus support the traditional view of its evolutionary origin from the membrane of the vacuole within which the symbiont was isolated from the host cell cytoplasm (McLean er al., 1988; Whatley et al., 1991). Furthermore, a clear parallel to this type of membrane continuity is provided in the giant, multinucleate amoeba, Pelomyxa palustris (Fig. 9). During most of the life cycle of the amoeba the symbiotic bacteria, each in its own vacuole, are dispersed throughout the cytoplasm but, just prior to nuclear division, the bacteria migrate and cluster around the nuclei (Whatley, 1976). At this time the vacuolar membranes become continuous with strands of ER, which, in turn, become continuous with the outer membranes of the nuclear envelopes, although the latter continuity is not shown in the section shown in Fig. 9. The pattern of intermittent continuity observed between the ER and the membranes of the bacteria-containing vacuoles in Pelomyxu closely resembles that between the ER and the outer membrane of the chloroplast envelope, which we have observed in green algae and land plants.

D. Ancestry In 1970 Raven suggested that the chloroplasts of green algae had evolved from a prokaryotic alga with chlorophylls a and b as the photosynthetic pigments. At the time no such alga was known but, during subsequent investigations of a variety of symbiotic partnerships (Figs. 10 and 1 l), just such an alga, Prochloron, was discovered independently by Lewin and Withers (1975) and by Newcomb and Pugh (1975). Several species are now known. They occur only as extracellular symbionts and are mainly associated with didemnids (Lewin and Cheng, 1989). Prochloron had previously been identified as a cyanobacterium. It was now found to have as its photosynthetic pigments both chlorophylls a and b and not chlorophyll a and phycobilins. Ultrastructural studies showed it to have the typical wall structure of a prokaryotic alga (Fig. 10). Interestingly, the thylakoids within the basic spiral were observed to be arranged mainly in pairs, but locally they formed irregular stacks of greater depth (Whatley, 1977). Since the discovery of Prochloron, two additional genera of prokaryotic algae with

FIG. 10 Prochloron didemni, a prokaryotic alga with chlorophylls a and b, an extracellular symbiont of a didemnid. Bar = 1 pm. FIG. 11 Chlorella, a green algal endosymbiont of Hydra. Bar = 1 p n . (Photograph courtesy of Dr. Chris Hawes, Oxford Polytechnic.)

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chlorophylls a and b have been found. These are the filamentous, freshwater P rochlorothrix hollandica (Burger-Wiersma et al., 1986) and the marine P rochlorococcus marinus (Chisholm et al., 1988).

E. Biochemical Features With the discovery of Prochloron, it seemed possible that a modem relative of the putative ancestral green chloroplast had now been found. Data from nucleotide sequencing soon cast doubt on this hypothesis (Seewaldt and Stackebrandt, 1982), although alternative proposals supporting the earlier hypothesis were also put forward (Van Valen, 1982). It is now generally considered that sequencing data support the view that none of the known prochlorophytes are ancestral to green chloroplasts. Instead it is believed that chlorophyll b has evolved more than once. Indeed chlorophyll b appears to have evolved on at least four occasions, leading to three distinct and unrelated prochlorophyte genera of cyanobacteria and to the chloroplasts of green algae and land plants (Palenik and Haselkom, 1992; Urbach et al., 1992; Lockhart et al., 1992). Nucleotide sequencing data have shown that green chloroplasts and the cyanelles of Cyanophora have a similar base composition. This has led to the proposal (a) that these organelles are more closely related to each other than they are to any of the cyanobacteria or prochlorophytes so far investigated and therefore (b) that they had a common origin. However, Lockhart and associates (Lockhart et al., 1991) have pointed out that evidence based on this and other molecular, biochemical, and ultrastructural data is conflicting. In their analyses of these data they show that although the majority of the best topologies link the cyanelles within the green chloroplast clade, these topologies are not significantly better than those linking the cyanelles outside the chloroplast clade. The authors conclude that the biochemical evidence in favor of grouping cyanelles with green chloroplasts is weaker than has been claimed. They further suggest that the similarity in base composition of the two organelles is due to the two distinct lineages evolving independently following the same pattern of direction of nucleotide change during their separate histories as endosymbionts rather than due to a common ancestry. It is only to be expected that the pattern of evolutionary change followed by endosymbionts would be different from that followed by free-living cyanobacteria. Further information will be needed to test this hypothesis. Meanwhile the ancestry of green chloroplasts is still unresolved.

IV. Cryptomonads and Chlorarachnion Ultrastructural studies carried out on the chloroplasts of cryptomonads were largely responsible for the introduction of the hypothesis that some chloroplasts

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evolved from eukaryotic algal endosymbionts (Figs. 11 and 12). This idea has been supported by later cytological investigations and is now widely accepted.

A. Pigments In cryptomonad chloroplasts the primary photosynthetic pigment is chlorophyll a and the accessory pigments include both phycobilins and chlorophyll c2. The proportion of phycoerythrin to phycocyanin varies in different general and in different habitats (Erata and Chihara, 1989). In general the phycobilins are slightly different chemically from those in cyanobacteria and red algae. Cryptomonads also contain small amounts of some simple carotenoids, which may also participate in photosynthesis (Liaaen-Jensen, 1978; Rowan, 1989).

6. The Structure of Chloroplasts Cryptomonads usually have one large cup-shaped chloroplast (Fig. 12). This chloroplast has several distinctive features. The envelope comprises four membranes, the outermost of which has ribosomes attached to it. The inner pair of membranes encloses a compartment containing the photosynthetic apparatus. The outer pair of membranes is usually called the chloroplast ER. Between the inner and the outer pairs of membranes lies a second compartment that is enlarged along the face of the chloroplast adjacent to the cell nucleus. In this area the outermost membrane of the chloroplast envelope is continuous with the outer membrane of the nuclear envelope (Coombs and Greenwoood, 1976; Greenwood et al., 1977; Gillott and Gibbs, 1980). The inner compartment contains thylakoids, prokaryotic-type strands of DNA, and a pyrenoid within a stromal matrix containing small (70S, i.e., prokaryotic) ribosomes. The thylakoids are mainly arranged in pairs, though deeper stacking is common toward the upper edges of the chloroplast cup. Although the cryptomonad pigments include phycobilins, the thylakoids have no protruding phycobilisomes. In thin sections of conventionally fixed tissue the thylakoid sacs appear electron dense (Fig. 12). Recent studies using immunogold labeling have confirmed that it is at the surface of this intrathylakoid space that the phycobilins (phycoerythrin-545) are located (Spear-Bernstein and Miller, 1985; Ludwig and Gibbs, 1989a; Rhiel er al., 1989). Interestingly, a few cyanobacterial mutants and red algal chloroplasts also lack phycobilisomes (Rowan, 1989). Ludwig and Gibbs (1989a) have suggested how the site of phycobilins might have been transferred from outside the thylakoids, as in cyanobacteria or in red algal chloroplasts, to inside, as in cryptomonads. They note that in red algae the genes for phycobiliproteins and the anchor polypeptide are located on the chloroplast DNA but those for the linker polypeptides are located on the nuclear DNA (Gibbs, 1990).

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The matrix of the outer chloroplast compartment contains large (80s or eukaryotic) ribosomes and it is here that starch grains are laid down. However, the most distinctive component of this compartment is the nucleomorph. This is an organelle enclosed in an envelope of two membranes that are interrupted by pores. Osmiophilic material inside the nucleomorph has been identified as eukaryotic-type DNA (Gillott and Gibbs, 1980; Morrall and Greenwood, 1982; Hansmann et al., 1985; Ludwig and Gibbs, 1985, 1989b). Thus the cryptomonad chloroplasts are distinguished from red and green algal chloroplasts in having two separate packages of DNA, one prokaryotic and one eukaryotic. This evidence provided convincing support for the earlier hypothesis (Whatley et al., 1979; Gibbs, 1981) that cryptomonad chloroplasts have evolved from a eukaryotic algal endosymbiont. Even before the presence of DNA was confirmed, it had been proposed that the nucleomorph was a modified nucleus and that the outer compartment with its nucleomorph, large ribosomes, and starch was the modified cytoplasm of a eukaryotic algal symbiont and the inner compartment was its (prokaryotic type) chloroplast. The presence of phycobilins as an accessory photosynthetic pigment and of starch deposited in the outer compartment beside the nucleomorph led to the further proposal that the eukaryotic ancestor of the cryptomonad chloroplast might have been a red alga (Whatley et al., 1979). By contrast, Cavalier-Smith ( 1982) considered a dinoflagellate (which, like cryptomonad chloroplasts has chlorophylls a and c2 but, unlike them, has no phycobilins) to be a more probable ancestor, on the grounds that there was no reason to suppose that red algae ever had chlorophyll c. However, just as chlorophyll b has evolved (and phycobilins have been lost) on at least four occasions, so may chlorophyll c have evolved more than once (as may be indicated by its presence in some green algae), perhaps even during the transformation of a red alga into a cryptomonad chloroplast.

C. The Derivation of Envelope Membranes If the cryptomonad chloroplast evolved from a red alga, then the inner pair of envelope membranes belongs to the envelope of the red chloroplast. The outermost of the four membranes with its attached ribosomes is considered to be derived from the endomembrane system of the precryptomonad host cell and its inner partner to be derived from the plasma membrane of the endosymbiotic red alga. Thus the two outermost membranes of the cryptomonad chloroplast show

FIG. 12 The cryptomonad Cryptomonas sp. The chloroplast is large and cup-shaped. Note the inner compartment (IC) containing the thylakoids and the outer compartment (OC) containing starch and a nucleomorph (N). Bar = 1 pm.

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exactly the same homologies as those in the traditional view of the two envelope membranes of red and green algal chloroplasts, but the former are associated with a eukaryotic and the latter with a prokaryotic symbiont. If the cryptomonad chloroplast evolved from a dinoflagellate (whose chloroplasts have an envelope of three membranes), then the homologies of the inner pair of the four envelope membranes are less clear.

0. Biochemical Features Douglas and associates (Douglas et al., 1991) investigated the cell nucleus and the nucleomorph in Cryptomonas @ and showed that the cryptomonad cells do indeed comprise two separate eukaryotes, each with its own distinctive transcriptional genes for the small subunits of rRNA. The tree based on this analysis shows the two types of cell to be widely separated, the nucleomorph component being in close affiliation with the nuclear genome of the red algae, Cracilariopsis sp. and Cracilaria tikvahiae, and the nuclear genome in closer affiliation with more recently emerging groups. These observations provide support for the proposal that a red alga was the ancestor of the cryptomonad chloroplast. In Cryptomonas @, as apparently also in red algae, but in contrast to green algae, both large and small subunits of Rubisco are encoded on the plastid genome. In addition, the available sequence analyses show that the cryptomonad gene for the small subunit resembles the cyanobacterial and cyanelle genes more closely than that in green algae and land plants (Douglas and Durnford, 1989).

E. Chlorarachnion The only other known species with a permanent chloroplast that has two separate compartments, each with its own package of DNA, is the monospecific amoeboid “alga” Chlorarachnion reptans (Hibberd and Norris, 1984; Ludwig and Gibbs, 1987, 1989b). However, the chloroplast in Chlorarachnion has chlorophylls a and b as its photosynthetic pigments and, in consequence, is believed to have evolved from a green algal symbiont. Essentially the chloroplasts of cryptomonads and Chlorarachnion are the eukaryotic equivalents of the (prokaryotic) cyanelles; both classes of photosynthetic organelle are living representatives of what may be intermediate or internipted stages of chloroplast evolution. The number and diversity of cryptomonad species indicate that their “chloroplasts” have remained in this state of evolution for a very considerable period of time.

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V. Heterokont Algae or Chromista Seven different algal phyla are included here among the heterokont algae or Chromista: they are the bacillariophytes or diatoms, the marine and freshwater chrysophytes, the eustigmatophytes, the phaeophytes or brown algae, the raphidophytes, the xanthophytes, and the prymnesiophytes. With the exception of the last of these phyla, all are distinguished by having heterokont flagella with tubular mastigonemes. Although prymnesiophytes are further distinguished by the presence of a haptonema, other structural features indicate that they should nevertheless be considered part of the general heterokont class. The heterokont phyla include both photosynthetic and nonphotosynthetic species and some members are phagocytic. A few of the nonphotosynthetic species contain plastids without thylakoids but others have no plastids at all. Two additional nonphotosynthetic phyla, that were formerly classified as fungi, are also generally included among the heterokont algae (Round, 1989; Williams, 1991a,b), namely the oomycetes, which are aplastidic, and the labyrinthulids, which may contain a modified plastid.

A. Pigments As in other algae, the primary photosynthetic pigment is chlorophyll a. Chlorophylls c,, c2, and c3 have been identified as accessory pigments among the bacillariophytes, chrysophytes, and prymnesiophytes, but only chlorophylls c , and c2 are seen in the phaeophytes, raphidophytes, and xanthophytes. However, there are exceptions to these generalizations. Jeffrey (1989) has suggested that those phyla with chlorophyll c3 are the most advanced. No chlorophyll c has been identified in any eustigmatophyte. Carotenoids can also act as accessory pigments. The major carotenoids are usually classified as belonging to two different biosynthetic pathways, the fucoxanthin pathway found in bacillariophytes, chrysophytes, phaeophytes, prymnesiophytes, and some raphidophytes and the vaucheriaxanthin pathway found in eustigmatophytes, xanthophytes, and the remaining raphidophytes (Bjqjmland and Liaaen-Jensen, 1989).

6 . The Structure of Chloroplasts The chloroplasts of heterokont algae have many ultrastructural features in common (Figs. 13 and 14). All have an envelope of four membranes with ribosomes attached to the outermost one. In contrast to the cryptomonad chloroplasts, the

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four envelope membranes are always closely associated and there is no conspicuous outer compartment. However, there is usually a slight enlargement of the space between the two pairs of membranes along one face of the chloroplast. This reduced outer compartment contains only a restricted membrane reticulum (Fig. 14). As in cryptomonads the outer pair of membranes is usually referred to as chloroplast ER and the inner pair is regarded as the true chloroplast envelope (Dodge, 1973; Coombs and Greenwood, 1976; Heywood, 1977; Hibberd, 1980a,b,c; Hara and Chihara, 1982; Hara el al., 1985; Clayton, 1989). It is now accepted that heterokont chloroplasts, like cryptomonad chloroplasts, evolved from eukaryotic algal symbionts. A few of the species of heterokont algae that are nonphotosynthetic have plastids that lack thylakoids. In those species that have functional chloroplasts, the thylakoids are most commonly arranged in extensive bands usually of three membranes, although stacking to a greater depth is found locally in eustigmatophyte chloroplasts (Fig. 13). In the chloroplasts of bacillariophytes, freshwater chrysophytes, phaeophytes. xanthophytes, prymnesiophytes, and those raphidophytes that follow the fucoxanthin biosynthetic pathway, the thylakoids are continuous with and encircled by an additional band of three membranes, the girdle lamella. In these chloroplasts (Fig. 14) there is a pattern of regularly positioned and precisely orientated single-membrane interconnections between adjacent pairs of thylakoid bands and between the outermost band and the girdle lamella (Coombs and Greenwood, 1976). The complexity and high degree of organization of this pattern of thylakoid linkage suggest a common origin for the chloroplasts in these particular phyla.

C. The Eyespot Almost all algal phyla have members which possess an eyespot apparatus. Those in heterokont algae and dinoflagellates in particular may provide clues to the pathways of chloroplast evolution. The eyespot apparatus comprises an assemblage of carotenoid globules, virtually always within the chloroplast, arranged in close conjunction with a modified portion of the base of one flagellum. An eyespot apparatus has been observed in some members of all the

FIG. 13 Part of a chloroplast of the eustgmatophyte Vischeria srellara. The thylakoids are arranged in unlinked bands of two or three, but occasionally they form larger stacks. There is no girdle lamella. Bar = 1 pm. FIG. 14 Part of a chloroplast of the brown alga FKCUS serratus. The thylakoids are arranged in bands of three and there are highly ordered interconnections between adjacent bands. Note the four membranes of the plastid envelope and the limited membrane reticulum in the outer compartment. Bar= 1 pm.

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heterokont phyla considered here, with the exception of the bacillariophytes, the raphidophytes, and the oomycetes. Three different types of eyespot apparatus have been described. The first, and the most common, comprises a single layer of carotenoid globules lying inside the chloroplast, immediately below the plasma membrane and adjacent to a swelling at the base of the smooth flagellum (Foster and Smyth, 1980; Hibberd, 1986; Smyth et al., 1988). This chrysophyte type of eyespot apparatus is found in some chrysophytes, xanthophytes, and phaeophytes as well as (in modified form) in the “aplastidic” labyrinthulids. In the latter, the “plastid” component contains globules but no thylakoids and the envelope comprises only two membranes (Whatley, 1989). The eyespot apparatus is a complex system involving both the plastid, derived initially from a symbiont, and the flagellum, provided by the host cell. The period of coevolution must have been lengthy. This suggests a common origin for this type of eyespot apparatus and hence for the plastids in those phyla in which it is found (Whatley, 1989). The second type of eyespot apparatus has been observed only in prymnesiophytes, and then only in some members of the Pavlovales (Green, 1980; Hibberd, 1980b). The layer of globules in this type of eyespot lies under a depression of the plastid envelope, which, in turn, lies beside an invagination of the plasma membrane. In most species the eyespot is associated with a long and complex swelling at the base of the smooth flagellum. A blue-green fluorescence has been observed in one of the two flagella of several heterokont algae with the chrysophyte type of eyespot. This characteristic fluorescence appears to be restricted to these heterokont algae, since it was not observed in E. gracilis, nor in the three chlorophytes, two cryptophytes, four dinoflagellates, and two raphidophytes included in the survey. Interestingly a similar fluorescence was, however, observed in a prymnesiophyte, although not in a species that has an eyespot today (Coleman, 1988; Kawai, 1988). Green (1980) and Hibberd (1980b) have suggested that the chloroplast component of the structurally distinctive prymnesiophyte and chrysophyte eyespot apparati may have evolved from two quite different endosymbiotic ancestors. However, because of the complexity and the presumably prolonged common history of the eyespot apparatus, the blue-green fluorescence, if associated with the smooth flagellum, could point to a common ancestry not only for the flagellar component of chrysophytes and prymnesiophytes, but also for their plastids. The third type of eyespot apparatus is found only in some eustigmatophyte zoospores. It is distinctive because the eyespot component lies outside the chloroplast and it is associated with the hairy but not the smooth flagellum (Hibberd, 1980~).The eyespot comprises a cluster of (carotenoid) globules lying in the cytoplasm at some distance from the chloroplast and not enclosed in any

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membrane envelope. Carotenoids are synthesized de n o w only in chloroplasts where, as far as is known, they are permanently retained inside the plastid envelope. The source of the eyespot globules in eustigmatophytes is therefore obscure. It is possible, although unlikely, that the globular cluster of the eyespot represents a much reduced relic of an earlier plastid population. A separate evolutionary origin for the eustigmatophyte chloroplasts has been proposed (Hibberd, 1979).

D. Biochemical Features Mapping of the chloroplast DNA has been carried out on several species of heterokont algae, but information is still limited. The plastid DNA of the raphidophyte Olisrhodiscus luteus is similar to that of land plants, both in size ( 1 50 kb) and in the presence of a large inverted repeat. In the other heterokont species so far studied, both the size of the DNA and the length of the repeat are generally smaller and there appears to have been considerable rearrangement of the siting of some genes. The chloroplast DNA also appears to be heterogeneous and complex in some taxa. In the brown alga Pylaiella littoralis, for example, the chloroplast DNA has been shown to comprise two molecules, one large (133 kb) and one small (58 kb). It is still too early to know whether these differences between heterokont phyla in the composition of their chloroplast DNA reflect a rapid rate of nucleotide substitution or a separate chloroplast ancestry, although the former scenario is the more probable (Cattolico and Loiseau-de Goer, 1989; Kowallik, 1989). As yet there have been few analyses of the genes encoding for Rubisco. Those for both the small and the large subunit have been found in the chloroplast DNA of the raphidophyte 0. lureus (Cattolico and Loiseau-de-Goer, 1989), but the site of the gene for the small subunit has not been determined in the other heterokont algae that have been investigated (Kuhsel and Kowallik, 1987; Cattolico and Loiseau-de-Goer, 1989). The Rubisco from Olisfhodiscus is similar to that of the red alga, G . pacifica, but very different from that of green algae and land plants (Newman ef al., 1989). The authors point out that these observations may reflect either a common origin for the red and raphidophyte chloroplasts or a similar pattern of evolution resulting from the encoding of both subunits inside the chloroplasts rather than the nuclear coding of the small subunit, as occurs in the chloroplasts of green algae and land plants. A partial amino acid sequence from the brown alga Fucus serratus shows extensive similarity to the sequence from the cryptomonad Cryptomonas @, another alga in which the encoding of the small subunit takes place in the chloroplast (Douglas and Durnford, 1989) and for which a red algal ancestry has been proposed.

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E. Chloroplast Relationships The presence in bacillariophytes, chrysophytes, phaeophytes, prymnesiophytes, and some raphidophytes of chloroplasts with both a girdle lamella and the fucoxanthin pathway of carotenoid biosynthesis have provided the basis for the suggestion that these chloroplasts had a separate origin from those in eustigmatophytes, xanthophytes, and the remaining raphidophytes, which have no girdle lamella and follow the vaucheriaxanthin biosynthetic pathway. This proposal would require a separate origin for the chloroplasts within the two branches of the raphidophytes (Hibberd, 1986). However, other criteria can suggest different phyletic groupings. The same type of eyespot apparatus, for example, is found in chrysophytes, which follow the fucoxanthin pathway of carotenoid biosynthesis, and in xanthophytes, which follow the vaucheriaxanthin pathway. In an evolutionary tree of cytochrome c6, for example, xanthophyte and phaeophyte species are grouped with a red alga, but are conspicuously separated from a prymnesiophyte (Hunt et al., 1985). An investigation of the fucoxanthin+hlorophyll a/c-protein complexes in the bacillariophyte Phaeodactylum tricornutum and the prymnesiophyte Pavlova gyrans showed them to be almost identical (Fawley et al., 1987). The authors suggest that this points to a common evolutionary origin for the chloroplasts in these two phyla. These, and other discrepancies between the various phyletic groupings, may well reflect the instability of the genome in heterokont algae. Clearly, with the restricted information available at the present time, any rigorous assessment of the ancestry of heterokont chloroplasts is impossible.

VI. Euglenoids Only about one-third of euglenoids contain chloroplasts. Many of the colorless species are phagotrophic. As with the heterokont algae, there has been uncertainty whether the group is primarily photosynthetic, the chloroplasts having been lost from most species, or (the more widely accepted view) whether it is primarily nonphotosynthetic, the chloroplasts having been a more recent acquisition (Walne and Kivic, 1990).

A. Pigments The main photosynthetic pigments of euglenoid chloroplasts are chlorophylls a and b, the same as in the chloroplasts of green algae. The carotenoids a-and pcarotene are present in small amounts. Interestingly the main xanthophylls,

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diadinoxanthin, diatoxanthin, and heteroxanthin, resemble those in the chloroplasts of some heterokont algae (Rowan, 1989).

B. The Structure of Chloroplasts and Membrane Homologies The thylakoids in euglenoid chloroplasts are commonly arranged in extended bands of three, a pattern which resembles that in the chloroplasts of some of the simpler green, as well as in heterokont algae (Dodge, 1973; Coombs and Greenwood, 1976). The chloroplast envelope comprises three membranes. Two hypotheses have been put forward to account for their derivation. The first proposal envisages the euglenoid chloroplasts as modified eukaryotic symbionts, possibly green algae, following a comparatively recent act of symbiosis. This hypothesis suggests that the inner pair of membranes corresponds to the two envelope membranes of the green chloroplasts, but there are two alternative interpretations of the source of the outermost membrane. Gibbs (1978) proposed that the euglenoid chloroplast had evolved from a green alga, that the outermost of the three membranes was derived from its plasma membrane, possibly fused laterally to the membrane of the phagocytic vacuole, and that the cytoplasm of this green alga was subsequently lost. Whatley (Whatley et al., 1979) suggested that the plastids evolved from chloroplasts isolated from a green alga, and taken up and retained as symbionts in the cytoplasm of the euglenoid host. This proposal envisages the outermost membrane of the chloroplast as homologous with the vacuolar endomembrane provided by the euglenoid host cell and an early rather than a late loss of the “green” cytoplasm. The late loss of cytoplasm as suggested by Gibbs is a more acceptable hypothesis, as it is most unlikely that chloroplasts immediately isolated from their own cytoplasmic environment would be capable of permanent semiautonomy. However, the concept of the host endomembrane system as the source of the outermost envelope membrane receives some support from the recent observation by Ehara and associates (Ehara et al., 1984, 1985; Osafune et al., 1985) that the outermost envelope of euglenoid chloroplasts can become continuous with other parts of the endomembrane system of the euglenoid cell. The second hypothesis suggests a prokaryotic origin for the chloroplasts. The innermost of the three envelope membranes is again considered to be the plasma membrane of a prokaryotic symbiont and the outermost to be a vacuolar membrane provided by the host cell. However, the central membrane is seen as being derived from the outer membrane layer of the symbiont’s Gram-negative cell wall (Cavalier-Smith, 1982). Cavalier-Smith has also suggested that the chloroplasts of euglenoids and green algae, red algae, and dinoflagellates have all evolved from the same cyanobacterium, following a single and ancient act of endosymbiosis (Cavalier-Smith, 1982, 1986).

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C. The Eyespot In many euglenoids, both photosynthetic and nonphotosynthetic, there is an eyespot apparatus. This comprises an eyespot component that, unusually, is extraplastidic. It is situated in the cytoplasm adjacent and perhaps connected to nearby microtubules and close to the swollen base of one flagellum. The swollen base or paraflagellar body is believed to be the photoreceptor for phototaxis and the associated pigment a flavoprotein. The eyespot comprises 10-15 carotenoid globules that appear to be enclosed within a single membrane (Walne, 1971; Kivic and Vesk, 1974; Kuinicki et al., 1990). It has been suggested that the membrane-bound eyespot containing carotenoid globules is the reduced remnant of a plastid belonging to an earlier chloroplast population (Taylor, 1979).

D. Biochemical Features The various proposals that the chloroplasts of euglenoids and of green algae had some form of common ancestry were based on the fact that both had chlorophylls a and b as their photosynthetic pigments. However, a difficulty has long been recognized with this proposal, since the carotenoids in euglenoids more closely resemble those of heterokont algae (Coombs and Greenwood, 1976). Nor is this ambiguity clarified by the chloroplast ultrastructure, since a somewhat similar arrangement of the thylakoids in bands of three with few interconnections can be found in some of the simpler green as well as in some heterokont algae. The information derived from nucleotide sequencing data is still limited and at present it, too, provides an ambiguous picture. Several analyses of data for both the large and the small subunit rRNAs indicate that Euglena is closely associated with the kinetoplastids Trypanosoma and Crithidia (Sogin and Gunderson, 1987; Perasso et al., 1989, 1990; Douglas et al., 1991), a finding supported not only by some other sequencing analyses (Wolters and Erdmann, 1986; Hori and Osawa, 1987) but also by cytological observations (Willey and Wibel, 1985). In a tree based on eukaryotic small subunit rRNAs, the euglenoids and kinetoplastids emerge near the base (Sogin and Gunderson, 1987). A similar position for kinetoplastids is given by Perasso and associates (Perasso et al., 1990) in all three of their published trees based on analysis of the eukaryotic large subunit rRNAs. By contrast, green algae and land plants, red and heterokont algae, and cryptomonads are consistently shown as emerging much nearer the tops of these trees and hence far removed from the kinetoplastids. However, sequencing data of the chloroplasts of these algae on the one hand, and of Euglena on the other, reveal a very different pattern.

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Delihas and Fox (1987) reported that homologies for chloroplast and cyanelle

5s RNA between Euglena and other species were all similarly low-the angiosperm Spinacia oleracea 53%, the heterokont alga Ochromonas danica 58%, and C . paradoxa 62%. More recently in their analysis based on SSU chloroplast rRNAs, Douglas and Turner (199 1) found the chloroplasts of green algae and land plants on the one hand, and those of Euglena, cryptomonads, red and heterokont algae, and cyanelles of C . paradoxa on the other, to form two separate lineages, both derived ultimately from cyanobactena. On the basis of their published trees, Douglas and associates (Douglas and Turner, 1991; Douglas et al., 1991) consider that the great distance between Euglena on the one hand, and green algae and land plants on the other, taken together with the shallow placement and the two separate lineages of their chloroplasts do indeed suggest an endosymbiotic origin for the euglenoid chloroplast that is recent and separate from that of the green chloroplast. A similar view has been expressed by Perasso et al. (1989, 1990), although the latter acknowledges that a single cyanobactenal ancestor for all chloroplasts followed by the separate secondary loss of chloroplasts from several groups cannot at present be completely ruled out. In this context it should be remembered that Wolters and Erdmann (1986) and Hori and Osawa (1987) have proposed a more recent origin for the euglenoids and kinetoplastids than for the green algae and land plants.

VII. Dinoflagellates Only about half of the dinoflagellates are photosynthetic. The chloroplasts in almost all of the photosynthetic species are similar in pigmentation and structure. However, there are a few species with chloroplasts that differ from the predominant dinoflagellate type in the number of their envelope membranes, their pigments, and the arrangement of their thylakoids. The dinoflagellates with these anomalous chloroplasts fall into two categories: ( a ) those whose chloroplasts belong to temporary or permanent alien algal endosymbionts; (h) those whose chloroplasts appear to be indigenous despite their anomalous pigmentation and ultrastructure.

A. Pigments of Most Dinoflagellate Chloroplasts Almost all dinoflagellate chloroplasts have chlorophyll a as their primary photosynthetic pigment and chlorophyll c2 as an accessory pigment. In this respect they resemble cryptomonad chloroplasts but they differ from them in having no phycobilins. However, chlorophyll c, has been identified in Prorocentrum

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(Exuviellu) cassubicum, a species in which the chloroplasts are not atypical in other features (Jeffrey, 1989). The main and uniquely characteristic carotenoid of dinoflagellates is peridinin, which also acts as an accessory photosynthetic pigment. Diadinoxanthin and p-carotene are also abundant (Rowan, 1989; Bjqmland and Liaaen-Jensen, 1989).

6 . The Structure of Most Dinoflagellate Chloroplasts In the vast majority of photosynthetic dinoflagellates, the plastid envelope comprises three membranes, although these are not always easy to distinguish. For the most part, the thylakoids are arranged in broad, usually unconnected, bands of three, aligned parallel to the plastid long axis (Fig. IS). In a few species the thylakoids are partially encircled by a peripheral lamella. There is considerable variation in the structure of the pyrenoid, which is internal in some species and stalked in others. Starch is accumulated in the cell cytoplasm and, in species with stalked pyrenoids, it often forms a capping sheath (Dodge, 1973, 1975, 1987).

C. The Chloroplasts of Alien Algal Endosymbionts In some dinoflagellates photosynthesis is carried out by algal symbionts and not by indigenous chloroplasts. These symbionts are intracellular in some species and extracellular in others; the symbionts can be prokaryotic or eukaryotic, structurally intact, or greatly reduced. The symbiotic association can be temporary or permanent, although this is sometimes difficult to establish. The range of variation is astonishing. 1. Permanent Endosymbionts Cyanobacteria with their characteristic pigmentation and structure have been observed as symbionts of several dinophysoid species. These symbionts most often lie outside the cytoplasm, but endosymbiotic cyanobacteria have been identified in Amphisolenia globifera (Lucas, 1991). The most common algae associated with dinoflagellates are eukaryotic. Few species have been investigated in the electron microscope. However, of the symbiotic partnerships that have been investigated, it is clear that some are

FIG. 15 A dinoflagellate. Glenodinium sp.. showing the peripheral chloroplasts. The arrangement of the thylakoids in unconnected bands of three is shown in greater detail in the insert. Bar = 1 pm.

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permanent and others are temporary, and that the two associated species exhibit varying states of interdependence. The best known dinoflagellates with permanent algal endosymbionts are Peridinium balticum and Glenodinium (P.)foliaceum (Tomas and COX,1973; Withers and Haxo, 1975). These dinoflagellates have fucoxanthin and not peridinin as the main carotenoid and the accessory photosynthetic pigments include chlorophyll c, as well as chlorophyll c2. The atypical chloroplasts that synthesize these pigments belong to an easily identified heterokont algal endosymbiont. This endosymbiont is separated from the dinoflagellate cytoplasm by only a single membrane. Its chloroplasts have an envelope of four membranes, lenticular pyrenoids which are simple and internal, and thylakoids arranged in parallel bands of three and enclosed within a girdle lamella, features that have led to the proposal that the endosymbiont is either a chrysophyte or a bacillariophyte. A green alga has been found as a permanent endosymbiont of the dinoflagellate Lepidodinium viride (Watanabe et al., 1987, 1990). Here the symbiont is separated from the dinoflagellate cytoplasm by two membranes and two membranes also form the chloroplast envelope. The thylakoids are arranged in parallel bands of three and the simple, internal lenticular pyrenoid has a paracrystalline matrix. Chlorophylls a and b have both been identified. A preliminary analysis of carotenoids found p-carotene but neither peridinin nor fucoxanthin. The algal endosymbiont is probably a prasinophyte. In contrast to the symbionts in P. balticum and G . foliaceum, the prasinophyte symbiont has no obvious nucleus or mitochondria. Two types of vesicles have been observed in the symbiont cytoplasm. Both have an envelope of two membranes, but in one type the membranes are interrupted by pores. Watanabe and associates have suggested that these vesicles might be much reduced mitochondria and nuclei. However, DAPI staining has so far failed to reveal the presence of any DNA. Thus, in contrast to cryptomonad chloroplasts but as with heterokont chloroplasts, the Lepidodinium “chloroplasts” appear to function and proliferate successfully in the absence of any obvious source of the eukaryotic symbiont’s DNA. 2. Temporary Endosymbionts Several dinoflagellates contain “chloroplasts” that, on the basis of their ultrastructure and their pigments, are clearly modified cryptomonads. Species containing these apparently temporary endosymbionts include Gymnodinium acidotum (Wilcox and Wedemayer, 1984), G . aeruginosum (Schnepf et al., 1989), G . eucyaneum (Hu et al., 1980), and Amphidinium poecilochrum (Larsen, 1988). The “chloroplasts” of A. wigrense (Wilcox and Wedemayer, 1985) probably also belong in this category although the cryptomonad cytoplasm is much reduced and the chloroplast stroma is separated from the dinoflagellate cytoplasm by three rather than by five membranes (Schnepf and

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Elbrachter, 1992).The chloroplasts in all these dinoflagellates have paired thylakoids with wide intramembrane sacs that are osmiophilic. The numbers of the “chloroplasts” and, indeed, their degree of structural integrity appear to vary from individual to individual. Schnepf and associates (Schnepf, 1992;Schnepf and Elbrachter, 1992) have concluded that in the species with these cryptomonad symbionts or cleptochloroplasts, the partnership is temporary, although in some it may persist for several months. Schnepf has also suggested that the cleptochloroplasts that show the greatest structural integrity will be those that have only recently been taken up by the dinoflagellate host, and that integrity is then progressively lost until, eventually, the cleptochloroplasts are digested.

D. “Indigenous” Chloroplasts with Anomalous Pigments 1. Chloroplasts with Fucoxanthin Fucoxanthin has been identified as the main carotenoid not only in species such as P. balticum and G. foliaceum with their heterokont algal symbionts, but also in six species of Gymnodinium and one of Gyrodinium, in which the chloroplasts appear to be indigenous. The fucoxanthin-related xanthophylls identified in these species include 19’-hexanoyloxy-fucoxanthin,a pigment at present known elsewhere only in Emiliana huxleyi, a prymnesiophyte (Bjqjmland and Liaanen-Jensen, 1989;Rowan, 1989). The chlorophylls of these atypical chloroplasts have not been analyzed, but it would be interesting to know whether they include chlorophyll c l , which is characteristic of heterokont algae but not of dinoflagellates. A recent study of antibodies prepared against the light-harvesting peridinin-chlorophyll a-protein complexes showed cross-reactivity between the 28 species of dinoflagellates from eight different genera that were tested, but no cross-reactivity with the fucoxanthin-chlorophyll a-protein complex of a chrysophyte (Govind er al., 1990). Among the dinoflagellate species tested was Gymnodinium nelsoni. It would be very interesting to know whether the dinoflagellate pigment-protein complexes cross-react with those in any of the six Gymnodinium species with fucoxanthin as an accessory pigment but with chloroplasts that appear to be indigenous. There is some uncertainty about the number of envelope membranes in these anomalous chloroplasts. In Gymnodinium breve there appear to be three membranes as in most dinoflagellate chloroplasts, but in the other species there may only be two. In Gyrodinium aureoliim the chloroplasts may lie within some of the many cytoplasmic vacuoles (Kite and Dodge, 1985, 1988;Dodge, 1989). If this is correct, then they may be reduced endosymbionts or cleptochloroplasts rather than true chloroplasts. In the chloroplasts that synthesize fucoxanthin, the thylakoids are characteristically arranged in parallel bands of three, but they vary somewhat in their

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degree of branching and of partial enclosure by a peripheral lamella. In a few species the ends of some thylakoids may be fused to the plastid envelope (Dodge, 1987; Kite and Dodge, 1988). The pyrenoids also vary in structure, but are generally different from those in most dinoflagellate chloroplasts. The simple, internal pyrenoids of Gymnodinium micrum and of two other species in this group closely resemble those in some of the heterokont algae that follow the fucoxanthin pathway of carotenoid biosynthesis, e.g., chrysophytes, bacillariophytes, and prymnesiophytes (Dodge, 1987;Kite and Dodge, 1988). However, only the prymnesiophytes have chloroplasts with the combined characteristics of both single stalked and simple internal pyrenoids, thylakoids that can be branched and that sometimes appear fused to the plastid envelope, no true girdle lamella, and DNA arranged in scattered nucleoids, as well as one species in which the unusual xanthophyll 19’-hexanoyloxy-fucoxanthinhas been identified. 2. Chloroplasts with Phycobilins? The most unusual indigenous chloroplasts are found in the dinophysoids, a group that includes many phagocytic species. These chloroplasts, some of which are very small, have a distinctive ultrastructure. The envelope clearly comprises only two membranes. The thylakoids are often of limited extent and are mainly arranged in pairs rather than in bands of three. More distinctively, the thylakoid sacs contain osmiophilic material and so resemble the phycobilin-containing thylakoids of cryptomonad chloroplasts. The pigments of these dinophysoids have not yet been analyzed in detail, but they do frequently show the orange autofluorescence characteristic of phycobilin pigments (Hallegraeff and Jeffrey, 1984; Hallegraeff and Lucas, 1988; Schnepf, 1992)and phycoerythrin has been identified in Dinophysis norvegica (Geider and Gunter, 1989).Interestingly, in samples from mixed plankton blooms dominated by two dinophysoids, Dinophysis acuminata and D.fortii, the main carotenoid was found to be the characteristic dinoflagellate pigment peridinin (Hallegraeff and Lucas, 1988).

E. The Eyespot Several different types of eyespot have been observed in dinoflagellates. These are of some interest with respect to plastid evolution. When it is present, the eyespot in most dinoflagellates takes the form of a single layer of globules lying inside one of the chloroplasts immediately under the envelope membranes adjacent to the sulcus and next to the longitudinal flagellum. In Woloszynskia coronata the eyespot occupies a similar site but it is extraplastidic and comprises a cluster of globules with no delimiting membrane (Dodge, 1973). In P. balticum and G . foliaceum photosynthesis is camed out in the chloroplasts of the heterokont algal symbionts. There are no chloroplasts in the dino-

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flagellate cytoplasm, but there is an organelle that occupies the site and has the appearance of an eyespot. It has an envelope of three membranes and a granular matrix containing many carotenoid globules but no thylakoids. The pigments include p-carotene and several carotenoid precursors, but not peridinin or fucoxanthin (Withers and Haxo, 1978). This eyespot may be all that remains of an earlier population of indigenous dinoflagellate chloroplasts whose photosynthetic activity has been replaced by the more recently acquired alien algal symbiont (Tomas and Cox, 1973; Whatley, 1989). The “eyespot” of some members of the mainly phagotrophic Warnowiaceae is of particular interest. It is a large and complex structure called an ocelloid and it may have a function in phototaxis (Foster and Smyth, 1980; Smyth et al., 1988) or as a rangefinder (Taylor, 1990). The ocelloid comprises an assemblage of modified organelles, which includes not only a plastid (the melanosome) but also an endomembrane vesicle, a mitochondrion, endoplasmic reticulum, fibrils, and microtubules. The plastid component has an envelope of two membranes and contains a retinal body and a ring of pigment globules. Its chloroplast derivation can only be deduced during the early stages of cell division at which time the thylakoids can be identified (Greuet, 1987). In these species the former photosynthetic activity of the now reduced plastid has not been replaced by that of a new algal endosymbiont.

F. Biochemical Features Dinoflagellates are now considered to be closely affiliated with ciliates, for whose cells there is some sequencing information available; to the best of my knowledge, the only similar analysis of a dinoflagellate has been for Prorocentrum micans (Sogin and Gunderson, 1987; Perasso et al., 1989, 1990, Douglas et al., 1991). There appear to be no published sequencing analyses for the chloroplasts. However, it has been reported that in several dinoflagellates, as apparently in all the other algae so far investigated except the greens, both the small and the large subunit of Rubisco are encoded inside the chloroplasts (J. Chesnick and R. A. Cattolico, unpublished data, but reported in Newman et al., 1989). This scarcity of information precludes its use in helping to determine the origin(s) of dinoflagellate chloroplasts.

G. Ancestry Because of the extraordinary range in the structure and pigmentation of the dinoflagellate chloroplasts, their source (or sources) presents a particularly intriguing problem. First, it remains uncertain whether the dinoflagellates are a basically photosynthetic or nonphotosynthetic class. Second, it is a matter of

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dispute whether the ancestral symbiont (or symbionts) was prokaryotic or eukaryotic-or perhaps even both! Third, the taxa to which the symbiont(s) belonged is particularly uncertain. A major problem is the source of the chloroplast envelope membranes. As with euglenoids it has been suggested that the ancestral symbiont was a eukaryote with the inner pair of membranes homologous with those of its plastid envelope and the third membrane homologous with either the plasma membrane of the eukaryotic symbiont (Gibbs, 1978) or the vacuolar membrane of its new host (Whatley et al., 1979). Alternatively it has been proposed that the two inner membranes were derived from the plasma membrane and the outer wall membrane of a prokaryotic symbiont and the third membrane from the host’s vacuolar membrane. This prokaryotic symbiont is envisaged either as a cyanobacterium with chlorophyll c2 as well as chlorophyll a and phycobilins (Cavalier-Smith, 1982) or as the original cyanobacterial symbiont that gave rise to all prokaryotic-type chloroplasts (Cavalier-Smith, 1986). Yet another possibility is that the chloroplasts evolved from symbionts that were ingested by myzocytosis rather than by phagocytosis (Schnepf and Elbrachter, 1988). About half of the dinoflagellates are phagotrophic. Schnepf and Elbrachter (1 992) have reviewed the many nutritional strategies of dinoflagellates and have discussed the relationship of these to the possible course of evolution of the plastids. In addition to the more conventional uptake of organisms into phagocytic vacuoles, the method generally viewed as the basic means of acquiring endosymbionts, they describe myzocytosis, the uptake of organisms by means of a variety of specialized feeding tubes. The tips of these tubes penetrate but do not rupture the plasma membrane of the alien organism before sucking out the chloroplasts and other cytoplasmic contents into food vacuoles. With phagocytosis two membranes are left between the ingested organism and the dinoflagellate cytoplasm. With myzocytosis the plasma membrane of the alien is lost and only the single membrane of the host vacuole separates the two organisms. Schnepf and associates have suggested that myzocytosis is the means by which the impermanent (cryptomonad) cleptochloroplasts are acquired (Schnepf, 1992; Schnepf and Elbrachter, 1992). Interestingly the permanent (heterokont) symbionts of P.balticum and G .foliaceum are separated from their hosts by only a single membrane, although it has been suggested, on the basis of particle distributions observed in freeze-fracture studies, that the intervening membrane is derived from the alien plasma membrane rather than the expected vacuolar membrane of the host (Schnepf and Elbrachter, 1992). Although they appear to be temporary components of the dinoflagellate cytoplasm, cleptochloroplasts can survive for several months and can be transmitted to daughter cells. It remains to be seen whether they or their hosts have ever succeeded in transferring or establishing the genetic capacity necessary for their establishment as chloroplasts on a self-perpetuating and permanent basis. Although Schnepf and colleague formerly suggested that the dinophysoid chloro-

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plasts might have evolved in this way from cryptomonad symbionts, they now consider a cyanobacterial origin more probable (Schnepf and Elbrachter, 1988, 1992; Schnepf, 1992). Despite the considerable amount of information that has been accumulated over the last 20 years, the important question whether dinoflagellates are basically a photosynthetic or a nonphotosynthetic group still cannot be answered and the ancestry of the dinoflagellate chloroplasts remains a mystery. Most analyses based on nucleotide sequencing show the ciliates (and dinoflagellates) as emerging after the euglenoids and closer to, but usually before, the welldefined clusters of the heterokont and other algae. Unfortunately there are still no sequencing data for dinoflagellate chloroplasts. Modem dinoflagellates are found in symbiotic association with many different algal species, both prokaryotic and eukaryotic. Until more information becomes available it is impossible to determine whether ancient dinoflagellates were similarly “indiscriminate” and whether, in consequence, they acquired chloroplasts from more than one source. As with the euglenoids, it is still impossible to tell whether the chloroplast ancestry was prokaryotic or eukaryotic-or both!

VIII. Conclusions The question of chloroplast origin(s) cannot be resolved at this time. There are essentially two main opposing points of view. The first hypothesis suggests (a) a cyanobacterial origin for the chloroplasts of red and green algae, following either one or two separate acts of endosymbiosis; (b)separate eukaryotic, possibly red algal, origins for the chloroplasts of cryptomonads and heterokont algae; and (c) additional separate origins for the chloroplasts of euglenoids, possibly from a green alga, and for those of dinoflagellates, from one or from several different sources (Gibbs, 1981; Whatley ef al., 1979; Whatley and Whatley, 1981; Douglas and Tumer, 1991). The second hypothesis suggests ( a )a single common cyanobacterial origin for the chloroplasts of red and green algae, dinoflagellates, and euglenoids and (b)a secondary common origin for the chloroplasts of cryptomonads and heterokont algae from a eukaryotic dinoflagellate in which the prokaryotic type of plastid was already established (Cavalier-Smith, 1986). The crux of the problem lies with the order in which the different modem algal phyla diverged from the basic line of cell evolution and which of the emerging phyla were initially photosynthetic or nonphotosynthetic. The euglenoids provide a useful illustration of the problem. One of the early analyses based on nucleotide sequencing data was that by Hori and Osawa (1987) who made use of the now less acceptable 5s rRNAs. They suggest a recent origin for the euglenoids and dinoflagellates and place red

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algae as much the earliest of the algal phyla, a positioning of the red algae which conformed with the earlier concept that nonflagellate classes preceded flagellates (Taylor, 1978). In their analysis of 16s rRNA Wolters and Erdmann (1986) also suggest a more recent origin for the euglenoids, but this contrasts with their tree based on cytological and biochemical data, which indicates an early origin. However, other analyses based on large and small subunit rRNAs all place the euglenoids as emerging early and the other phyla as emerging significantly later and fairly close together (Sogin and Gunderson, 1987; Perasso et al., 1989, 1990; Douglas et al., 1991). There is also some morphological evidence, fairly widely accepted, that the euglenoids and their close relatives, the kinetoplastids, emerged as a nonphotosynthetic group (Walne and Kivic, 1990; but see Cavalier-Smith, 1986). If, then, the euglenoids were primarily nonphotosynthetic and if they emerged early, as the more recent analyses suggest, then the euglenoid chloroplasts evolved following an act of symbiosis that took place after the euglenoid divergence from the main line of cell evolution. In their analyses of small subunit rRNAs, Douglas and Turner (1991) point out that the deep placement (early origin) of the euglenoid and kinetoplastid cells compared with the shallow placement (late origin) of the Euglena chloroplasts suggests a late and separate origin for the euglenoid plastid. The alternative proposal suggests (a) that the source of all chloroplasts except those in cryptomonads and heterokont algae was a cyanobacteriurn taken up in a single act of endosymbiosis; (b) that this took place long before the euglenoids emerged as a distinct group; ( c ) that the euglenoid chloroplast evolved by divergence from the ancestral cyanobacterium; and (d)that chloroplasts were then lost from many euglenoids and from their kinetoplastid relatives, as well as from many other phyletic groups (Cavalier-Smith, 1986). This view of the origin of euglenoid plastids is not in agreement with either the concept of the euglenoids as a basically aplastidic group or the chloroplast sequencing data. Similar opposing views have been expressed about dinoflagellates, but the impressive diversity of their chloroplasts and the general lack of biochemical information at present preclude a realistic analysis of the origin(s) of their chloroplasts. The view that dinoflagellates, like euglenoids, are primarily aplastidic (Loeblich, 1976; Bujak and Williams, 1981; Dodge, 1987; Whatley, 1989), together with the range of variation in chloroplast structure and pigmentation, provides the basis for the hypothesis that dinoflagellate chloroplasts had a polyphyletic origin. The apparent ease with which modem dinoflagellates take up as symbionts both the prokaryotic cyanobacteria and a variety of different eukaryotic algae provides an interesting parallel to this proposal, although of course it has no direct bearing on what took place in the past. If dinoflagellate chloroplasts had a polyphyletic origin, then the proposed ancestor of the majority could have been either a cyanobacterium or a red alga. For those dinoflagellates with indigenous chloroplasts that synthesize fucoxanthin, a heterokont (prymnesiophyte ?) ancestry must be considered. The dino-

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physoid chloroplasts with their putative phycobilins could have evolved either from a cyanobacterium or from a cryptomonad. There is, at present, no way of telling which, if any, of these conjectures is correct. However, a better understanding of the ancestry of dinoflagellate chloroplasts could well provide important clues to the problem of chloroplast origins in general. The cryptomonads and heterokont algae are also commonly seen as primarily aplastidic, although Cavalier-Smith ( 1982, 1986) again considers this condition to be the result of the loss of an earlier chloroplast population. He also suggests that the cryptomonad chloroplasts evolved from endosymbiotic dinoflagellates and that the heterokont algae evolved by divergence from cryptomonads. The opposing view is that the chloroplasts of both cryptomonads and heterokont algae evolved from red algae (Gibbs, 1981, 1990, Whatley and Whatley, 1981; Whatley, 1989). The latter concept of a red algal origin for cryptomonad chloroplasts is supported by the sequencing analyses of Douglas and associates, which show close affinity between the cryptomonad nucleomorph and the nuclei of red algae. These authors consider that their analyses also indicate an origin for heterokont chloroplasts from one or more red algae (Douglas and Turner, 1991; Douglas et af.,1991). A polyphyletic origin for the chloroplasts of heterokont algae has been proposed by several authors. Indeed, at one time or another, nine separate acts of endosymbiosis have been proposed (Whatley, 1989). However, as suggested in Section V,E, the overlapping groupings of phyla that have particular chloroplast features in common make a monophyletic origin of all heterokont chloroplasts more probable. The red and green algae and land plants are the only phyla in which aplastidic species are unknown. They are also the only phyla about whose chloroplast ancestor (a cyanobacterium) there is general agreement. Two separate chloroplast lineages have recently been identified (Douglas et al., 1991). The first lineage is restricted to the chloroplasts of green algae and land plants; the second lineage includes the chloroplasts of all other algae. However, this pattern of separation leaves unresolved the question whether the two chloroplast lineages evolved following a single act of endosymbiosis with subsequent divergence, or following two separate acts. The original hypothesis of Schimper (1 883) envisaged a cyanobacterial ancestry for all chloroplasts and this view is now generally accepted. As a result of ultrastructural and cytological evidence not available to Schimper, the hypothesis has now been expanded to incorporate the concept of a secondary origin for some chloroplasts from a eukaryotic algal symbiont in which a primary (cyanobacterial) chloroplast was already established. It is to be anticipated that the expanding and varied molecular information that is now being accumulated will help to resolve the problems that remain, the numbers and identities of the different endosymbionts involved and the precise means by which the cell nucleus established overall control of the variously evolving symbiotic partnerships.

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Acknowledgments I thank Dr. Barbara McLean, Dr. Chris Hawes, and The New Phyrologisr for permission to use their micrographs; Professor R. A. Lewin, Scripps Institute of Oceanography, San Diego, and Professor J. D. Dodge, Royal Holloway and Bedford New College, University of London, for providing resin blocks of algal tissues; and Professor K. W. Jeon, University of Tennessee, Professor E. Schnepf, University of Heidelberg, and Dr. C. J. Howe, University of Cambridge, for providing preprints of their publications.

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Index

A Abnormally functioning spindle, mitosis and, 230,232 Acunthumoebu. cytoskeletal dynamics and, 97-98, 109 Acetylcholine. avian salt glands and, 161, 165-167.203-204 Acetylcholinesterase. avian salt glands and, 159 Acheta, karyosphere and, 18, 2 I Acid phosphatase, avian salt glands and, 144. 147 Acrosomal processes, cytoskeletal dynamics and. 97 Actin cytoskeletal dynamics and current concepts, 96-98, 100, 102-103 cytoskeletal components, 103. 105, 108 Dictyosrelium, I 10-1 12 mechanism, 120 theories, 87-89,94 karyosphere and, 4 I Actin-binding proteins (ABP). cytoskeletal dynamics and current concepts, 103 cytoskeletal components, 104-108 Dictyostelium, I 10-1 12 theories, 88-89 a-Actinin, cytoskeletal dynamics and, 96, 105-106, 111-112 Actomyosin, cytoskeletal dynamics and current concepts, 94, 102 cytoskeletal components, 105-109 Dictyostelium, 110, I 1 2 mechanism, 119 theories, 87-88 Adaptation, avian salt glands and, 158. 161, 192 Adenylate cyclase, avian salt glands and, 200-20 1

301

ADP, avian salt glands and, 135 Adrenal gland, avian salt glands and, 189, 191-192 Adrenergic innervation, avian salt glands and, 167-168.200 Adrenoconicotrophic hormone (ACTH). avian salt glands and, 192, 198 Aedes. karyosphere and, 9, 18, 36 Agrohucrerium, plant-pathogen interactions in, 62-63.66 Aldosterone, avian salt glands and, 192 Algae, see also Green algae: Heterokont algae; Red algae chloroplasts and, 259-260,295 cryptomonads. 275 dinoflagellates, 286, 288-289, 291 mitosis and, 221-222, 224 Alleles, Arahidopsis thalianu and, 56, 58. 62-63,67-68 Amiloride. avian salt glands and, 176 Amino acids Aruhidopsis rhalianu and, 73.75 chloroplasts and. 281 cytoskeletal dynamics and. 97, 106-108 Amoeba, chloroplasts and, 270 Amoeba, cytoskeletal dynamics in, see Cytoskeletal dynamics in Amoeba Amphibians avian salt glands and, 129 karyosphere and, 26-27 mitosis and, 247 Amphidinium

chloroplasts and, 288-290 mitosis and, 223 Amylopectin. chloroplasts and, 265 Anus plutyrhynchos, avian salt glands and, 130, 133 Anastomoses, avian salt glands and, 145-146, I48 Angiogenesis, avian salt glands and, 15 1

302

INDEX

Angiotensin 11, avian salt glands and, 189, 192-196. 199 Anlagen of the central body (ACR), karyosphere and, 35 Annuli, karyosphere and, 9 Anopheles gunibiue, karyosphere and, 9-10, 18 Antibodies avian salt glands and, 171, 196 chloroplasts and, 260 cytoskeletal dynamics and, 95. 99, 108-109 mitosis and, 237,246, 252 Antidiuretic hormone (ADH), avian salt glands and, 178, 180, 182, 184 Antimotic drugs, 225,229, 237 Antiobiotics, mitosis and, 235 Anurans, karyosphere and. 27-36 Apical cells, avian salt glands and, 140, 147, 171-173 Apical tight junctions, avian salt glands and, 138-139 Aplystu, cytoskeletal dynamics and. 100 Aruhidopsis thuliuna, 53-54 biochemistry, 72-76 genetic model, 60-64 models plant defense, 76-77 resistance genes, 76 systemic acquired resistance, 77-78 pathogens, 54-55 biochemistry, 59-60 genetics, 55-59 phenotypes bacteria, 66-69 fungi, 64-66 nematodes, 7 1-72 viruses, 69-71 Arginine vasotocin (AVT), avian salt glands and, 189, 198-199 Ascuris suumi, cytoskeletal dynamics and, 9 1 Aspergillus. mitosis and, 243, 245, 252 Aspergillus niduluns, mitosis and, 226. 228-229,232,248,251 ATP avian salt glands and, 135, 144, 173 cytoskeletal dynamics and, 88, 96, 98. 103, 116-118

mitosis and, 246 ATPase avian salt glands and, 147 cytoskeletal dynamics and current concepts, 94, 97, 103

cytoskeletal components, 106, 108-109 theories. 88 mitosis and, 234 Atrial natriuretic factor (ANF), 196-198, 203 Atropine, avian salt glands and, 161-162, 165, 203-205 Autonomic nervous system, avian salt glands and, 155 Avian salt glands, 129-130, 205 blood supply microvasculature, 148-15 1 secretion, I5 1-154 hormonal control, 189, 199-200 angiotensin 11, 192-195 arginine vasotocin, 198-1 99 atrial natriuretic factor, 196-198 pituitary, 19 1-192 prolactin, 198 innervation adrenergic. 167-1 68 cholinergic, 159- I62 nerve supply, 155-159 vasoactive intestinal polypeptide, 162-167 receptive systems, 177 neural integration. 188-190 tonicity, 177-184 volume, 185-188 secretory mechanism, 168-169 current models, 175-176 early models. 172-175 Na-K-ATPase. 169-172 secretory tissue duct system, 145-148 enzymes, 144-145 fine structure, 133-140 hypertrophy, 140-144 ZOOlOgy, 130-133 stimulus-secretion coupling, 200 calcium, 203-205 cyclic AMP, 200-202 cyclic GMP, 201-203 protein phosphorylation, 205 Avirulence, Arubidopsis thuliuna and, 75, 77 pathogens, 54,56-57 phenotypes, 67, 69 u w gene, Arubidopsis rhaliuna and biochemistry, 73,75 genetic model, 61 models, 76-71 pathogens, 56-57 phenotypes, 67,69 Axoplasm, avian salt glands and, 159

303

INDEX

B Bacillariophytes. chloroplasts and, 280, 282. 290 Bacteria, see also Cyanobacteria Arahidopsis thaliana and biochemistry. 73-75 models, 78 pathogens, 56-58 phenotypes, 65-69 chloroplasts and, 259, 270 Barley, plant-pathogen interactions in, 58 Basal lamina, avian salt glands and, 135, 159 Basement membranes, avian salt glands and, 138, 149. 159 Basophils. karyosphere and. 25 Beetles, karyosphere and, 18-25 Birds avian salt glands and, 129-1 30, 205 blood supply. IS 1 hormonal control, 191. 195, 198 secretory mechanism, 177-178 secretory tissue, 131-132, 140-141, 147 karyosphere and. 25, 37-38 Elaps. karyosphere and, 19,21,25 Blood-brain barrier. avian salt glands and, 138. 182. 184, 195 Blood supply, avian salt glands and innervation, 157. 168 microvasculature, 148-1 5 1 receptive systems, 182, 184-1 85 secretion. 151-154 secretory mechanism. 173, 175-176, 178 secretory tissue, 131-132. 141 Eomhvx mori. mitosis and, 225 Bone marrow, mitosis and. 225 Brain avian salt glands and hormonal control, 195, 197 receptive systems. 180. 182, 184, 187. 189 cytoskeletal dynamics and, 109-1 I0 Brainstem, avian salt glands and. 187-188 Brassica. plant-pathogen interactions in, 65. 69 Bumetanide, avian salt glands and, 154

C Calcium avian salt glands and, 184,200.203-205 cytoskeletal dynamics and. 86, 88, 102. 106-108

mitosis and, 251 Culliphora, karyosphere and, 42 Calmodulin cytoskeletal dynamics and, 97, 105 mitosis and, 251 Camalexin. Arabidopsis fhaliano and, 75 Cumelina suriva, Arahidopsis thaliana and, 75 Capping, cytoskeletal dynamics and. 112-1 13. 1 I8 Capping proteins. cytoskeletal dynamics and, 96. 107 Carbachol, avian salt glands and, 161 Cardiovascular system. avian salt glands and. 188-189. 192 Carotenoids. chloroplasrs and dinoflagellates, 286, 288. 290-291 euglenoids, 282, 284 green algae. 265 heterokonr algae, 277-278. 280-282 red algae, 261, 265 Cauliflower mosaic vims, Arabidopsis thaliana and, 62-63.67 cDNA Arahidopsis rhaliana and, 74 cytoskeletal dynamics and, 106-108 Cecidomyiidae. karyosphere and. 1 1 Cell division arrest, mitosis and, 228 Cell fusion, mitosis and, 252 Central nervous system, avian salt glands and, 155, 178. 193 Centrioles, mitosis and, 224, 237-238 Centromeres, mitosis and. 222, 230, 235, 239-240.244 Centrosomes cytoskeletal dynamics and, 94 mitosis and, 217 dissociation of events, 23 1. 237-238. 240-241,243,250 mitotic events, 219-220, 224 Cerebrospinal fluid, avian salt glands and, 182, 184, 195 Chaos chaos. cytoskeletal dynamics and. I 15 Chelonia, avian salt glands and, 188 Chelonia mydas. avian salt glands and, 147 Chemotaxis, cytoskeletal dynamics and, 85, 88. 110-111, 117 Chironomus, karyosphere and, 41 Chitinase, Arabidopsis rhaliana and, 74 Chlorarachnion, chloroplasts and, 276 Chloride, avian salt glands and, 129, 205 blood supply, 154 hormonal control. 196-197

304 innervation, 155, 157 secretory mechanism, 173, 175-176 secretory tissue, 131-133, 139 stimulus-secretion coupling, 200-20 1 Chlorophyll, chloroplasts and, 25-260. 284 cryptomonads, 273. 275 dinoflagellates, 285, 288-289, :!92 green algae, 265, 270, 272 heterokont algae, 277. 282 red algae, 261, 263, 265 Chlorophytes, endosymbiosis and, 265. 280 Chloroplasts, endosymbiotic origin of, 259-260,293-295 cryptomonads, 272-273 biochemistry, 276 Chlorarachnion, 276 envelope membranes, 275-276 pigments, 273 structure, 273-275 dinoflagellates, 285 alien aIgal endosymbionts, 286. 288-289 ancestry. 291-293 biochemistry, 291 eyespot, 290-291 fucoxanthin, 289-290 phycobilins, 290 pigments, 285-286 structure, 286-287 euglenoids, 282 biochemistry, 284-285 eyespot, 284 pigments. 282-283 structure, 283 green algae, 265 ancestry, 270-272 biochemistry. 272 envelope membranes, 266, 268-270 pigments, 265 structure, 266-267 heterokont algae, 277 biochemistry, 28 1 chloroplast relationships, 282 eyespot, 278, 280-281 pigments, 277 structure, 277-279 red algae, 261-264 biochemistry, 263-264 cyanelles, 264-265 glaucophytes. 264 pigments. 261 structure, 26 1-263

INDEX Cholinergic innervation, avian salt glands and, 175, 195 innervation, 159-163, 165, 167 stimulus-secretion coupling, 20 I , 203-204 Cholinesterase, avian salt glands and, 157-161, 167 Chromatid linkage protein (CLiP), mitosis and, 244-245 Chromatids, mitosis and, 217 chromosomes, 238-239,241,244-245, 251 dissociation of events, 235, 237 genetics, 23 I , 233 mitotic events, 220-221, 225 Chromatin karyosphere and, 8, 15, 21, 23, 39 mitosis and chromosomes, 239-240. 246-247, 249-250 dissociation of events, 235, 238 genetics, 232-233 mitotic events, 219-220.222. 224 Chromatography avian salt glands and, I63 chloroplasts and, 265 karyosphere and, 41 Chromista, chloroplasts and, 277-282 Chromosomes Arahidopsis thaliana and, 62-63, 67 cytoskeletal dynamics and, 113 karyosphere and, 1,42-45.47 nuclear matrix, 41 oocytes of insects, 9, 11, 15, 19. 21. 23, 25 vertebrates, 25-28, 30.33, 35-39 mitosis and, 217-218 dissociation of events. 227, 229-252 mitotic events, 218-223. 225 Chrysopa, karyosphere and, 1 I , 15, 18, 23,42 Chrysophytes, chloroplasts and, 280, 282, 288-290 Ciliates, chloroplasts and, 293 Circumventricular organs (CVO), avian salt glands and, 182, 195, 197 Cleptochloroplasts, endosymbiosis and, 289, 292 Clones Arahidopsis thaliana and biochemistry. 73-74 genetic model, 61-63 models, 76 pathogens, 56-57

305

INDEX phenotypes. 67,71-72 cytoskeletal dynamics and, 107-108 Colcemid, mitosis and. 240, 244. 246 Colchicine cytoskeletal dynamics and, 109 mitosis and, 230-23 I , 235-236.240, 245-246, 259 Coleoptera. karyosphere and, 8 Collagen, avian salt glands and, 149 Compactin, cytoskeletal dynamics and, 89-91 Concanavalin A, cytoskeletal dynamics and, 99, 113 Connective tissue. avian salt glands and. 133, 137, 149, 159-160 Contractile systems. cytoskeletal dynamics and, 96 Contraction, cytoskeletal dynamics and, 86-88, 93-96 Coprinus, mitosis and, 222 Cortical expansion, cytoskeletal dynamics and. 88-89.93 Cortical flow, cytoskeletal dynamics and, 99-101 Cortical network, cytoskeletal dynamics and, Ill Cortical tension, cytoskeletal dynamics and. 115-1 17 Corticosterone, avian salt glands and, 19 1-192 Cranial nerve. avian salt glands and, 155. 158 Crickets, karyosphere and, 18 Crypthecodinium cohnii, mitosis and, 223 Cryptomonads, chloroplasts and, 260. 264, 272-273.293-295 biochemistry, 276 dinoflagellates, 285, 288-290. 292-293 envelope membranes, 275-276 euglenoids, 284-285 heterokont algae, 277-278, 281 pigments, 273 structure, 273-275 Cryptomonas @, chloroplasts and, 276. 28 1 Cryptophytes. chloroplasts and. 280 Culex pipiens, karyosphere and, 9 Cyanelles, chloroplasts and, 264-265. 272, 276,285 Cvonidiuni cddarium, chloroplasts and, 264 Cyanobacteria, chloroplasts and, 259-260, 294-295 cryptomonads. 273, 276 dinoflagellates, 286, 292-293 euglenoids, 283, 285

green algae, 268, 270 red algae, 261, 263-265 Cyonophora. chloroplasts and, 264-265, 272 Cyanophora paradoxu, chloroplasts and, 264, 285 Cyclic AMP avian salt glands and, 169. 200-201 cytoskeletal dynamics and, 89, 102-103. 110-11 I Cyclic GMP avian salt glands and, 200-203 cytoskeletal dynamics and, 103 Cyclins, mitosis and, 249-25 I Cystrones, karyosphere and, 18 Cytochalasin, cytoskeletal dynamics and, 88 Cytochalasin B cytoskeletal dynamics and, 100 mitosis and, 234 Cytokinesis cytoskeletal dynamics and, 91 mitosis and dissociation of events, 227-235 mitotic events, 220-22 I , 224-226 Cytoplasm avian salt glands and. 138-140, 204 chloroplasts and, 270, 280 dinoflagellates, 286, 288, 291-292 euglenoids, 283-284 red algae, 261. 263-265 cytoskeletal dynamics and, 102, I 1 1, 120 cytoskeletal components. 106, 109 mechanism, 117, 119 theories, 87-88 karyosphere and, I, I 1,42,44 mitosis and chromosomes, 242-243, 245-246, 25 I dissociation of events, 230, 238 mitotic events, 220, 222-223, 226 Cytoskeletal dynamics in Amoeba, 85-86, I20 Amoeba proteus, 87, 9 1, 93 current concepts. 94 contraction, 94-96 cortical flow, 99-101 projection, 97-99 signal transduction. 101-103 cytoskeletal components, 103-104, I10 actom yosin, 105- 109 microtubules, 109-1 I0 Dictyostelium

actomyosin, 110-1 13 capping, 112-1 14

306 microtubules, 113-1 14 substrate exploration, 112 mechanism biased friction, 1 17-1 20 cell motility, 114-1 15 motor proteins, 116-1 17 theories, 86 amoeboid movement, 86-92 cell division, 91 93-94 Cytostatic factor, mitosis and, 247 Cytovillin, cytoskeletal dynamics and, 106

Deadaptation, avian salt glands and, 140-141 Defense, plant, Arahidopsis thaliana and, 53-55,73,76-77 Deletion, Arahidopsis rhaliuna and. 57, 63 Denervation, avian salt glands and, 158 Dephosphorylation, mitosis and, 248-249, 252 Depolarization, avian salt glands and, 157 Depolymerization cytoskeletal dynamics and, 109-1 10 mitosis and, 243, 245 Desmosomes, avian salt glands and. 135 Diabetes, avian salt glands and. 198 Diacylglycerol avian salt glands and, 200. 203 cytoskeletdl dynamics and, 102 2.6-Dichloroisinicotinic acid (INA), Arahidopsis thaliana and, 78 Dicryosteliumi, cytoskeletal dynamics and, 85 actomyosin, 110-1 13 current concepts, 96-98, 101, 103 cytoskeletal components, 103-106. 108-1 09 mechanism, 116-1 17 microtubules, 113-114 theories. 86, 88-89, 94 Dictynsteliuni discoideum, cytoskeletal dynamics and, 95 Dinitrophenol, mitosis and, 246 Dinoflagellates, chloroplasts and. 2175-276, 283, 285-295 Dinophysis, chloroplasts and, 290 Dinophysoids, chloroplasts and, 290, 292, 294-295 Diptera, karyosphere and, 8-1 I Disease resistance, Arahidopsis thaliana and. 53-54 genetic model, 61, 64

INDEX pathogens, 54-56,58-60 phenotypes, 65-68, 72 Dissociation of mitotic events chromosomal events, 238-252 cytokinesis, 233-235 experimental evidence, 235-238 genetic evidence, 226-233 DNA Arahidopsis thaliuna and, 57, 63, 67 avian salt glands and, 141, 143, 158 chloroplasts and, 259, 263, 281 cryptomonads, 273, 275-276 dinoflagellates, 288, 290 karyosphere and, 8,41-43,45 oocytes of insects, I I , 14, 17-19. 21, 23, 25 vertebrates, 26, 28, 32, 35-38 mitosis and chromosomes, 240-241,243,248-249 dissociation of events, 226-228, 231, 238 mitotic events, 222, 225 DNP, karyosphere and, 42, 45 Dosage, avian salt glands and, 167 Drosophilu cytoskeletal dynamics and, 97 karyosphere and, I I , 43 mitosis and, 252 dissociation of events, 226, 230-235, 237. 245 mitotic events, 225-226 Drosophila melanogaster, karyosphere and, 8 Drugs, mitosis and. 225, 229, 237, 246 Ducks, avian salt glands and, 130 blood supply, 15 I , 154 hormonal control, 193, 196, 199-200 innervation, 155, 158-159. 161, 168 receptive systems, 177-178, 180, 182, 185-186, 189 secretory mechanism, I72 secretory tissue, 131-133, 135. 137. 140-141, 143, 145-146 stimulus-secretion coupling, 200, 203 Dynein. cytoskeletal dynamics and, 109-1 10 Dystrophin, cytoskeletal dynamics and, 106

E Ectoplasm, cytoskeletal dynamics and, 86-87 EDTA cytoskeletal dynamics and, I09

307

INDEX karyosphere and, 21.45 ein. Arabidopsis rhaliuna and, 75-76 Elasmobranchs. avian salt glands and, 147-148, 177, 196 Electron density avian salt glands and, 139, 159 cytoskeletal dynamics and, 96 karyosphere and, 21, 38 Electron microscopy avian salt glands and blood S U P P ~ Y ,148-149 innervation, 158, 163, 167 receptive systems, 180. I82 secretory tissue, 133, 143-1 44 chloroplasts and, 268. 286 karyosphere and. 7 oocytes of insects, 8, 13-17, 19, 21-25 vertebrates, 37 Electrophoresis cytoskeletal dynamics and, 105, 109 karyosphere and, 40 Elongation avian salt glands and, 135 cytoskeletal dynamics and, 108. I13 Elongation factors, cytoskeletal dynamics and, 107 Embryos, karyosphere and, 1 I , 38-39, 42 Endomitosis, 225, 239 Endoplasm, cytoskeletal dynamics and. 87, I10 Endoplasmic reticulum avian salt glands and, 140. I7 1, 203 chloroplasts and, 268, 270, 273, 278, 291 mitosis and, 219-220, 222, 225, 245 Endopolyploidy, mitosis and, 225. 240-24 I Endoreduplication, mitosis and. 225, 23 I 240 Endosymbiotic origin of chloroplasts, see Chloroplasts. endosymbiotic origin of Endothelium, avian salt glands and, 138-1 39, 149. 151. 160 Enzymes Arabidopsis rhaliuna and, 72-73 avian salt glands and, 159, 189, 193 secretory mechanism. 169. 171-172 secretory tissue, 138, 144-145 mitosis and, 250-25 1 Epinephrine. avian salt glands and. 167. 200 Epithelium avian salt glands and. 149, 163, 176. 200. 205 hormonal control, 196-197 secretory tissue. 135, 138-140. 143. 146-148

cytoskeletal dynamics and, 101. 113 Equatorial contraction, cytoskeletal dynamics and, 93-94 Ereniia velos, karyosphere and, 37 Erwinia, plant-pathogen interactions in, 57 Erysiphe cruciferarum, plant-pathogen interactions in, 65 Erysiphe fischeri, plant-pathogen interactions in, 58-59 Ethmoidal ganglion, avian salt glands and. 158, 163 Ethylene, Amhidopsis rhaliaria and, 75 Euchromatin, mitosis and, 222, 230 Euglena chloroplasts and. 284-285, 294 mitosis and, 222 Euglena jiracilis, chloroplasts and, 264, 280 Euglenoids, chloroplasts and, 264, 282-285, 293-294 Eukaryotes chloroplasts and, 259-260, 263, 295 cryptomonads, 275-276 dinoflagellates. 286, 292-293 euglenoids, 283-284 green algae, 268, 270 heterokont algae, 278 mitosis and, 237 Eukaryotes, higher, mitosis and. 219, 224-226 Eukaryotes. lower, mitosis and, 252 dissociation of events, 226-230, 233 mitotic events, 219-224 Eustigmatophytes. chloroplasts and, 277, 280-282 Evolution, Arabidopsis thaliana and, 54, 58-59.64,67 Extension. cytoskeletal dynamics and, 89-91 Extrdcehlar fluid, avian salt glands and, 185-187 Extracellular fluid tonicity (ECFT), avian salt glands and, 129, 177, 180 Extracellular fluid volume (ECFV). avian salt glands and, 129, 20 I hormonal control, 195-196 receptive systems, 177, 185-1 89

F F-actin. cytoskeletal dynamics and current concepts, 96-98, 100-101 cytoskeletal components, 106-108

308

INDEX

Dictynstelium, 1 1 1 - 1 13 mechanism, 116, 118

Fertilization, mitosis and, 241 Fibrils chloroplasts and, 291 karyosphere and oocytesof insects, 11, 14-15, 17,21 vertebrates, 25, 30, 32, 37-09 Fibroblasts, cytoskeletal dynamics and, 88, 96, 100, 103, 106, 115 Filamin, cytoskeletal dynamics and, 96, 106 Fish avian salt glands and, 129 karyosphere and. 25 Fission zone, mitosis and, 228 Flagella chloroplasts and, 277-278, 280, 284, 289 mitosis and, 224 Flies, karyosphere and, 9, 43 Fluorescence chloroplasts and, 280 cytoskeletal dynamics and, 99, 101 karyosphere and, 37 Fluorescence microscopy, cytoskeletal dynamics and, 1 I3 Fodrin, cytoskeletal dynamics and, 108 Folic acid, cytoskeletal dynamics and, 110-111 Forskolin, avian salt glands and, 200-201 Fragmin, cytoskeletal dynamics and, 107 Freeze-fracture studies avian salt glands and, 138 chloroplasts and, 292 Fucoxanthin, chloroplasts and, 278, 282, 288-290,294 Fttrus serratus, chloroplasts and, 281 Fungi Aruhidopsis thaliana and, 61, 74, 76 pathogens, 56.58-59 phenotypes, 64-66 chloroplasts and, 277 mitosis and, 221-222, 224 Furosemide, avian salt glands and, 154, 162, 173,201

G G-actin, cytoskeletal dynamics and, 101-102 Gametocytes, karyosphere and, 2-7, 15.40 Gametogenesis, karyosphere and, 7-8,41

Ganglia, avian salt glands and, 155, 157-159, 163, 193, 196 Geese, avian salt glands and, 131, 153, 155, 158-159, 162 Gelation, cytoskeletal dynamics and, 86, 88. 105 Gelsolin, cytoskeletal dynamics and, 107 Gene amplification, karyosphere and, 27 Gene expression, Arahidopsis thalianu and, 74 Gene functions, mitosis and, 252 Genes chloroplasts and, 263, 281 cytoskeletal dynamics and, 94, 104, 109-110, 119-120 Genetics Arubidopsis thaliana and biochemistry, 72-74 genetic model, 60-64 models, 76-77 pathogens, 55-60 phenotypes, 66, 68, 70 mitosis and, 226-233, 252 Genotype, Arahidopsis thaliana and, 64, 74 pathogens, 55.57-58 phenotypes, 65-70, 72 Germinal vesicle breakdown, mitosis and, 247 Germinal vesicles, karyosphere and, 44, 46-47 Giant nucleus mutant, mitosis and, 230-23 I Glandular hypertrophy, avian salt glands and, 140-14 1 Glaucophytes, chloroplasts and, 264 Glenodinium foliaceum, chloroplasts and, 288-290,292 Glia, avian salt glands and, 182 Glorin, cytoskeletal dynamics and, 102 P-Glucanase, Arahidopsis thaliana and, 73. 77 Glucose, avian salt glands and, 178, 180, 192 Glucose-6-phosphate dehydrogenase, avian salt glands and, 145, 147 Glycoprotein cytoskeletal dynamics and, 99, 107 karyosphere and, 41 Gnaptnr spinimanus, karyosphere and, 19 Golgi apparatus avian salt glands and, 135, 140, 143-144, 147, 171 mitosis and, 219-220, 234 Green algae, chloroplasts and, 260, 264-265, 276,293,295 ancestry, 270-272 biochemistry. 272

309

INDEX cryptomonads, 276 envelope membranes, 266, 268-270 euglenoids. 282-285 heterokont algae, 28 1 structure. 266-267 Griffrthsia pocifira. chloroplasts and, 263. 28 I GUS reporter gene, Arahidopsis thaliana and, 71, 73 Gvmnodiniunt. chloroplasts and, 288-290 Gvrodinium. chloroplasts and. 289

H Haliaecfus leucogaster, avian salt glands and, I47 Heavy meromyosin. cytoskeletal dynamics and, 94 HeLa cells. cytoskeletal dynamics and, 95 Hemorrhage, avian salt glands and, 185 Heritability, Arahidopsis rhaliana and, 56 Heterochromatin, mitosis and, 222, 230 Heterodera schachfii. plant-pathogen interactions in, 72 Heterokont algae, chloroplasts and, 260. 277, 293-295 biochemistry. 281 dinoflagellates, 288, 290, 293 euglenoids, 283-286 eyespot, 278,280-281 relationship, 282 structure, 277-279 Hexamethonium, avian salt glands and, 157 Hexokinase, avian salt glands and, 145 Hexose monophosphate, avian salt glands and, 145 Histochemistry. avian salt glands and, 159-161. 167, 171 Homology Arahidopsis rhaliana and, 68, 74. 76 avian salt glands and, 130. 176 chloroplasts and, 261, 263, 276, 283, 285, 292 cytoskeletal dynamics and current concepts, 97-98, 103 cytoskeletal components, 105-107. 109 theories, 94 karyosphere and, 11 mitosis and, 228. 23 1, 250 Hormones hahidopsis thaliana and, 72, 75

avian salt glands and, 130-1 3 I , 168, 188, 20 1 control, 189-200 hrp genes, Arahidopsis thaliana and, 57 Hybridization Arahidopsis fhaliana and, 63, 67, 76 karyosphere and, 1 1 Hydrogen, avian salt glands and. 173 Hydrolysis, cytoskeletal dynamics and, 88, 116. 118 Hyperglycemia, avian salt glands and, 192 Hyperkalemia, avian salt glands and, 192 Hyperplasia, avian salt glands and. 141. 143 Hypersensitive response (HR), Arahidopsis thaliana and, 75. 78 pathogens, 54, 57 phenotypes, 65.67-69 Hypersensitivity, Arahidopsis thaliana and, 59-60 Hypertonic stimulation, avian salt glands and. 182, 184-186, 198,200 Hypertrophy, avian salt glands and, 140-144. 151. 158, 191-192 Hypophysectomy, avian salt glands and, 192, 198 Hypothalamus, avian salt glands and hormonal control, 197-198 receptive systems, 180. 182, 184, 188-1 89

I Immunization. Arahidopsis thaliana and. 77-78 Immunofluorescence, cytoskeletal dynamics and, 95-96, 110-1 1 I Immunology, avian salt glands and, 162-163, 196 Infection, Arahidopsis thaliana and, 55, 60, 64-66,71-72 Inhibitors avian salt glands and hormonal control, 192-193, 195, 198-199 innervation, 157, 161-162, 165, 168 receptive systems, 178, 180, 182, 187-189 secretory mechanism, 169 stimulus-secretion coupling, 201, 204-205 cytoskeletal dynamics and, 94, 105, 107, 1 I3 mitosis and. 217, 252 chromosomes, 24 1,243-246,249

310

INDEX

dissociation of events, 227-228, 234-237 mitotic events, 218 Inner centromeric proteins (IN-CENPS), mitosis and, 244-245 Innervation, avian salt glands and, 131, 189, I95 adrenergic system, 167-168 cholinergic system, 159-162 nerve supply, 155-159 vasoactive intestinal polypeptide, 162-167 Inositol phosphate, avian salt glands and, 200, 205 1,4,5-Inositol triphosphate (IP3) avian salt glands and, 200, 203, 205 cytoskeletal dynamics and, 102 Insects, karyosphere and, 25. 43 Diptera, 8-1 1 Neuroptera, 11-18 tenebrionid beetles, 19-25 Interstitial fluid volume (ISFV), avian salt glands and, 185, 187-188 Isocitric dehydrogenase. avian salt glands and, 145. 147 Isoosmotic glucose, avian salt glands and, 178. 180 Isoproterenol, avian salt glands and, 167

K Karyokinesis, mitosis and dissociation of events, 228, 230, 232-235 mitotic events, 224-226 Karyosomes, vertebrates and, 25 Karyosphere, 1-8 chromosome assembly, 42-43 function, 44 gametogenesis, 8 mammals, 38-39 nuclear matrix, 40-41 nuclei, 43-44, 46-47 oocytes of insects, 8 Diptera, 8-1 1 Neuroptera, 11-1 8 tenebrionid beetles, 19-25 protein metabolism, 41-42 substructural elements, 44-46 vertebrates, 25-27 anurans. 27-36 birds, 37-38 reptiles. 36-37

Kidney avian salt glands and, 129-130, 176, 188. 199 cytoskeletal dynamics and, 106 Kinesin, cytoskeletal dynamics and, 109 Kinetics, Arahidnpsis thaliana and, 60, 70 Kinetochores, mitosis and, 217 chromosomes, 239-241,243-247 dissociation of events, 227, 230, 237-238 mitotic events, 2 19-220, 222-223 Kinetoplastids, chloroplasts and, 264, 284-285. 294

L Lacertu muralis, karyosphere and, 37 Lactic dehydrogenase, avian salt glands and, 145 Lamella chloroplasts and, 263, 266, 278, 282, 286, 290 cytoskeletal dynamics and, 94, 98, 101. I 15 Lamina pores. karyosphere and, 30,40 Larus, avian salt glands and, 133, 140-141 Lepidodinium iiride, chloroplasts and, 288 Leukocytes, cytoskeletal dynamics and, 99, 102 Ligands avian salt glands and, 165, 168 cytoskeletal dynamics and. 102 Light microscopy avian salt glands and, 133, 148, 167, 180. 182 karyosphere and, 11, 18, 39, 45 mitosis and, 225, 230, 232 Lignin, Aruhidnpsis thaliana and, 60 Linkage, Arahidopsis thaliana and, 63 Lipids avian salt glands and, 141, 144. 169. 204 chloroplasts and, 268 cytoskeletal dynamics and, 99 karyosphere and, 28.41 Lithium, avian salt glands and, 184 Liver karyosphere and, 4 1 mitosis and, 225 Lizards avian salt glands and, 133. 147, 155 karyosphere and, 37 Lorodes striatus, karyosphere and, 36

INDEX

31 1 M

Macrophages, cytoskeletal dynamics and, 99 Magnesium avian salt glands and, I7 1 , I84 cytoskeletal dynamics and, 88, 97. 103. 106. I08 Major resistance genes (R genes). Arabidopsis rhaliana and, 56, 58, 60, 66. 68. 76 Malic enzyme, avian salt glands and, 145, 147 Mannitol, avian salt glands and, 183-184 Mapping Arahidopsis thuliana and, 63-68 chloroplasts and, 265, 281 cytoskeletal dynamics and, 106, 109 Maturation promoting factor, mitosis and, 218, 229.23 I , 242,245,247-250, 252 Megakaryocytes, mitosis and, 225 Meiosis, 230. 247 karyosphere and, I , 7-8, 42,44 oocytes of insects, 9, I 1 vertebrates, 28, 36, 38-39 Mdoidogvnr incognitu. plant-pathogen interactions in, 72 Membrane-binding proteins, cytoskeletal dynamics and, 107-108 Messenger RNA Arahidopsis thuliana and. 73-74 karyosphere and. 42 Metacholine, avian salt glands and. 161 Microfilaments, karyosphere and, 4 I Microsomes, avian salt glands and. 205 Microtubule organizing center (MTOC), cytoskeletal dynamics and. I 10, I 13 Microtubules chloroplasts and, 284. 291 cytoskeletal dynamics and, 94. 100. 109-1 10. 113-1 14 mitosis and. 217 chromosomes, 244-246.25 I dissociation of events, 232-233. 236-238 mitotic events. 21 9-224 Microvilli avian salt glands and, 138-139, 147, 172, 182 cytoskeletal dynamics and. 97, 106. 116 Mildew, Arahidopsis thaliuna and, 76 Mitochondria avian salt glands and innervation, 159 secretory mechanism, I7 I

secretory tissue, 135, 139-1 41, 144, I47 chloroplasts and, 259. 288. 291 Mitosis, 2 17-2 18, 252 avian salt glands and. 135, 143 cytoskeletal dynamics and, 91, 107, 1 13 dissociation of events chromosomal events, 238-252 cytokinesis, 233-235 experimental evidence, 235-238 genetic evidence, 226-233 mitotic events higher eukaryotes, 224-226 history, 218-219 lower eukaryotes, 220-224 standard type, 219-220 Monoclonal antibodies, cytoskeletal dynamics and, 109 Monomer-binding proteins, cytoskeletal dynamics and, 108 Morphogenesis cytoskeletal dynamics and. 1 1 2-1 13 karyosphere and, 7-8, 38,45-47 Morphology Arabidopsis thuliana and, 72 avian salt glands and, 133, 155, 163, 167, 171. 182 cytoskeletal dynamics and, 113 karyosphere and, 1 , 7,40, 45 Mosquitoes, karyosphere and, 9-1 1. 18, 30.44 mRNA, see Messenger RNA Mucocytes, avian salt glands and, 147 Muscarinic agonists, avian salt glands and, 200 Muscarinic receptors, avian salt glands and innervation, 161-162, 165. 167-168 secretory mechanism, 176 stimulus-secretion coupling, 203, 205 Muscle. cytoskeletal dynamics and, 96. 98, 105- I06 Mutagenesis, Arabidopsis fhaliana and biochemistry, 74 genetic model, 62 models, 76-77 phenotypes, 66, 70, 72 Mutants Aruhidopsis thalianu and, 53, 57 biochemistry, 74-75 genetic model, 61-63 models, 16-77 phenotypes, 68-72 chloroplasts and, 273

31 2 cytoskeletal dynamics and, 94. I 1 1-1 12, 119-120 karyosphere and, 9 , 4 3 mitosis and chromosomes, 242,245, 248-249, 25 1-252 dissociation of events, 226-244 Myosin, cytoskeletal dynamics and current concepts, 95-98, 100, 103 cytoskeletal components, 103, 105, 108-109 Dic/yosreliurn, 110-1 13 mechanism, 116-1 19 theories, 86-89.94 Mytilus, cytoskeletal dynamics and, 97 Myzocytosis, chloroplasts and, 292

NADPH, avian salt glands and, 147 Na-K-ATPase, avian salt glands and secretory mechanism, 169-172, 175-176 secretory tissue, 140-141 stimulus-secretion coupling, 203, 205 Nematodes, Arahidopsis rhaliana and, 7 1-72 Neural integration, avian salt glands and, 188-189, 193 Neuromodulators, avian salt glands and, 162, 165, 189 Neuroptera, karyosphere and, 8, 11-18 Neurotransmitters, avian salt glands and, 131, 165 Nocodazole, mitosis and, 235, 246 Nonhistone proteins, mitosis and, 249 Nonhost resistance, Arahidopsis ihaliana and, 58.69 Norepinephrine, avian salt glands and, 167-168, 189,200 Nuclear envelope, mitosis and, 2117 chromosomes, 240-243.245-247 dissociation of events, 230. 232-233, 235, 238 mitotic events, 218-225 Nuclear envelope breakdown, mitosis and, 249, 25 I Nuclear lamina, mitosis and, 245-246, 249 Nuclear matrix, karyosphere and, 40-41 Nucleoids, chloroplasts and, 290 Nucleolus-like bodies (NLBs), karyosphere and, 46-47 oocytes of insects, 19. 21, 23, 25

INDEX vertebrates, 39 Nucleomorphs, chloroplasts and, 275-276, 295 Nucleotides avian salt glands and, 201-205 chloroplasts and, 260, 272, 281, 284, 293 Nucleus tractus solitarii (NTS), avian salt glands and, 188-189, 195 Nutrimental oogenesis, karyosphere and, 13

0 Oceanodroma leucorrhoa, avian salt glands

and, 133 Octopus, cytoskeletal dynamics and, 107

Okadaic acid, mitosis and, 235, 245, 249. 251 Oligonucleotides, Arahidopsis thaliana and, 64 Olisrhodiscus lureus, chloroplasts and, 28 1 Oocytes karyosphere and, I , 43-44, 46-47 Diptera, 8-1 1 Neuroptera, 11-1 8 tenebrionid beetles, 19-25 vertebrates, 26-38 mitosis and, 247 Oogenesis, karyosphere and, I , 7.41 anurans, 27-36 birds, 37-38 insects. 13, 17 mammals, 38-39 reptiles, 36-37 vertebrates, 25-27 Organum vasculosum laminae terminalis (OVLT), avian salt glands and, 182, 197 Osmoregulation, avian salt glands and, 189. 197-198 Osmosensitivity, avian salt glands and, 183-184, 188 Osmosis. avian salt glands and, 129, 151, 155 hormonal control, 192, 196 receptive systems, 177-178, 180, 184, 187 secretory tissue, 131, 139, 141, 144 Osmotic pressure, cytoskeletal dynamics and. 89 Ouabain, avian salt glands and secretory mechanism, 169, 171, 173. 175-176 stimulus-secretion coupling, 201. 203-204 Oxidation, Arahidopsis thalianu and, 60 Oxygen Arahidopsis thaliana and, 59 avian salt glands and, 151, 154, 161, 176, 201

INDEX

313 P

PAL. Aruhidopsis thuliunu and. 72-73. 77 Parasites, Aruhidopsis thulianu and, 54-55. 7 I

Paraventricular nucleus (PVN), avian salt glands and, 182, 188-189, 195 Parenchymal cells, avian salt glands and, 143 blood supply, 148-149 hormonal control, 193, 196 innervation, 158-159, 162-163. 168 receptive systems, 180, 189 Pathogenesis-related protein, Aruhidopsis rhuliunu and, 59, 74. 78 Pathogens. Arubidopsis thuliana and, see Aruhidopsis rhulianu Prlunfis, avian salt glands and. I39 Pe1oniy.w. chloroplasts and. 270 Penicillum griseofirlvuni, mitosis and. 235

Peridinin, chloroplasts and, 286. 288-290 Peridinium hulticum, chloroplasts and, 288-290,292 Pemnasporu purasitim, plant-pathogen interactions in, 65. 76-78 pH. cytoskeletal dynamics and, 86, 105. 108 Phaeophytes, chloroplasts and, 277-278. 282 Phagocytes chloroplasts and, 277. 290, 292 cytoskeletal dynamics and, 1 I I Pharmacology, avian salt glands and, 161-164. 167-169,205 Phenotype Aruhidopsis rhulianu and. 54 bacteria, 66-69 biochemistry, 74 fungi, 64-66 genetic model, 61-63 models. 77-78 nematodes. 7 1-72 pathogens, 56-57. 59-60 viruses, 69-7 1 cytoskeletal dynamics and, 120 mitosis and, 228 Phenoxybenzamine. avian salt glands and, 168 Phentolamines, avian salt glands and. 168 Phosphatidylinositol 4.5-bisphosphate. cytoskeletal dynamics and, 102 Phosphofructokinase. avian salt glands and, 145 Phosphoinositols, avian salt glands and. 203-205 Phospholipase C, cytoskeletal dynamics and, 102-103

Phospholipids avian salt glands and, 144. 169, 203 cytoskeletal dynamics and, 98. 102 karyosphere and. 4 1 Phosphorylation avian salt glands and, 169. 205 cytoskeletal dynamics and. 96. 103, 108, 117 mitosis and, 2 18. 247, 249-25 1 Photosynthesis, chloroplasts and. 259-260, 270.277.293 cryptomonads, 273,275-276 dinoflagellates. 285-286, 288, 290-29 I , 293 euglenoids, 282. 284 red algae, 261. 263-265 Phototaxis, chloroplasts and, 284. 291 Phragmoplasts. mitosis and, 220 Phrynocephalus. karyosphere and, 37 Phycobilins. chloroplasts and, 259-260, 295 cryptomonads, 273,275 green algae, 266, 270 heterokont algae. 285,290. 292 red algae, 26 I , 263-265 Phycobilisomes. chloroplasts and. 263, 265-266.273 Phycocyanin. chloroplasts and, 261. 263 Phycoerythrin, chloroplasts and, 261, 263, 273. 290 Physurum, mitosis and, 221, 224. 234 Phvsururn pulycephulum

cytoskeletal dynamics and, 107, 115 mitosis and, 226. 235 Phytoalexins. Aruhidopsis thuliunu and, 59, 73, 75 Pituitary-adrenal axis, avian salt glands and, 189. 192-192 Plant defense, Aruhidopsis rhuliunu and, .53-55.73,76-77 Plant defense genes. Arahidopsis thuliunu and. 60. 75 Plant pathology in Aruhidopsis thaliunu. see Arubidopsis rhuliunu

Plasma membrane avian salt glands and, 129. 157. 196, 203-204 secretory mechanism, 169. 17 I secretory tissue. 135. 139-140, 143-144 chloroplasts and, 280, 283, 292 cryptomonads. 275 green algae. 266. 268 red algae, 26 I cytoskeletal dynamics and. 96, 98-100, 118

314 mitosis and, 220 Plasmodium, mitosis and, 224, 234 Plastids, chloroplasts and, 268, 276. 284, 293-294 dinoflagellates, 286, 290-292 heterokont algae, 277, 280-281 Polar relaxation, cytoskeletal dynamics and, 91.93 Pollen, Arahidopsis thaliuna and, 62 Polymerization cytoskeletal dynamics and, 85., 110. 116 current concepts, 97, 102-103 cytoskeletal components, 105, 107 theories, 86, 89 karyosphere and, 9 mitosis and, 235, 237 Polypeptides chloroplasts and, 272 cytoskeletal dynamics and, 109 mitosis and, 245, 250 Polvphagu, karyosphere and, 25 Polyploidization, mitosis and, 225, 230-231, 235-237.239 Ponticulin, cytoskeletal dynamics and, 107-108, 111 Porphyra yewensis, chloroplasts and, 263 Porphyridium aeruginosum, chloroplasts and, 264 Potassium, see also Na-K-ATPase avian salt glands and, 132. 134, 192, 201 cytoskeletal dynamics and, 109 Potato. plant-pathogen interactions in, 72 Potato spindle tuber viroid (PSTV), Arabidopsis thaliana and, 7 I Prasenophytes, chloroplasts and, 266, 288 Pru!ylenchus penetrans, plant-pathogen interactions in, 72 Prematurely condensed chromosomes (PCCs), mitosis and, 238, 241-243 Prochlornn, chloroplasts and, 260, 270, 272 Prochlorophytes, chloroplasts and, 260, 272 Profilin, cytoskeletal dynamics and, 101-103, I ox Projection, cytoskeletal dynamics and, 97-99 Prokaryotes chloroplasts and. 259-261.263.283. 294 cryptomonads, 273,276 dinoflagellates, 286, 292-293 green algae, 266, 268 mitosis and, 226 Prolactin, avian salt glands and, 198

INDEX Propranolol, avian salt glands and, 168 Prorocentrum micans, chloroplasts and, 29 1 Protein Arabidopsis thaliana and, 59-60, 68, 70. 74, 78 avian salt glands and, 161, 192, 205 secretory mechanism, 169. 172 secretory tissue, 141, 143 chloroplasts and. 263. 282, 289 cytoskeletal dynamics and, 85-86, 89, 120 current concepts, 96-97 cytoskeletal components, 104-109 Dicryostelium, 110-1 I I mechanism, 116-1 17 karyosphere and. I. 40,42-45 oocytes of insects, 19 vertebrates, 25, 28, 32, 37-39 mitosis and chromosomes. 241.243-247,250-25 I dissociation of events, 2 18. 23 I Protein kinase avian salt glands and, 205 cytoskeletal dynamics and, 97 mitosis and, 227, 249-252 Protein kinase C. cytoskeletal dynamics and, 102 Protozoa, cytoskeletal dynamics and, 98 Prymnesiophytes, chloroplasts and, 277-278. 280,282,289-290 P seudomonas. plant-pathogen interactions in, 57. 73. 76 Pseudomonas syringae. plant-pathogen interactions in, 66-69, 74-75, 78 Pyluiellu littoralis, chloroplasts and, 281 Pyrenoids, chloroplasts and, 263. 288, 290

Q Quinuclidinyl benzilate (QNB), avian salt glands and, 161-162

R Race specificity, Arahidopsis thaliana and, 54, 56-58.65.73 Rana ridihunda, karyosphere and. 32-36 Rona temporaria, karyosphere and, 47 nuclear matrix, 41 vertebrates, 27-28, 30, 33. 35

INDEX Raphidophytes, chloroplasts and. 277-278. 280-282 rDNA. karyosphere and, I I , 17.37 Rectal glands. avian salt glands and blood supply, 149, 154 hormonal control, 196-197 innervation, 157. 163, 165 receptive systems, 177. 185, 187 secretory mechanism, 169. 171, 176 secretory tissue, 131. 140. 147-148 stimulus-secretion coupling. 201, 203 Red algae, chloroplasts and, 260-266, 293-295 biochemistry, 263-264 cryptomonads, 273.275-276 euglenoids, 283, 285 heterokont algae, 281 pigments, 26 I structure, 261-263 Renin-angiotensin system. avian salt glands and, 192 Replication Amhidopsis thaliana and, 70-7 I mitosis and chromosomes, 240, 243, 248-249 dissociation of events. 227-228. 23 1, 238 mitotic events, 219, 225 Reptiles avian salt glands and, 129. 160. 187 blood supply, 149 secretory tissue. 131-132. 139. 147-148 karyosphere and. 25.36-37 Resistance, disease, see Disease resistance Resistance genes, see Major resistance genes (R genes) Respiration, avian salt glands and, 144. 176 Restriction fragment length polymorphism, Arahidopsis thaliana and, 62-65, 67, 76 Rhodopsin, cytoskeletal dynamics and, 107 Ribosomes avian salt glands and, 135. 141, 172 chloroplasts and. 263. 275, 277 karyosphere and. 18 RNA Arahidopsis thaliana and. 70. 74 avian salt glands and, 141, 143, 158. 172 chloroplasts and. 285 karyosphere and. I , 1 I . 19. 30. 42 RNP. karyosphere and, 15.42-43.45 Rough endoplasmic reticulum. avian salt glands and, 139. 143

315 R P M l , Arahidopsis thaliana and, 67-68. 74 rRNA, chloroplasts and, 265,276, 284-285, 293-294 Rubisco, chloroplasts and, 263, 276, 281, 291

S Sac~charomvces cytoskeletal dynamics and, 97 mitosis and. 222 Sac~c~haromyces cerevisiae cytoskeletal dynamics and, 103 mitosis and, 226, 228. 230. 250-251 Salivary gland, avian salt glands and, 165, 167 Salt glands, avian, see Avian salt glands Sarcomeres, cytoskeletal dynamics and. 89. 96, 115, 119 Saionmalus ohesus, avian salt glands and, I49 Scanning electron microscopy, avian salt glands and, 180. 182 Schiiosaccharomyces pomhe. mitosis and, 226, 228,23 I , 250-25 1 Second messengers, avian salt glands and, 200-20 I , 203 Secretion. avian salt glands and blood supply, 151-154 duct system, 145-148 enzymes, 144-145 fine structure, 133-140 hormonal control, 196, 199 hypertrophy, 140-144 innervation, 155, 157-163. 165, 167 mechanism. 168-176 receptive systems, 177-190 stimulus-secretion coupling, 200-205 Loology. 130-1 33 Senecio vulgaris. plant-pathogen interactions in, 58-59, 64 Septation, mitosis and, 229 Sequences Amhidopsis thalianu and, 63, 7 I , 74 chloroplasts and, 260, 29 I. 293-295 cryptomonads, 276 euglenoids. 284 green algae, 272 heterokont algae, 281 cytoskeletal dynamics and. 97-98, 105-109 Severin, cytoskeletal dynamics and. 107, I I 1

316 Shark rectal glands, see Rectal glands Short-circuit current (SCC), avian salt glands and hormonal control, 197 innervation, I6 I , 163 secretory mechanism, 175-176 stimulus-secretion coupling, 201, 204 Signal transduction Arahidopsis thaliana and, 56, 62, 68, 73 avian salt glands and, 13 I , 188 cytoskeletal dynamics and, 101-103 Skeletal muscle, cytoskeletal dynamics and. 96. 106 Snakes, avian salt glands and, I 3 1. 139 Sodium, avian salt glands and, 129, 154. 192, 205, see also Na-K-ATPase innervation,,lSS, 157, 161 receptive systems. 178, 183-185. 187 secretory mechanism, 173, 175-176 secretory tissue. 131-133. 135, 139. 141 stimulus-secretion coupling, 2(N-20 I Sodium chloride, avian salt glands and, 178, 182, 184-185, 187 Solation, cytoskeletal dynamics and, 86. 88 Sonlateria niollissinta. avian salt glands and, 130, 141 Somatostatin, avian salt glands and, 154 Spectrin, cytoskeletal dynamics and, 108 Spermatocytes. karyosphere and. I , 8 Spindle pole body, mitosis and, 226 Squalus acanthias, avian salt glands and, 163, 20 1 Steroids. avian salt glands and, 191-192 Stimulus-secretion coupling. avian salt glands and, 200-205 Stvloc~ephalus.mitosis and, 22 1 -;!22 Subfornical organ, avian salt glands and, 195, I97 Substance P3avian salt glands and. 199 Succinate dehydrogenase, avian salt glands and, 145. 147 Sucrose, avian salt glands and, 183-184 Supraoptic nucleus (SON), avian salt glands and, 182. 195 Susceptibility. Arahidnpsis thaliana and, 68-69,12-73 Swelling pressure, cytoskeletal dynamics and, 89 Symbionts. chloroplasts and. 260, 295 cryptomonads, 275-276 dinoflagellates, 286. 288-289. 292-293

INDEX euglenoids, 283 green algae, 268, 270 heterokont algae, 278. 280 Symbiosis. chloroplasts and, 261, 283, 294 Synapses, avian salt glands and, 158-159 Synaptonemal complex (SC), karyosphere and, 7,40.43-45 oocytes of insects, 9. 11, 17. 21, 23 vertebrates, 36 Syncytium. mitosis and, 23 I , 234 Systemic acquired resistance, Amhidupsis thalianu and, 59. 77-78

T Taxol. mitosis and, 229, 236-237, 246 Ta.nrs hrevifiolia, mitosis and, 236 Teleosts avian salt glands and, 129 karyosphere and, 25 Temperature-sensitive mutants, mitosis and, 226-227,229-233.238,255 Tenebrionid beetles, karyosphere and, 19-25 Tensin, cytoskeletal dynamics and, 103 Tentvrianomas tuurica. karyosphere and, 19. 21 Tetraethylammonium (TEA), avian salt glands and, 175-176 Tetruhymena, mitosis and, 228 Tetrodotoxin. avian salt glands and. 157, 165 Thylakoids, chloroplasts and, 261, 263, 266, 273 dinoflagellates, 285, 288-29 1 euglenoids, 283-284 heterokont algae, 277-278 Thymidine, avian salt glands and, 143 Thyroid hormones, avian salt glands and, 199 Tipula, karyosphere and, 18 Tissue specificity, Arabidopsis rhaliana and. 70 Tobacco, plant-pathogen interactions in, 7 I Tolerance, Aruhidnpsis thulianu and. 7 1, 76 Tomato, plant-pathogen interactions in. 72, 74 Tonicity. avian salt glands and, 157. 177-185 Topoisomerase. mitosis and, 227 Tracheloraphis totevi. karyosphere and, 36 Traction, cytoskeletal dynamics and, 89-91 Transcription Arahidopsis thaliuna and. 59.73. 77 avian salt glands and, 192 karyosphere and, 44-45

INDEX

317

oocytes of insects, 18-19, 23 vertebrates, 26-27. 38 Translocation cytoskeletal dynamics and, 89. 100, 110, 118 mitosis and, 220 Transmission electron microscopy, avian salt glands and, I38 Tridihexethylchloride, avian salt glands and, 162 Triricum, mitosis and, 225 Trypanosoma, chloroplasts and, 284 Tubulin cytoskeletal dynamics and, 109 mitosis and, 227. 235, 238 Turnip crinkle virus (TCV). Arahidopsis thalrana and, 70 Turnip yellow mosaic virus (TYMV). Arahidopsis rhalrana and, 70-7 I Turtles, avian salt glands and, 147, 161, 17 1, 188

innervation, 159, 162-163, 165, 167 secretory mechanism, 169, 172 cytoskeletal dynamics and, 97-98, 100, 1 I I karyosphere and, I , 19. 38.44. 46-47 mitosis and, 219,234,245, 247 Villin, cytoskeletal dynamics and, 107 Vinblastine, mitosis and, 235 Vincristine. mitosis and, 235 Virulence. Arahidopsis thaliana and, 54, 57, 67, 69, 73. 75 Viruses, Arahidopsis thaliana and, 69-7 1 Vittelogenesis, karyosphere and, 15, 21

X

Uromasryx acanthinurus, avian salt glands and, 139, 147 Urrrhu. avian salt glands and, 130

Xanthomonas, plant-pathogen interactions in, 57 Xanthomonas campestris, plant-pathogen interactions in. 66, 69, 75 Xanthophylls, chloroplasts and, 265, 282, 289-290 Xanthophytes, chloroplasts and, 277-278, 282 Xenopus karyosphere and, 45 mitosis and, 218, 250 Xenopus laevis, karyosphere and, 30, 40

v

Y

U

Vacuoles chloroplasts and, 261, 266, 270, 283, 292 cytoskeletal dynamics and, 98 Vascular volume, avian salt glands and. 185-187 Vasoactive intestinal polypeptide (VIP), avian salt glands and, 162-1 67, 197, 201 Vasoconstriction. avian salt glands and. 154 Veratridine, avian salt glands and, 165 Verkbrdtes, karyosphere and, 25-27 anurans. 27-36 birds, 37-38 mammals. 38-39 reptiles. 36-37 Vesicles avian salt glands and, 144

Yeast cytoskeletal dynamics and, 97 mitosis and chromosomes, 245, 247-248, 256 dissociation of events, 218, 226-228, 232, 237 Yeast artificial chromosomes (YAC), Arahidopsis thaliana and, 63, 67

Z Zonulae adherentes, avian salt glands and, 135, 138-139 Zonulae occludentes. avian salt glands and, 135, 138-139

ISBN 0-12-364547-6