International Review of Cytology: A Survey of Cell Biology, Volume 114

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME 114

SERIES EDITORS GEOFFREY H. BOURNE JAMES F. DANIELLI KWANG W. JEON MARTIN FRIEDLANDER

1949-1988 1949-1984 19671984-

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN DEAN BOK GARY G. BORISY BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DIBERARDINO DONALD K. DOUGALL BERNDT EHRNGER CHARLES J. FLICKINGER NICHOLAS GILLHAM M. NELLY GOLARZ DE BOURNE YUKIO HIRAMOTO MARK HOGARTH

KEITH E. MOSTOV AUDREY MUGGLETON-HARRIS ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC W. J. PEACOCK LIONEL I. REBHUN JEAN-PAUL REVEL L. EVANS ROTH JOAN SMITH-SONNEBORN WILFRED STEIN RALPH M. STEINMAN HEWSON SWIFT ALEXANDER L. YUDIN

INTERNATIONAL

Review of Cytology A SURVEY OF CELL BIOLOGY

Editor-in-Chief

G. H. BOURNE (Deceased)

Editors

K. W. JEON

Department of Zoology University of Tennessee Knoxville. Tennessee

M. FRIEDLANDER Jules Stein Eye Institute UCLA School of Medicine Los Angeles, Calrornia

VOLUME 114

ACADEMIC PRESS, INC. Harcourt Brace Jovnnovich, Publishers

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0 1989 BY ACADEMIC PRESS.

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Contents CONTRIBUTORS ................................................................

ix

Fertilization in Paramecium: Processes of the Nuclear Reorganization K . HIWATASHI AND K . MIKAMI I. I1 . I11. IV. V VI .

.

Introduction .......................................................... Processes of Conjugation and Nuclear Reorganization in Ibmmecium ........ Nuclear Activation and Meiosis ......................................... Exchange of Gametic Nuclei and Formation of Synkaryon .................. Differentiation of Germinal and Somatic Nuclei ........................... Summary and Perspectives .............................................. References ............................................................

1 2 4 7 9 16 18

Characteristics of Microtubules at the Different Stages of Neuronal Differentiation and Maturation VINCENT MEININGER AND STEPHANE BINET

.

I I1. 111. IV

.

Introduction .......................................................... Microtubules during Neurogenesis: Morphological Analysis ................. Microtubules during Neurogenesis: Biochemical Analysis ................... Summary and Conclusions .............................................. References ............................................................

21 28 51 69 71

Generation of Cell Diversity during Early Embryogenesis in the Nematode Caenorhabditis elegans SUSANSTROME

I . Introduction

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

11. Analysis of Cell Fate Determination by Embryo Manipulation

111. The Generation of Zygotic Asymmetry and Partitioning of Maternal Components to the Early Blastomeres ......................... V

81 89

99

vi

CONTENTS

1v. Genetic Approaches to Analyzing Early Development ...................... V. Summary .............................................................

VI . Future Perspectives .................................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

112 118

i19 121

Uracil-DNA Glycosylases and DNA Uracil Repair N . V. TOMILIN A N D 0. N . APRELIKOVA I . Introduction If . I l l.

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

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

I v.

V. Physiological Variations of UDG Activity and lntracellular dUTP Pool . . . . . . . VI . Biological Role of DNA Uracil Repair-Speculations and Discussion ......... V1I . Conclusions ........................................................... References ...........................................

125 127 134 I47 154 164 170 171

Age-Related Changes in DNA Methylation: Do They Represent Continued Developmental Changes? LAURA L. MAYS-HOOPES 1. Introduction ......................... .............................. 11. Methylation of the Overall Genome ......................................

............... Ill . Methylation of Highly Repetitive Sequences ........... 1v. Methylation of Proviral and Interspersed V. Methylation of X-Linked Genes .............. ........... V I . Methylation of Globin Genes ........................................... VII . Methylation of the Chicken Vitellogenin I1 Gene . . . . . VIII . a-Fetoprotein and Albumin ............................................. IX. Methylation of Several Other Genes in D X . Final Synthesis and Conclusions ......................................... References ............................................................

181

184 189 191 197

201 205 208 210 213 214

Epithelial-Capillary Interactions in the Eye: The Retinal Pigment Epithelium and the Choriocapillaris GARVE . KORTE. MARGARET S. BURNS.AND ROYW. BELLHORN

I . lntroduction

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

11. Histologic Evidence of Epithelial-Capillary Interactions .................... 111. Epithelial-Capillary lnteractions in the Eye ...............................

I v. Mechanisms of RPE-Choriocapillaris Interactions ......................... V . Conclusion ........................................................... References ............................................................

221 221 224 239 244 244

CONTENTS

vii

Dinoflagellate Sexuality Lois A . PFIESTER

.

I I1. I11. IV. V.

.

VI VII .

Introduction .......................................................... Selected Life Cycles .................................................... Nuclear Phenomena ................................................... Cyst Formers versus Non-Cyst Formers ................................... Environmental Control of Sexuality ...................................... Sexuality: Its Function and Significance .................................. Future Research ....................................................... References ............................................................

249 254 262 267 268 269 270 270

Water Exchange through the Erythrocyte Membrane GHEORGHE BENGA I . Introduction .......................................................... I1. Osmotic and Diffusional Permeability of Red Blood Cells .................. 111. Characterization of Diffusional Water Permeability in Human RBC and Ghosts ............................................. IV. Conditions for Inhibition of Water Diffusion in RBC and Ghosts ........... V. Uptake and Binding of ['"'HglPCMBS by RBC ........................... VI Identification of Membrane Proteins Involved in the Water Permeability of Human RBC ................................ VII . Electron-Microscopic Studies ............................................ VIII . Alterations of Water Permeability of Human RBC in Disease Processes ................................................... IX. Conclusions on the Mechanisms of Water Exchange in RBC ................ References ............................................................

273 274

INDEX .......................................................................

317

.

279 290 296 299 304 307 309 313

This Page Intentionally Left Blank

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

0. N . APRELIKOVA (125), Laboratory of Chromosome Stability, Institute of Cytology, Academy of Sciences of the Union of Soviet Socialist Republics, 194064 Leningrad, USSR ROYW. BELLHORN (221), Department of Surgery, University of California at Davis, School of Eterinary Medicine, Davis, California 95616 GHEORGHE BENGA(273), Department of Cell Biology, Medical

and Pharmaceutical Institute Chj-Napoca,Faculty of Medicine, 3400 Cluj-Napoca,Romania STEPHANE BINET(21), Laboratoire GYnatomie, UER Biomkdicale des Saints-Peres et Bmussais-Hdtel-Dieu,F-75270 lhris Cedex 06, France MARGARETS. BURNS(221), Department of Ophthalmology, University of Calfornia at Davis, School of Medicine, Davis, California 95616 K. HIWATASHI (l), Biological Institute, Tohoku University, Sendai 980, Japan LAURAL. MAYS-HOOPES (181), Department of Biology, Occidental College, Los Angeles, Calfornia 90041 GARYE. Kom (221), Department of Ophthalmology, MonteBore Medical Center and Albert Einstein College of Medicine, Bronx, New York 10467 ix

X

CONTRIBUTORS

VINCENT MEININGER(2 l), Laboratoire dxnatomie, UER

Biomddicaledes Saints-Pkreset Broussais-Hatel-Dieu,F-752 70 Paris Cedex 06, France K . MIKAMI (l), Research Institutefor Science Education, Miyagi University of Education, Sendai 980, Japan LOISA. P FIESTER (249), Department of Botany-MicrobioIogy, University of Oklahoma, Norman, Oklahoma 73019 SUSANSTROME(81), Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, Indiana 4 7405 N . V. TOMILIN (129, Laboratory of Chromosome Stability, Institute of Cytology, Academy of Sciences of the Union of Soviet Socialist Republics, I94064 Leningrad, USSR

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 114

Fertilization in Paramecium: Processes of the Nuclear Reorganization K. HIWATASHI" AND K. M I K A M I ~ *Biological Institute, Tohoku University, Sendai 980, Japan and ?Research Institute f o r Science Education, Miyagi University of Education, Sendai 980, Japan

I. Introduction

The process of fertilization in Paramecium is usually called conjugation. Conjugation of Paramecium is a biological phenomenon that has attracted many investigators since the organism was discovered (see Wichterman, 1986). Descriptions of cytological details of the process of conjugation in Paramecium were almost complete by the end of the last century (Hertwig, 1889; Maupas, 1899; Calkins and Cull, 1907). Modern experimental work on conjugation in Paramecium, however, began with the discovery of mating types by Sonneborn (1937). This discovery not only gave a clear proof that conjugation in Paramecium results in true fertilization, a process leading to genetic recombination, but also made conjugation in Paramecium a subject of reproducible and orderly manipulations for modern studies in cell biology. Since the first review on analytical studies on conjugation in Paramecium was published by Metz (1954), sexual cell interactions in Paramecium have been reviewed by many authors (Hiwatashi, 1969, 1981; Nanney, 1977; Miyake, 1978; Cronkite, 1980; Hiwatashi and Kitamura, 1985). In those reviews, however, the main emphasis is often put on the cell-cell interactions during the mating process. Nuclear phenomena during the sexual phase of ciliates including Paramecium have been reviewed at the structural and ultrastructural levels by Raikov (1982), and at the molecular level by Steinbriick ( 1986). Nuclear phenomena in Paramecium, however, have not been treated extensively, probably because only a few nuclear phenomena had been studied in any depth. Many advances in the study of nuclear changes during conjugation of Paramecium, especially using the nuclear transplantation technique, have made possible the discovery of much interesting new evidence. This review will focus on nuclear phenomena, especially of germinal micronucleus, during and after conjugation in Paramecium. I Copyright Q 1989 by Academic Press. Inc.

All rights of reproduction in any form reserved.

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K. HlWATASHl A N D K . MIKAMI

11. Processes of Conjugation and Nuclear Reorganization in Paramecium

Processes of conjugation including nuclear events have been described (Sonneborn, 1947; Hiwatashi, 1969, 1981; Miyake, 1980; and others), but a summary of the major nuclear events may be useful for understanding the rest of this review. Although the series of nuclear changes in conjugation varies among species of Paramecium, certain basic events are common and can be described as seven sequentially occurring processes according to Miyake (1981): (1) meiosis, (2) formation of haploid gametic nuclei. (3) exchange of gametic nuclei between mates, (4) formation of diploid synkaryon, ( 5 ) division of synkaryon, (6) differentiation of new micro- and macronuclei. and (7) fragmentation and degeneration of the old macronucleus. Purumecium, like other ciliate protozoans, has two different nuclei: somatic macronucleus and germinal micronucleus. It is the latter that undergoes meiosis during conjugation. By mixing highly mating-receiving cells of complementary mating types, we can easily obtain a large number of conjugating cells in which meiosis occurs nearly synchronously. Thus, Purumecium cells offer good material for analyzing the process of meiosis experimentally. In the meiotic prophase of Purumecium, the micronucleius forms a conspicuous structure called the “crescent,” where the spherical micronucleus elongates and forms a large spindlelike structure. This stage was once thought to be comparable to the bouquet stage (zygoten stage) in the meiosis of higher organisms (Raikov, 1982), but later studies showed that the “crescent” stage in Paramecium is more likely the pachytene or even the diplotene stage. The meiosis consists of two successive nuclear divisions as in other organisms, and the first division is heterotypic (Sonneborn, 1947). Meiosis produces four haploid nuclei in an exconjugant cell in P. cuirduturn and P. bursariu, 8 nuclei in P. uureliu complex, and 16 or more in P. multimicronuclecztirm. In any case, only one of those meiotic products enters the paroral region and survives, but the other products degenerate and are resorbed. The survival versus degeneration of meiotic products provides an interesting basic problem and will be discussed in detail later. The surviving meiotic products undergo a mitotic division, producing migratory “male” and stationary “female” pronuclei. The migratory pronucleus penetrates into the partner cell and fuses with the stationary pronucleus there, forming diploid synkaryon. During or immediately after the formation of synkaryon, the conjugating pairs separate. The synkaryon divides two or three times depending on the species, and the division products differentiate into new micro- and macronuclei. What controls

FERTILIZATION IN Paramecium: NUCLEAR REORGANIZATION

3

FIG. I . An exconjugant soon after mating-pair separation. A synkaryon divides and gives rise to four nuclei. The old macronucleus (Ma) begins to transform from ovoid shape to ribbonlike structure.

the differentiation of the same mitotic products into two completely different nuclei within a same cell is another interesting problem in nuclear events in conjugation of Paramecium and other ciliates. This problem will be discussed later in detail. In exconjugants of Paramecium except for P . bursaria, the old macronucleus transforms to a thick ribbonlike structure called “skein” and then fragments into many pieces. Figures 1-3 depict exconjugate cells stained by Feulgen reaction and counterstained with fast green. Old ma-

FIG.2. Critical stage of the nuclear determination. Four nuclei are localized at the anterior region of the cell and the other four at the posterior region. The old macronucleus (Ma) is at the stage of skein formation.

4

K. HlWATASHl A N D K. MlKAMl

FIG.3 . Development of macronuclear anlagen (An). Four macronuclear anlagen which are stained weakly by Feulgen reaction and well with fast green are observed in an exconjugant. The old macronucleus (Ma) fragments into small pieces.

cronuclear fragments usually degenerate and are resorbed. In some cases, especially when new macronuclear primordia (usually called macronuclear anlagen; the singular is anlage) are damaged or lost, fragments of old macronucleus regenerate and become a complete macronucleus. This phenomenon is called macronuclear regeneration (Sonneborn, 1947), abbreviated as MR. Macronuclear regeneration never occurs when exconjugants contain healthy macronuclear anlagen. This suggests that DNA synthesis in the old macronucleus is controlled in some way by the macronuclear anlagen. Thus, differentiation of new macronuclear anlagen versus regeneration of old macronuclear fragments provides us an intereting problem on the regulation of DNA synthesis within a single cell. Nuclear events seen in the course of conjugation in Pummecirrm involve many important and interesting problems of basic cell biology, and in the following section we will review how these problems have been approached and what answers have been obtained.

Ill. Nuclear Activation and Meiosis A. EARLY MICRONUCLEARMIGRATIONA N D COMMITMENT TO NUCLEARREORGANIZATION

When mating-reactive cells of complementary mating types are mixed. the earliest nuclear change observed in the cells is the early micronuclear migration (EMM) (Fujishima and Hiwatashi, 1977). In vegetative cells of Pnruriiecirrm cuirdatirm, the micronucleus usually rests in a concavity of

FERTILIZATION IN Paramecium: NUCLEAR REORGANIZATION

5

the macronucleus. At meiosis, the micronucleus is always at some distance from the macronucleus, so that before the start of meiosis the micronucleus detaches from the macronucleus (Calkins and Cull, 1907; Wichterman, 1940). This detachment of micronucleus from the macronucleus was found to occur as early as 10-15 minutes after the onset of the mating reaction and was called the EMM. The EMM occurs only in the cells ready to conjugate and when cells are activated for conjugation. Thus, EMM is observed not only when mating-reactive cells of complementary mating types are mixed and induced to agglutinate, but also when cells are stimulated for conjugation by conjugation-inducingchemicals (Miyake, 1968). The EMM never occurs when mating-incompetent cells in the period of sexual immaturity are stimulated by the conjugation-inducing chemicals (Fujishima and Hiwatashi, 1977). Cronkite ( 1979) succeeded in inducing EMM when mating-reactive cells of P. cuudutum were cooled in an ice bath for 60 minutes and then rapidly warmed to room temperature. Since cooling parameciums in an ice bath is known to allow Ca” accumulation inside the cell (Browning and Nelson, 1976), Cronkite interpreted his results to be due to one of two alternate mechanism:

1 . The accumulation of Ca” may induce EMM, but in the cold, energy may not be available for the EMM. Return to room temperature would make possible an already triggered EMM to take place. 2. Not the accumulation of Ca2’ itself but the increase of Ca” in cold temperature followed by the decrease upon returning to room temperature may trigger the EMM. He induced EMM also by treating cells with the calcium ionophore A23187. Sexually immature cells did not respond either to the cold treatment or to ionophore with EMM. Those results clearly show that the EMM is a phenomenon intimately related to activation of cells for conjugation and suggests that concentration of free Ca” inside the cell has an important role in the activation process. The EMM, however, does not irreversibly commit cells to meiosis, because when cells once activated for conjugation were prevented from proceeding to conjugating union, the micronucleus in the EMM returned to the concavity of the macronucleus (Fujishima and Hiwatashi, 1977). EMM seems to have a close relationship with the strong cyclosis which occurs immediately after mating reaction (M. Fujishima, unpublished). B. INDUCTION OF MEIOSIS

When complementary mating types of P. cuudutum are mixed, mating agglutination continues for about an hour and then the holdfast unions,

6

K . HIWATASHI A N D K . M l K A M l

pairs united at the anterior ends, are released from the agglutinates. Until this stage. micronuclei cannot be microscopically distinguished from those in the cells of stationary phase and are called stage I micronuclei. In 2 hours after mixing mating types, cells unite much more tightly at their paroral regions. These tight unions are usually called the paroral unions. The process is considered to be under the control of the macronucleus, since amacronucleate cells retain mating reactivity for a few hours after removal of the macronucleus but never proceed to paroral union (K. Mikami, unpublished). In this stage, the micronucleus swells to three to four times larger in diameter and is called the stage I 1 micronucleus. This is the stage when the micronucleus undergoes premeiotic DNA synthesis. The premeiotic DNA synthesis in this stage of P. cuuduturn was confirmed both by microspectrophotometry and by autoradiography with 'H-dUMP (Fujishima, 1983). A characteristic feature of the DNA synthesis in this stage is that it occurs exclusively in the micronucleus without concomitant occurrence in the macronucleus, while concomitant occurrence in both nuclei is the rule in premitotic DNA synthesis. How only one of the two nuclei in a cell can undergo DNA synthesis is unknown. However, an interesting difference between premitotic and premeiotic DNA syntheses was reported by Fujishima (1983). His experiments showed that in the premitotic DNA synthesis deoxythymidylic acid is formed both by the de novo and salvage pathways, but in the premeiotic DNA synthesis mostly by the de novo pathway. This suggests that the activity of thymidine kinase is low in cells in the early conjugating stage.

C. SURVIVAL VERSUS DISINTEGRATION OF MEIOTICPRODUCTS Positional information seems to be very important for nuclear differentiation during a sexual cycle. After meiosis, only one of the four nuclei produced remains as a gametic nucleus while the other three degenerate. In this nuclear process of survival versus disintegration, the intracellular position of the micronucleus plays an important role, since the surviving nucleus always lies in the special region of the cytoplasm called the paroral region, which is the region around the cytostome. The nucleus in the paroral cone is known to attach to the surface membrane (Hertwig, 1889; see Sonneborn. 1954). The nucleus seems to be anchored to the membrane directly or by other structural components, because the nucleus in the paroral region was difficult to remove by micropipetting although removal wits easy for nuclei outside the region (Yanagi. 1987). On the other hand, the remaining three which lie outside the region always degenerate (Wichterman, 1940; Sonneborn, 1954; Skoblo and Ossipov, 1968). Sonneborn and colleagues showed that in an abnormal strain of P . ciurelici all mi-

FERTILIZATION IN Paramecium: NUCLEAR REORGANIZATION

7

cronuclei produced at meiosis invariably degenerate and disappear, instead of all but one. In this abnormal strain, nuclei never got into the paroral cone and thus did not attach to the surface membrane (Sonneborn, 1954). The interpretation on the cause of survival versus degeneration of nuclei has been that the cytoplasm or the surface membrane of the paroral cone differs from the rest of the cell, and this difference determines survival or degeneration of the nuclei. Yanagi (1987), however, pointed out that this interpretation is a bit too simple and naive. When he transplanted the nucleus in the early conjugating cells (stage 11) into the cell at the stage when three meiotic products were degenerating, it did not degenerate but divided. On the other hand, when one of the meiotic products lying outside the paroral region and thus destined to degenerate was transplanted into the cell of meiotic prophase (stage IV), it did not survive but degenerated. When the surviving nucleus in the paroral region was removed microsurgically, one of the three nuclei lying outside the paroral region and destined to degenerate moved into the paroral region and survived. From these results, he concluded that the nuclei after meiosis were destined to degenerate but could be rescued from degeneration by the special environment of the paroral region (Yanagi, 1987). How one of the haploid micronuclei migrates to the particular region of the cell has not been fully analyzed. A possible interpretation is that the micronuclei are moving with cyclosis and one of them is accidentally trapped by and occupies the region when it comes close. The degeneration of micronuclei must be under the control of the macronucleus like the other micronuclear phenomena. When the macronucleus is eliminated at early conjugational stages in P. cuudutum no micronuclei degenerate, but when it is removed after the first meiotic division all of the micronuclei degenerate (K. Mikami, unpublished). The evidence implies that some information directing the disintegration of micronuclei is given from the macronucleus before the first meiotic division.

IV. Exchange of Gametic Nuclei and Formation of Synkaryon The nucleus surviving at the paroral cone divides once and produces a migratory nucleus and a stationary nucleus. The migratory nucleus lies close to the boundary of mating cells and anchors tightly to the cortex or the membrane. Therefore, it is not easy to remove the nucleus in this stage by micropipette (K. Mikami, unpublished). What is the mechanism of nuclear migration to the partner cell? The apertures of intercytoplasmic communication between mating partners are present nearly all through the united zone, including the paroral zone where the pronuclei migrate

8

K . HlWATASHl AND K . MlKAMl

(Vivier. 1974). Sections viewed with the electron microscope show many openings of maximum 0Spm in diameter, at various points of cell-cell junction between the mates. The openings may be able to vary in length and seem to function as passages for the migratory nucleus. In electron micrographs of P. mirlrimicronucleatum, Inaba er al. ( 1966) observed pronucleus migrating through the boundary. In order for the nucleus to pass through the openings, some driving apparatus would be necessary. In Terrahymena, Orias et al. (1983) revealed a basketlike structure, consisting of a meshwork of microtubules, associated with each migratory pronucleus. This microtubule meshwork is thought to be functioning in the transfer of gametic pronuclei across the junction. Numata ct a / . (1985) reported an interesting finding in Tetrahvmena that a protein which resembles the intermediate-filament proteins from mammalian cells is involved in several nuclear events in conjugation, including production of four haploid nuclei by prezygotic divisions (meiosis), selection of one surviving nucleus from the four meiotic products. formation of the gametic pronucleus by the mitotic division of the surviving meiotic product, transfer of the gametic pronucleus across the cell-cell junction, and zygote formation by fusion of pronuclei. They speculated that the intermediate-filament protein has a crucial role in the transfer of gametic pronuclei across the cell-cell junction in association with the microtubular meshwork. Some genetic information necessary for the migration of the gametic nuclei may be supplied from the macronucleus at some stage between the first and second meiotic division (K. Mikami, unpublished). When the whole macronucleus was eliminated soon after the first meiotic division of the micronucleus, micronuclei underwent the second meiotic division, and then one of the four division products migrated into the paroral region and divided, but none of the division products migrated into the partner cell. The evidence shows that the macronucleus is indispensable for sending the migratory nucleus into the partner cell. On the contrary, the macronucleus is unnecessary for receiving the migratory nucleus from the partner. When both the macronucleus and the micronucleus were removed by micropipetting from one of the conjugating cells at the stage of micronuclear crescent, the gametic nucleus of the normal partner migrated into the anucleate cell (K.Mikami, unpublished). How does the migratory nucleus recognize the stationary nucleus in the partner cell and fuse with it? Unfortunately, there seems to be no evidence available to answer this question. A connective structure composed of intermediate-filament proteins is formed between migratory and stationary nuclei after reciprocal exchange of the migratory nuclei in Tetrahymena (Numata et al., 1985). Jurand (1976) reported associations be-

FERTILIZATION I N Purumeciuni: NUCLEAR REORGANIZATION

9

tween microtubules and a migratory nucleus in Paramecium. Mechanism of the exchange of gametic nuclei and the formation of synkaryon in Paramecium may be in some way similar to those discovered in Tetrahymena.

V. Differentiation of Germinal and Somatic Nuclei A. POSITIONAL CONTROL OF MACRONUCLEAR DIFFERENTIATION New macro- and micronuclei develop from division products of a synkaryon. Two working hypotheses on the mechanism of nuclear differentiation can be proposed (Raikov, 1982): (1) the specific cytoplasm affects the postzygotic nuclei to determine their differentiation, and (2) the differentiation factor is not in the cytoplasm but in the nuclei themselves. Long ago, Maupas (1889) pointed out the importance of nuclear localization within the cell for nuclear differentiation. Many workers (Calkins and Cull, 1907; Egelhaaf, 1955) have supported hypothesis (1) since the work of Maupas. In Tetrahymena, centrifugation studies suggested that local cytoplasm plays a crucial role in the determination of the nucleus (Nanney, 1953). Sonneborn (1954) also presented some results against the intranuclear segregational mechanism of the nuclear differentiation in P . aurelia. Positional control of nuclear differentiation have now been clearly demonstrated by microsurgical technique in P . caudatum (Mikami, 1980) and with a mutant clone in P . tetraurelia (Grandchamp and Beisson, 1981). In P . caudatum, new macronuclear anlagen and a micronucleus differentiate after the fertilization nucleus (synkaryon) divides three times. The determination of macro- and micronuclei does not occur before the third (last) postzygotic division. This conclusion was drawn from the following results. When all nuclei but one were eliminated by microsurgery after the first or the second postzygotic division, the remaining one divided twice in the former case and once in the latter, and then differentiated macronuclear anlagen, two in the former case and one in the latter, and a micronucleus. Moreover, a microsurgical analysis revealed that the stage of determination of the two kinds of nuclei is immediately after the third (last) nuclear division. As mentioned earlier (Fig. 2), immediately after the third nuclear division, four nuclei localize near the anterior end of the cell and the other four near the posterior end for a short time (15-30 minutes at about 27°C) (Mikami, 1980). When all of the anterior nuclei were removed, exconjugants had developed a normal number of macronuclear anlagen but all clones derived from the operated exconjugants were amicronucleate (Mikami, 1982). On the other hand, no macronuclear anlagen developed when all of the posterior nuclei were removed (Mikami, 1980).

10

K. HIWATASHI A N D K. M l K A M l

If one to three of them were removed, a decreased number of anlagen developed. These experiments clearly demonstrate that the anterior nuclei are determined to be germinal micronucleus and the posterior nuclei to develop into macronuclear anlagen. Thus, the microsurgical results revealed that the determination of the nuclear differentiation occurs immediately after the last division of the synkaryon in close association with a brief localization of the nuclei near the opposite ends of the cell. In P. trtrriiireliri, Grandchamp and Beisson (1981) revealed the same conclusion from their results. obtained using mutants which form abnormal numbers of macronuclear anlagen and micronuclei. The anteroposterior localization of the daughter nuclei produced by the third division is due to the orientation of the nuclear spindles, which lie parallel to the longitudinal axis of the cell, to their extension to opposite ends of the cell, and probably also a marked shortening of the cell length (Mikami, 1980; Grandchamp and Beisson, 1981)and assembly of an array of subcortical microtubule bundles (Cohen er al., 1982). One important question here is whether ( I ) the synkaryon is undetermined “neutral” nucleus which may be determined to be micro- or macronuclei. or (2) the synkaryon itself is micronucleus and the determination is only for macronucleus. The fact that the synkaryon underwent normal mitosis and retained its ability as a germinal micronucleus when transplanted into vegetative cells (Harumoto and Hiwatashi, 1982) shows that the synkaryon is not different from a vegetative micronucleus. When the vegetative micronucleus was transplanted into an early exconjugant in P. tetraurelia, it occasionally differentiated into a new macronucleus (Mikami and Ng, 1983). The result clearly shows that the micronucleus can differentiate directly into a macronucleus without going through a series of nuclear events of meiosis, mitosis, and synkaryon formation. Thus, vegetative micronucleus, synkaryon, and postzygotic division products have the same competence and can be altered by the stage-specific cytoplasmic environment for macronuclear anlagen. Do the cytoplasmic environments act directly upon nuclear determination or do they induce a critical nuclear division resulting in the production of the different nuclei‘? In P. caudurum, there is no intrinsic difference between nuclei localized anteriorly and those situated posteriorly. Both were found to retain their nature as germinal micronuclei when they were implanted into vegetative cells (Mikami, 1985). When one of the posterior nuclei was transplanted into an amicronucleate cell at vegetative phase, the nucleus was able to divide at every fission of the recipient cell and after several fissions had DNA content nearly equal to or less than ordinary micronuclei. When such heterokaryons were then conjugated with amicronucleates, macronuclear anlagen developed from the division products of the implanted nuclei and thereafter the caryonides derived

FERTILIZATION IN Paramecium: NUCLEAR REORGANIZATION

11

from the conjugation were true to express the marker gene of the implanted nuclei (Mikami, 1985). Thus, the nuclei localized posteriorly after the third postzygotic division retain functions of the micronucleus, and up to this stage they are not committed irreversibly to becoming a macronucleus. Therefore, the third nuclear division itself does not produce the nuclear difference but merely transports the daughter nuclei into cytoplasmic or cortical environments differentiated anteroposteriorly, and some determinants in the posterior region of the cell direct the nuclei toward macronuclear anlagen. The area where the nuclei are determined to be macronuclear anlagen is not confined to the extremely posterior end of the cell but covers rather a large posterior area, because in P . caudatum, the nuclei immediately after the third division often localize not in the end of the cell but in a relatively interior part of the cell when the oval shape of the cell is viewed from the side (Mikami, 1980). In P . tetraurefia,an amputation of the posterior one-third of the cell made half an hour before the polar positioning of the nuclei inhibited macronuclear differentiation, while similar amputations made 2 hours before the polar positioning did not inhibit the differentiation (Grandchamp and Beisson, 1983). What, then, is the anteroposterior differentiation of the cytoplasm? Is the cortex involved? The determination of the primordial germ cells in some metazoa is known to be dependent on the localized germ cell determinants, for example, in Drosophila (Mahowald et al., 1979) and in amphibians (Blacker, 1958). As mentioned before, survival of the meiotic products in the paroral cone seems to depend on its attachment to the surface membrane. However, it is not clear whether the nuclei localized in the posterior region attach to the cortical layer of the cell. Is there then any possibility that the determinants are in the endoplasm? Cyclosis in Paramecium is very noticeable during the vegetative phase. If this cyclosis remains active during conjugation as in the vegetative phase, the determinants should not be localized in the posterior part of the endoplasm. At the stage of nuclear determination, however, the cyclosis was found to calm down (K. Mikami, unpublished). Thus, the possibility that localization of the determinants is in the posterior endoplasm cannot be ruled out. As to the difference between anterior and posterior parts of the Paramecium cell, two opposite gradients of the distribution of mechanoreceptor ion channels are known in Paramecium: increasing depolarizing mechanosensitivity involving Ca” channels toward the anterior pole, and increasing hyperpolarizing mechanosensitivity involving K channels toward the posterior pole (Ogura and Machemer, 1980). On the basis of these gradients of mechanoreceptor ion channels, Gandchamp and Beisson (1983) performed an interesting experiment. They modified intracellular ionic environments using Ca” and K’ ionophores when the nuclei were

12

K . HIWAIASHI A N D K. MlKAMl

lying at the opposite poles of the cell. The K' ionophore valinomycine (in the presence of 50 mM KCI) induced an excess of macronuclear anlagen (four macronuclear. no micronuclear), while the Ca' ' ionophore A23 I87 induced a reverse effect, producing an excess of micronuclei (no macronucler anlagen. four micronuclei). From these results they concluded that polarity of the concentration gradients of Ca" and/or K' within the cell might control nuclear differentiation. When the old macronucleus of P . cuudaturn was eliminated soon after the first division of the synkaryon, the postzygotic micronuclei showed no sign of differentiation into macronuclear anlagen, although they divided once (Mikami, 1980). This shows that macronuclear gene products are necessary for determination or differentiation of the macronucleus. Berger ( 1987) reported a temperature-sensitive mutant which does not develop macronuclear anlagen in the stringent temperature. A maternal effect was observed when this mutant was crossed to wild type. This suggests that gene products of an old macronucleus are necessary for the development of the new macronucleus. However, how the gene products act only on the nuclei lying in the posterior part of the cell still remains as an open question. The number of micronuclei varies among species of Parameck4m. Paramecium c-uudatum has only one micronucleus. In the third postzygotic nuclear division of P . caudatum, however, four candidates for the micronucleus are localized in the anterior region of the cell. All of the four anterior nuclei seem to retain equal potentiality as a micronucleus. This is supported by two experiments. When any three of them were removed from exconjugants, the remaining nucleus was able to divide at each postconjugational fission and the clones derived from the cells became unimicronucleate clones (Mikami, 1982). When any one of the four nuclei which have localized near the anterior region at the stage of nuclear differentiation was transplanted to an amicronucleate cell at vegetative phase, the clones derived from the recipient cells became unimicronucleate (Mikami, 1985). So far, there is no evidence showing nucleocytoplasmic interactions in the elimination of supernumerary presumptive micronuclei. Questions remaining to be answered are how one presumptive micronucleus is chosen to survive, and at what stage the degeneration of the other nuclei is determined. B. DEVELOPMENT OF MACRONUCLEAR ANLAGENA N D FUNCTIONING OF Pwzucorrc MACRONUCLEUS

In most ciliates, including Paramecium, cytoplasmic and nuclear events during conjugation, except for stomatogenesis, are controlled by the macronucleus throughout the whole process of conjugation. (For the micro-

FERTILIZATION IN Paramecium: NUCLEAR REORGANIZATION

13

nuclear function during conjugation, see Ng, 1986; Mikami, 1988.) The nuclei determined to be macronuclear anlagen seem to grow through a sequence of developmental stages into mature (new) macronuclei under the control of a prezygotic (old) macronucleus. In the postzygotic micronuclei determined to be macronuclear anlagen in P. caudatum and P . bursaria, parts of the chromosomes are uncoiled, and other parts are condensed and partly fused, producing heterochromatic aggregates (see Raikov, 1982). In P. caudatum cultured at 27°C heterochromatic aggregates appear about 1 hour after the anteroposterior localization of the nuclei, that is, the critical stage of the nuclear determination. About 9 hours after the determination, the heterochromatic aggregates assemble into 10 granular bodies. Thereafter, the grains migrate to the central part of the anlage. In P. bursaria, the pericentromeric regions of the chromosomes are preserved in the anlage, while the chromosome arms detach and disintegrate (Schwartz, 1978). After the disappearance of chromatic aggregates, the anlage is faintly stained by the Feulgen reaction in most Paramecium species (see Raikov, 1982). In P . caudatum, the aggregates of chromsomes gradually disintegrate and disappear about 12 hours after the determination. In this stage, a Feulgen-negative area appears in the central part of the anlage. The anlage increases in volume and is stained very weakly by Feulgen reaction but well with fast green (Fig. 3). In P . tetraurefia, Berger reported the appearance of the nucleolus in this stage (Berger, 1973a). In P. caudatum, the postzygotic nucleus at the stage of the macronuclear determination has the same amount of DNA as the G, micronucleus (2C), while the mean macronuclear DNA content is about 40 times as much as that of the micronucleus. The DNA content of macronuclear anlagen does not increase during the early stages of macronuclear development. About 15 hours after nuclear determintion, when chromatic aggregates disappear completely, most of the anlagen still have the 2C DNA content. In the next stage, macronuclear anlagen increase their DNA content. Cytophotometric studies indicate that DNA increases progressively in the developing macronuclear anlagen of P . caudatum (Dupy-Blank, 1969) and P. tetraurefia (Berger, 1973a). In the latter species, four or more discontinuous rounds of DNA synthesis occur in the macronuclear anlage during the first cell cycle after conjugation (Berger, 1973a). Discontinuous rounds of DNA synthesis also occur in P. caudatum (Mikami, 1987). In most macronuclear anlagen of P. caudarum, the first duplication of DNA seems to finish about 24 hours after the determination. The amount of DNA after the duplication, however, seems to be slightly lower than twice (4C) the original 2C value (Mikami, 1987).The macronucleus of P. caudatum contains DNA of about 80C (Mikami, 1987). If the micronucleus duplicates its entire genome at each round of DNA synthesis, the content should be 64C after five rounds and 128C after six rounds, but never 80C. This par-

14

K. HlWATASHl A N D K. M I K A M I

adox of DNA content may be solved by differential replication or partial elimination of DNA (Mikami, 1987). During the development of the macronuclear anlagen, partial elimination of the micronuclear DNA has been shown in the ciliates Eidplotes, S t y lonychiu, Oxvtricha, and Tetruhymenu (Steinbruck. 1986). The macronucleus of P. hursarirr contains shortened chromosomal fragments rather than whole chromosomes (Schwartz, 1978). In this species, the DNA content of the macronuclear anlage increases initially up to 6-fold, and then decreases by half temporarily at the "achromatic" stage prior to resumption of DNA synthesis (Schwartz and Meister, 1975). When do the chromosomes of the macronuclear anlage start their transcriptional activity? In the case of the pwA gene of P. tetrurrrelici, the phenotypic expression of macronuclear anlagen occurred late in the first postconjugational cell cycle or during the first part of the second cell cycle (Berger, 1976).In P. c~uitdutum,phenotypes of the genes +"'"'and were expressed very early during the first round of DNA synthesis after the macronuclear determination. Since the gene action was observed by phenotypic expressions, considering the time lag necessary for the accumulation of gene products, the genes may be transcribed even before the first round of DNA synthesis. Hence, at least with regard to these genes, a certain chromosomal or intranuclear reorganization from the genetically inert micronuclear type to the transcriptionally active rnacronuclear type must occur by the first round of DNA synthesis (Mikami, 1987). +If'"-'

C. MACRUNUCLEAR REGENERATION (MR) OR NUCLEAR R EORGA N IZ A T ~ O N ( N R ) During or soon after conjugation in Purumerium except for P . birrsaria, the old macronucleus comes loose into a ribbonlike form (skein formation) and then fragments into dozens of small nuclei. The fragmented nuclei disintegrate eventually after the development of macronuclear anlagen. The time of macronuclear disintegration varies among species. In P. fetruiireliu, autolysis of the fragments is more rapid in starved exconjugants than in well-fed ones (Berger. 1973a. 1974). In P. cnirdutum the fragments persist for a long time even in starved exconjugants (Mikami, 1979). In the ordinary process of conjugation, the old macronuclear fragments eventually cease their function and are absorbed in the cytoplasm, and new macronuclear anlagen develop to mature macronuclei. When normal macronuclear anlagen are not formed in the cell after conjugation, however. the old macronuclear fragments regenerate. This is called macronuclear regeneration (Sonneborn. 1947) and abbreviated as MR. On the

FERTILIZATION IN Paramecium: NUCLEAR REORGANIZATION

15

other hand, ordinary development of macronucleus from the macronuclear anlage is called NR (nuclear reorganization). The skein formation and fragmentation of the old macronucleus do not necessarily mean its irreversible change to disintegration. In the old fragmented macronuclei, nucleoli are actively formed soon after conjugation (Egelhaaf, 1955, in P. bursaria; Jankowski, 1966, in P. putrinum; Berger, 1973a, in P. tetraurefia),and the rate of RNA synthesis per unit volume of the nucleus is much greater in the fragmented macronuclei than in the anlagen in P. aurefia (Berger, 1973a). Moreover, the beginning of DNA synthesis in the old macronuclear fragments is recognized late in the first postconjugational cell cycle in the ordinary NR process of P. caudatum. In the second or third postconjugational cell cycles, the DNA synthesis is as active as that in the macronuclear anlagen, so that the amount of DNA in the fragments doubles during the second cell cycle after conjugation (Mikami, 1979). Thus, the fragmented macronuclei are capable of functioning as a macronucleus and so can regenerate into a complete macronucleus even several fissions after conjugation. In P. caudatum, the induction of MR by removal of macronuclear anlagen is possible until the third cell cycle after conjugation. Degeneration of the fragmented macronuclei has been known to depend on the presence of new macronuclei or macronuclear anlagen (Sonnebom, 1940, 1947; Berger, 1974). DNA synthesis in old macronuclear fragments of P. caudatum is not yet depressed until the third cell cycle after conjugation, but is depressed at the fourth or fifth cell cycle. If the macronuclear anlagen is pipetted out of the cell, the rate of DNA synthesis in the macronuclear fragments starts to increase. This suggests that the macronuclear anlagen is inhibiting DNA synthesis in the fragments. In P. tetraurelia, Berger (1973b) injected cytoplasm taken from NR cells into MR cells and succeeded in inhibition of the DNA synthesis in the fragment nuclei. From this result, he proposed a hypothesis that degeneration of the old fragmenting macronuclei is a result of action of a specific inhibitor of DNA synthesis released from the developing macronuclear anlage. In P. caudatum, when cells in the second postzygotic cell cycle were kept in a nonnutrient medium, the macronuclear anlage grew predominantly and growth of macronuclear fragments was suppressed. When cells were supplied with sufficient nutrients, however, both kinds of nuclei grew together (Mikami, 1979). This result suggests competition for a limited pool of DNA precursor between macronuclear anlage and the fragmented macronuclei. There is other evidence to show this competitive relationship. When the cells lack macronuclear anlagen, the rate of [3H]thymidineincorporation into regenerating macronuclear fragments was greater than that into macronuclear anlage in normal cells with both kinds of nuclei.

16

K. HlWATASHl A N D K . MlKAMl

A similar result has been reported in P . tetraurelia (Berger, 1973b). Macronuclear anlagen seem to be superior to macronuclear fragments with regard to the utilization of the limited precursor. The suppression of DNA synthesis in the old macronuclear fragments could be explained by this competition hypothesis, because if new anlagen are superior to the old fragments in the utilization of the precursor, the latter would be deprived by the new anlagen of the limited DNA precursor. Why, then, does the suppression of DNA synthesis in the fragments occur even under conditions of sufficient supply of nutrient? If there is a limitation of the rate of DNA precursor supply to nuclei even in the condition of sufficient nutrient and the macronuclear anlage has a high demand for the precursor, DNA synthesis in the old macronuclear fragments would be eventually depressed. Nevertheless, there remains the question of the mechanism of the superiority of the new anlage in the precursor utilization. In P. caudatum, the existence of the inhibitor as reported in P. tetraurdiu by Berger has not been proven by microinjection of the cytoplasm (K.Mikami, unpublished). However, once the suppression of DNA synthesis occurs. macronuclear fragments can hardly regenerate if the anlagen are removed (Mikami, 1979). This irreversibility of the suppression observed in P. cauduturn seems to favor the inhibitor hypothesis. We have no hypothesis to explain the mechanism of selective disintegration of the old macronuclei.

VI. Summary and Perspectives The process of nuclear reorganization during conjugation in Parumrcium provides an excellent system for the study of nuclear behavior and nuclear differentiation. Since conjugation of Paramecium can be induced simply by mixing cells of complementary mating types and the sequence of nuclear changes proceeds almost synchronously in induced conjugating pairs, it provides u s with a reproducible and easily manipulatable system for the study of nuclear behavior. In conjugation of Paramecium. the processes of nuclear change can be grouped into three categories: ( I ) migration of the nucleus from one place to the other, (2) disintegration of some nuclei and survival of others, and (3) degradation of DNA in some nuclei and synthesis in others. Migration of nuclei is observed in the EMM, in the migration of one meiotic product to the paroral region and in the migration of pronucleus to the partner cell. Evidence for the involvement of microtubules in those nuclear migrations is being accumulated, but almost nothing is known about what controls the direction of migration. These phenomena in ciliate

FERTILIZATION IN Paramecium: NUCLEAR REORGANIZATION

17

nuclear behavior share a common problem with directed movement of sperm pronucleus toward egg pronucleus in the fertilization of metazoan eggs (Chambers, 1939; Hamaguchi and Hiramoto, 1980). Disintegration versus survival of nuclei is observed in the meiotic products and in the postzygotic products of the third nuclear division situated in the anterior portion of a cell. These processes are in some way homologous to the polar body formation in the maturation of metazoan oocytes. In the polar body formation, positions of spindles determine the survival versus disintegration (as polar body nuclei) of meiotic products. For the survival versus disintegration of meiotic products in Paramecium, the role of the position of spindle is denied but positional control is still evident, although the mechanism is unknown. For the postzygotic third-division products, what controls the survival as a micronucleus or degradation is still unknown. The positional control of macro- and micronuclear differentiation has the same biological meaning as the determination of the germ cell by local cytoplasmic determinants in insect and amphibian eggs (Mahowald et al., 1979; Blacker, 1958). In insect and amphibian eggs, germ cell determinants have already been isolated (Davidson, 1986; Okada and Kobayashi, 1987). In germ line soma determination in Paramecium, an ionic mechanism has been suggested but has not yet been proven. Differential control of DNA synthesis among three different nuclei is observed in exconjugant cells. In the micronucleus, DNA synthesis is controlled so as to keep the 2C-4C cycle of DNA amount coupled with the cell division. In the new macronuclear anlagen, DNA synthesis continues until the completion of the highly polygenomic mature macronu&us. In the old macronuclear fragments, on the other hand, DNA synthesis is suppressed and the DNA is decomposed and resorbed. How these different processes are controlled without confusion within the same cell is of great interest. Competition between the new macronuclear anlagen and old fragments for utilization of DNA precursor was suggested, but molecular mechanisms controlling the DNA synthesis and degradation in the exconjugant cells remain unclear. Molecular reorganization during nuclear differentiation in ciliate conjugation has become a hot target of molecular biologists (see Steinbruck, 1986; Brunk, 1986; Blackburn and Karrer, 1986; Yao, 1986). The main subject in the study of molecular reorganization of ciliate nuclei is to discover the molecular difference between a genically inert micronucleus and genically active macronucleus and how that difference is brought about. While Tetrahymena and hypotrichous ciliates are mostly used for these molecular studies, Paramecium should be used also, since it has the longest history in the study of nuclear reorganization during conjugation.

18

K. HIWATASHI AND K. MlKAMl ACKNOWLEDGMENT

Part of the manuscript was prepared while K. Hiwatashi was staying in Prof. K. Heckmann's laboratory as a guest professor at the University of Munster (Federal Republic of Germany). Prof. Heckmann's hospitality is acknowledged with gratitude.

REFERENCES Berger. J. D. (l973a). Chrc~rnosoma42, 247-268. Berger. J. D. (1973b). Chronrosoma 44,3 3 4 8 . Berger. J . D. (1974). J. Protoawl. 21, 145-152. Berger. J. D. (1976). Cenrr. Res. 27. 123-134. Berger. J. D. (1987). I n t . Ciliuie Mol. Gemt. Conf:. 2nd. August 2-6. 1987. Abstr. Blackbum. E. H.. and Karrer. K. M. (1986). Annu. Rev. Genet. 20, SOI-S?l. Blacker. A. W. (1958). J. Embryo/. Exp. Morphol. 6, 491-503. Browning, J. L., and Nelson. D. L. (1976). Narurc (London) 259, 491-494. Brunk. C. F. (1986). I n r . Re%..Cvrol. 99, 49-84. Calkinr. G . N., and Cull. S. W. (1907). Arch. Protisfenkd. 10, 375415. Chambers. E. L. (1939). J. Exp. Bio/. 16, 409-424. Cohen. J.. Adoutte. A,. Grandchamp. S.. Houdebine. L.-M., and Beisson, J. (1982). Biol. CoN. 44, 33-44. Cronkite, D. L. (1979). I n "Biochemistry and Physiology of Protozoa" ( M . Levandowsky and S. H. Hunter. eds.), 2nd ed., Vol. 2. pp. 221-273. Academic Press, New York. Davidson. E. H. (1986). "Gene Activity in Early Development.'' 3rd ed. Academic Press. Orlando. Florida. Dupy-Blank. J . ( 1%9). Proristologica 5. 239-248. Egelhaaf. A. (1955). Arch. Prurisrenkd. 100, 447-514. Fujishima. M. (1983). J. Crll Sci. 60, 51-65. Fujishima. M., and Hiwatashi. K. (1977). J . Exp. Zuul. 201, 127-134. Grandchamp, S., and Beisson, J . (1981). Dev. B i d . 81, 336-341. Grandchamp. S.. and Beisson. J . (1983). Eur. Conf. Ciliara Biol.. 5th. September 5-9. 1983. Abstr. Hamaguchi, M. S.. and Hiramoto. Y. (1980). Da r... Gruwrh Di#>r. 22, 517-530. Harumoto. T.. and Hiwatashi. K. (1982). Exp. Ce/I Res. 137, 476481. Hertwig, R. (1889). Ahh. R n w r . Akad. Wiss.. Malh.-NNtrir,c,iss. K I . 17, 151-233. Hiwatashi. K. (1y69). In "Fertilization" (C. B. Metz and A. Monroy. eds.). Vol. 2. pp. 255193. Academic Press. New York. Hiwatashi. K. (1981). I n "Sexual Interactions in Eukaryotic Microbes" (D.H. O'Day and 1'. A. Horgen. eds.). Vol. I , pp. 351-378. Academic Press. New York. Hiwatashi. K, and Kitamura. A. (1985). I n "Biology of Fertilization" (C. B. Metz and A. Monroy eds.), pp. 57-85. Academic Press. New York. Inaba. F. lmamoto. K., and Suganuma. Y. (1966).Pruc. J p n . A w d . 42, 394-398. Jankowski. A. W. (1966). T.sirolugiyu 8. 725-735. Jurand. A. (1976). J. Gen. Micmhiol. 94. 193-203. Mahowald. A. P.. Allis. C. D.. Karrer. K. M.. Underwood. E. M.. and Waring, G. L. ( 1979). In "Determinants of Spatial Organization" (S. Subtelny and I. K . Konigsberg. eds.). pp. 127-146. Academic Press. New York. Maupas. E. (1889). Arch. Zoo/. Exp. Grn. 27, 149-517.

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Metz, C. B. (1954). In “Sex in Microorganisms” (D.H. Wenrich. ed.), pp. 284-334. Am. Assoc. Adv. Sci., Washington, D.C. Mikami. K. (1979). Chromosoma 73, 131-142. Mikami, K . (1980). Dev. Biol. 80, 46-55. Mikami, K . (1982). J . Cell Sci. 56, 453-460. Mikami, K . (1985). Dev. Growth Dqfer. 27, 21-27. Mikami, K . (1987). Dev. B i d . 123, 161-168. Mikami, K . (1988). In “Paramecium” (H.-D. Gortz, ed.), pp. 120-1 30. Springer-Verlag. Berlin and New York. Mikami, K., and Ng, S. F. (1983). Exp. Cell Res. 144, 25-30. Miyake, A. (1968). J. Exp. Zool. 167, 359-380. Miyake, A. (1978). Curr. Top. Dev. Biol. 12, 31-82. Miyake, A. (1981). In “Biochemistry and Physiology of Protozoa” (M. Levandowsky and S. H. Hunter, eds.), 2nd ed., Vol. 4, pp. 125-198. Academic Press, New York. Nanney, D. L. (1953). Biol. Bull. (Woods Hole, Mass.) 105, 133-148. Nanney, D. L. (1977).In “Microbial Interactions” (J. L. Reissig, ed.). pp. 351-397. Chapman & Hall, London. Ng. S. F. (1986). Prog. Protisfol. I , 215-286. Numata, O., Sugai, T., and Watanabe. Y. (1985). Nature (London) 316, 192-194. Ogura, A., and Machemer, H. (1980). J. Comp. Physiol. 135, 233-242. Okada, M., and Kobayashi, S. (1987). Dev., Growth Differ. 29, 185-192. Orias, J. D., Hamilton, E. P., and Orias. E. (1983). Science 222, 181-184. Raikov, I . B. (1982). “The Protozoan Nucleus,” 2nd ed. Springer-Verlag. Berlin and New York. Schwartz, V. (1978). Arch. Protistenkd. 120, 255-277. Schwartz, V., and Meister, H. (1975). Arch. Protistenkd. 117, 60-64. Skoblo, I . I . , and Ossipov. D. V. (1968). Acfa Protozool. 5, 273-290. Sonneborn, T. M. (1937). Proc. Natl. Acad. Sci. U.S.A. 23, 378. Sonneborn, T. M. (1940). Anar. Rec. 78, 53-54. Sonneborn, T. M. (1947). Adv. Genet. I , 263-358. Sonneborn, T. M. (1954). Caryologia, Suppl. pp. 307-325. Steinbriick, G . (1986). Results Problems Cell Dgfer. 13, 105-174. Vivier, E. (1974). In “Paramecium-A Current Survey” (W. J. Wagtendonk, ed.), pp. I89. Elsevier, Amsterdam. Wichterman, R. (1940). J. Morphol. 66, 423-451. Wichterman, R. (1986). “The Biology of Paramecium,” 2nd ed. Plenum, New York. Yanagi, A. (1987). Dev. Biol. 122, 535-539. Yao, M.-C. (1986). In “The Molecular Biology of Ciliated Protozoa” (J. G. Gall, ed.), pp. 179-201. Academic Press, Orlando, Florida.

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INTERNATIONAL REVIEW OF CYTOLOGY,VOL. 114

Characteristics of Microtubules at the Different Stages of Neuronal Daerentiation and Maturation VINCENTMEININGER AND STEPHANE BINET Laboratoire d'Anatornie, UER BiornPdicaie des Saints-Perm et BroussaisHbtei-Dieu, F-75270 Paris Cedex 06, France

I. Introduction

The importance in cell organization of extensive arrays of linear polymers that could extend for long distances was suggested in the 1970s. Among the three major cytoplasmic polymers-microtubules, intermediate filaments, and actin filaments-it became increasingly clear that microtubules took up a great deal, particularly with regard to their highly dynamic behavior, their abundance in the neuron, and the high heterogeneity of their components, which confer a large range of structural and functional capabilities on these organelles. In the present review, we approach the question of the microtubules (MT) in the developing nervous system by considering the morphological and biochemical organization of the MT and their components at the different stages of neurogenesis. We first review these different stages and the morphogenetic events which occur during these stages. Then we review the most significant studies on the dynamics of MT. Next, we consider the morphological aspects of MT in the different cell types which are involved in neurogenesis and in the two major differentiating domains of the neurons, that is, the axonal and the dendritic-perikaryal domains. Finally, we examine the biochemical correlates of these morphological aspects with regard to the two main components of the MT, the tubulin and the microtubule-associated proteins (MAP). A. NEUROGENESIS

Schematically, it is possible to discriminate during neurogenesis distinguishable events which allow the transformation of the neural plate, containing at the most several thousand primordial cells, into a mature central 21 Copyright Q 1989 by Academic Press. Inc.

All rights of reproduction in any form reserved.

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VINCENT MEININGER AND STEPHANE BlNET

nervous system ((3“)composed of many billions of highly organized neuronal and glial cells. These events include the following: I . The proliferation of an appropriate number of neurons and glial cells takes place from a unique cell type. Proliferation implies that the primordial cell undergoes complete mitotic cycles. 2. The migration of the future young neurons is necessary, since in the developing nervous system neurons are generated in sites different from those in which they later reside. Migration implies that cells use motile forces and move through an environment which seenis to play a role in the process of differentiation. The migration follows an orderly sequence starting with the large neurons first, then intermediate-sized neurons, and finally small neurons (Jacobson, 1978). 3. The differentiation of different cell types occurs at the end of migration. Differentiation implies an orderly sequence of events with a tendency for neurons to differentiate before the glial cells in any particular region of the brain. The neuron acquires a very spatial organization and two clearly distinct domains, the axonal and the perikaryal-dendritic(Lasek and Brady, 1982a). During this process occur morphogenetic movements, axons coming up and growing prior to dendrites. 4. The interconnection of neurons takes place during the growth of neuronal processes. It leads to the formation of the synapses and to the establishment of a specific set of afferent and efferent connections. Synapse formation implies mechanisms of cell recognition and inhibition of cell movements to form stable intercellularjunctions. 5. The regulation of the number of cells, of synapses, and of cell processes is important, since there is an overproduction of neurons, of cell processes, and of synapses (“multiinnervation”) during the earliest stages of development (Cowan, 1979). B. MICROTUBULES

It is well known that MT are spatially and functionally associated with a wide range of eukaryotic cell activities. Their appearance as stiff, springy fibers able to resist bending in an elastic fashion and to transmit tensile and compressive forces along their lengths has suggested that they are involved in the establishment and maintenance of cell shape. This role seems essential in neurons, which are highly polarized cells. Through the interconnections that they are able to establish by different types of material to several organelles and fibrous elements of the cell, MT participate actively in the spatial organization of the cytoplasm and contribute to intracellular transport activity, which is very important in neurons, particularly in the axonal domain (the axonal

MICROTUBULES IN DIFFERENTIATION AND MATURATION

23

transport) both in growing and mature axons (Lasek and Brady, 1982b). The spatial relations of MT with cell membranes suggest that they plan an active role in the regulation of cell surface components as membrane receptors. Microtubules are not only rigid, “skeletal” structures. In contrast to the specialized, stable MT of cilia and flagella, the great majority of cytoplasmic, interphasic, and mitotic MT are labile, dynamic polymers which exchange subunits rapidly with a soluble subunit pool. Microtuble dynamics is essential for rapid intracellular processes, such as the reorganization of the MT cytoskeleton at the transitions between the mitosis and interphase stages of the cell cycle and also during morphogenetic events requiring elongation or shortening of cell processes, which are essential morphogenetic events during neurogenesis. 1. Microtubules and Microtubule-Organizing Centers

Microtubules have a cylindrical wall 24 nm in diameter composed of 13 protofilaments. Each protofilament is made of tubulin with two monomers, the a and the (3 tubulin, arranged in the form of a dimer. The arrangement of these dimers in the MT lattice is polarized, giving the MT an intrinsic structural polarity. The initial assembly of MT in vivo is organized by a specific region of the cell, the microtubule-organizing center (MTOC) (Brinkley, 1985). Centriole, basal bodies,and kinetochore are considered as the major organizers of the cytoskeleton of the cell (McIntosh, 1983). The orientation of the MT in relation to the MTOC determines its intrinsic polarity. In vivo, most if not all interphasic MT are oriented with their pole of assembly, the plus end ( + 1, distal to the centriole and their pole of disassembly, or minus end (-), proximal to the centriole. The intrinsic polarity of tubulin dimers and of MT gives the plus and minus ends unique structural, functional, and assembly characteristics. 2. Mechanisms of Assembly of M T Assembly of MT in vitro occurs by nucleated, linear condensation-polymerization reactions (Hill and Kirschner, 1982). Various models have been proposed to explain the dynamics of this assembly. Microtubules initially were viewed as polymers in simple equilibrium with a fixed concentration of tubulin subunits (Johnson and Borisy, 1979). Margolis and Wilson (1978) found that in presence of GTP, MT were not in true equilibrium, but were in steady state. They interpreted these data as indicating that there was a net addition of tubulin at one pole of the MT, the plus end (+), and an exactly balanced net loss of subunit at the other pole, the minus end (-). They designed this behavior as “tread-

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milling” or head-to-tail polymerization driven by GTP hydrolysis (Bergen and Borisy, 1980; Margolis and Wilson, 1981). Other suggested the existence of tubulin exchange at sites all along the length of MT (Inoue, 1981). This mechanism of exchange of tubulin subunits has been the matter of extensive discussion, but recent studies seem to discard this possibility at least in interphasic M T (Sammak et a / . , 1987) and in most mitotic MT (Salmon et a / . , 1984). More recently, a different kind of behavior of MT in vitro was described and called “dynamic instability” (Mitchison and Kirschner, 1984a,b; Kristoffersen e t a / . , 1986). It seems that the dynamicinstability model is the most accurate interpretation of the dynamic of assembly of MT in vitro and from pure tubulin. In this model, the individual MT in a given population exhibit three different dynamic phases (Cassimeris et a/., 1987): no growth at a nucleation center; elongation, or growing phase, usually at constant velocity; rapid shortening, or shrinking phase, which occurs very rapidly and provides new subunits for growth. The separate growing and shrinking phases of MT in the same population, with rather infrequent transitions between them, defined the “dynamic instability” (Kirschner and Mitchison, 1986). It seems that the probability for a shrinking MT to revert to a growing M T and vice versa is very low. Salmon et a / . ( 1984) define three abrupt transitions between the different phases: nucleation is the abrupt transition for no growth to elongation, catastrophe is the abrupt transition from elongation to rapid shortening, and rescue is the rapid transition from rapid shortening to elongation before complete depolymerization back to the nucleation center. The mechanistic basis for the putative phase transitions is unknown, but Kirschner and Mitchison (1986) proposed a “GTP cap model” based on the work of Carlier and Pantaloni (1981) and Hill (19115). In this model, tubulin-GTP adds to the elongating MT, and some time after the incorporation, GTP is hydrolyzed to GDP, resulting in a MT with an unstable core of tubulinGDP. and a “cap” of tubulin-GTP stabilizing the elongating end. Loss of the GTP cap results in a rapid and extensive depolymerization, the catastrophe. The development of appopriate molecular probes showed that in cell culture, many MT are remarkably dynamic and their behavior is in accordance with the dynamic-instability model (review in Kirschner and Mitchison, 1986; Sammak et a l . , 1987). If treadmilling cannot explain most of the properties of MT in vivo, and particularly rapid movements, it seems that this behavior could be critical for certain other aspects of MT function in cells, for example, the growth polarity of MT. On the other hand, dynamic instability is incompatible with data obtained both with MAP-rich MT and MAP-depleted MT, which indicate that M T at polymer mass steady state attain stable length distribution and exhibit treadmilling (Margolis and Wilson, 1978, 1981; Farrell

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et al., 1979;Cote and Borisy, 1981).Farrel et al. (1987) have subsequently showed that the extent of MAP-rich MT is small, and MAP-depleted MT exhibit extensive dynamic instability behavior; moreover, under steadystate conditions of constant polymer mass and stable MT length distribution, both MAP-rich and MAP-depleted MT preparations exhibit treadmilling behavior. So it becomes clear that MAP play a major role in the structural organization and stability of MT in cells. They also seem to be involved in the determination and control of MT function. These authors proposed a phase-dynamic model in which the balance between the GTP cap and the MAP regulates the behavior of the MT: treadmilling or dynamic instability.

C. MICROTUBULES AND NEUROGENESIS The highly specified functions of the different cell types observed during neurogenesis suggest that different types of MT exist, with specific biochemical, functional, and probably structural properties. To approach this question, it is necessary to try to correlate biochemical and morphological analyses of the structure of MT with the various events which occur during neurogenesis. Fairly simple at the beginning (i.e., at the time of the closure of the neural plate), the organization of the developing nervous system becomes more and more obscure due to the progressive intricacy of different events and of different cell types. This intricacy explains the difficulty in correlating a specific morphogenic event, and/or a specific cell type with the structural analysis of MT, and that the more mature the CNS the more difficult the correlation. Also explaining the difficulty of precise analysis is that the correlation of the structural analysis of MT with a specific morphogenetic event precludes the use of the whole nervous system, because a spatiotemporal gradient of maturation exists from the caudal to the cranial pole (Jacobson, 1978). This explains why at a given stage of embryonic maturation, different regions, each at a specific stage of development, correspond in the whole embryonic nervous system. Despite important variations among the patterns of development, it is possible to describe successive patterns of development in the CNS (Fig. 1).

I . During the earliest stages, neuroepithelium is composed of a single type of cells. We proposed to call this cell the “bipolar neuroepithelial cell” (BNC) (Repetto-Antoineand Meininger, 1982),as this term permits definition of the orientation and nature of these cells. Their bipolar orientation-perpendicular both to the internal, or ventricular, surface and

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v I

2

FIG.I . Schematic drawings illustrating the development of the neural plate. In drawing I , the neural plate is composed only of bipolar neuroepithelial cells at various stages of the cell cycle. mitotic cells are at the contact with the ventricular surface, and the neural plate consists of only a Ventricular (V) zone. Drawing 2 shows the aspect of the neural plate when the intermediate zone (I) appears, marked by the tangential neurons and axons; the marginal zone (M) is apically located, and the ventricular zone (V) basally. In drawing 3, the neural wall has enlarged; the perikarya of the radial glial cells occupy the ventricular zone (V). Mitotic cells are located above in the subventricular zone (Sv); migrating neurons ascend along the radial glial fibers. The intermediate zone (1) has thickened, and perikarya of additional cells have crossed the intermediate zone toward the cortical plate (Cp).

external, or pial, surface (Fig. 2)-allows a polarity to be defined. We shall refer to the basal pole as the pole delineating the ventricular surface and to the apical pole as the pole attached to the basal membrane of the pial surface. Mitotic figures are seen close to the ventricular surface. These figures correspond to BNC in mitotic phase of the cell cycle. These mitotic cells do not differ from the BNC, but their MT are in a different state. In our descriptions, we shall distinguish between the BNC and the mitotic cells. 2. Cells which do not reenter the cell cycle detach from the ventricular surface and start to ascend toward the pial surface. We refer to these cells as migruting young neurons. 3 . At the end of their migration they stop near the pial surface, and these postmigratory young neurons start to grow their axon tangentially to this surface. Postmigratory young neurons and axons contribute to

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FIG.2. Schematic drawing to illustrate a possible sequence for the generation cycle of BNC. Cells were reconstructed from the tectal plate of mouse embryo on the tenth day of gestation (E10). Bottom line represents the ventricular surface; top line the apical, or pial, surface. Microtubules (MT) are not shown. Cell 4 is in prometaphase. In cells 5-7, the nucleus ascends toward the pial surface. In cell 5 , ciliogenesis occurs. In cell 6. the primary cilium is mature. In cell 6, the primary cilium starts to resorb. In cells 1-3, the nucleus comes back toward the ventricular surface, and its long axis becomes tangential to the ventricular surface; centriologenesis occurs during these stages.

forming a tangential layer cutting the radial orientation of the BNC and delineating three zones (Fig. 1): the intermediate zone, which is the layer of axons and young neurons, the marginal zone apically and the ventricular zone basally. The marginal zone contains no cells and only the apical part of the BNC. The ventricular zone contains stages of the BNC cell cycle marked by the position of the nucleus. 4. Soon after the appearance of the first differentiating neurons, the number of cells increases and all the major events of neurogenesis are intermingled and contribute to progressively obscuring the morphology. Above the intermediate layer, in the cortical anlage, the cortical plate appears due to the apical migration of the cells, beyond the intermediate layer. Between the intermediate zone and the ventricular zone the subventricular zone appears, containing proliferating cells which give rise to the macroglia and various classes of neurons, probably most of the intermediate-sized and small interneurons.

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All these zones are uniquely developmental structures, disappearing or becoming so transmuted with time that they are not found in the mature nervous system.

11. Microtubules during Neurogenesis: Morphological Analysis

A. MT A N D T H E BNC Whatever the time of appearance of the first neurons, it is assumed that all neurons as well as glia arise from a common stem cell, the BNC. 1. Morphology of the BNC

The perikaryon looks like a radially oriented channel in which a large nucleus moves, ascending and descending during the cell cycle (the toand-fro movements) (Fujita, 1963) (Fig. 2). The apical, or pial, process located between the apex of the nucleus and the pial surface is a transient structure which is detached from the pial surface during the G , phase of the cell cycle (Cohen, 1987). Between the basal pole of the nucleus and the ventricular surface is the basal, or ventricular, process, which is a stable structure since it remains attached to the ventricular surface throughout the various cell cycles and its detachment marks the end of the mitotic process and the beginning of the migratory process (Hinds, 1979; Repetto-Antoine and Meininger, 1982). The ventricular processes from adjacent cells are tightly joined, and the junctional zone between them exhibits very narrow intercellular clefts which seem to be totally occluded in places (Saunders and Mollgard, 1981). At the earliest stages of development, these junctional complexes seem very active in inhibiting the transfer of proteins in the intercellular space (Saunders and Mollgard, 1981), suggesting that most of the traffic occurs through the BNC and that MT may be involved in this traffic. The ventricular process exhibits a characteristic primary cilium with a thick base and a long, thin tip (Cohen and Meininger. 1987). The base has a reduced ciliary necklace and a 9 + 0 pattern of MT doublets. In the tip, the pattern decreases from 7 + 0 to 2 + 0. This cilium is associated with a basal body and a centriole. The basal body differs from that of the motile cilium in three ways: ( 1 ) internal and external sheets interconnecting the nine sets of three MT. the triplet sets, from base to apex; (2) the constant existence of an accessory basal foot, and (3) the scarcity of a ciliary rootlet. We have suggested that the ciliary apparatus of the BNC is a rigid, polarized structure, and the cilium may be involved in the possible exchanges between the ventricular fluid and the developing nervous system. This cilium is transient and present only during the G , and the initial S phase

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of the cell cycle (Cohen, 1987) (Fig. 2). At the end of mitosis, the centrioles migrate toward the ventricular process of the neuroepithelial cell, near the ventricular surface. The centriole, located near the ventricular surface, matures and forms a basal body, since its tip is capped by a vesicle, probably originating in the cytoplasm. This vesicle fuses with the plasmalemma and the cilium growths by the centrifugal extension of the nine sets of MT doublets. These nine sets invade the thick base of the cilium, which is initially capped by a ball-shaped tip, giving the appearance of a mushroom cilium. The secondary extension of MT doublets contribute to form the tip of the mature cilium. Centriologenesis occurs later, before mitosis, and is concomitant with the progressive resorption of the cilium. The daughter centrioles, or procentriole, begins to take on form near the tips of fibrils that extend perpendicularly and at a short distance from the wall of the parent centriole. Osmiophilic material accumulates around these fibrils, and gives rise to the MT of the mature daughter centriole. These centrioles formed by a centriolar process are further engaged in mitosis, after the total resorption of the cilium. This pattern of development suggests that in the BNC centnologenesis and ciliogenesis are two independent phenomena (Cohen et al., 1988). 2 . Interphasic MT Until recently no studies were devoted to the analysis of the MT in the BNC. To address the question of the location and the possible significance of the interphasic MT in the BNC, we used immunofluorescent staining of these MT with a tubulin-specific antibody (Repetto-Antoine et al., 1984). Analysis in situ was allowed in the embryonic tissues by the introduction of a water-soluble embedding medium, the high molecular weight polyethylene glycol (PEG), which permits semithin sections to be obtained very easily (Wolosewick et al., 1984) after glutaraldehyde fixation. In semithin PEG-embedded sections, interphasic MT are radially oriented and appear to extend the whole length of the BNC (Fig. 3). In both the ventricular and apical processes, the appearance exhibited by MT is related to the morphology of the process and to the location of the moving nucleus. Our results suggest that when the cell nucleus ascends toward the pial surface (during the G, and initial part of S phase), the apical process enlarges progressively in front of the nucleus, and the ventricular process narrows behind, exhibiting progressively the appearance of a thin rope. An opposite situation is observed when the nucleus moves back toward the ventricular surface (during the late part of S and G 2phases). During the G , phase, the ventricular process remains attached to the ventricular surface, whereas the apical process is detached from the apical surface (Fig. 2). The spatial organization of MT in the cytoplasmic channel of the

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FIG.3. PEG-embedded 0.5-em semithin sections in the frontal plane of the tectal plate of mouse embryo at EIO. On the left, tubulin immunostaining; o n the right, phase-contrast micrograph of the corresponding region. The radially oriented MT of the BNC are seen both

in the ventricular process (small arrowheads) and in the apical process (large arrowhead). The large arrow points t o a tangential young neuron, exhibiting a diffuse fluorescence suggesting the existence in the perikaryon of unpolymerized tubulin ( x 1200).

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BNC is explained by the movements of the nucleus. In the thin, ropeshaped processes, MT are organized in densely packed bundles, intensely fluorescent, located mainly in the central core of the process. In the enlarged, column-shaped portion of the processes, MT are arranged at the periphery of the process, probably in contact with the cortex of the cell. At the level of the moving nucleus, MT are drawn laterally in a thin strip of cytoplasm between the nucleus and the plasma membrane. These aspects suggest that in the BNC, MT are organized in dense bundles extending from the ventricular surface to the tip of the apical process, and show the appearance of a columnar framework surrounding the nucleus during its to-and-fro movements. N o information is available on the structures which organize these MT. However, the centriolar apparatus which is present during the whole cell cycle and located near the ventricular surface seems to be a good candidate for being the MTOC. In all cells, MT seem to range between two extreme categories, labile and stable, so far as control of their lengths and/or their rate of depolymerization (Schulze and Kirschner, 1987) is concerned. Besides this definition of stability in vivo, stability also refers to a particular behavior of MT after cold treatment and when exposed to drugs. Some observations (Mandelkow and Mandelkow, 1985) suggest that cold treatment induces disassembly proceeding from both ends and from inside, at least in vitro, whereas drugs, at least antimitotic drugs, induce disassembly from one or both ends, depending on the dose, but not from inside (Dustin, 1984). In cold-treated embryos, we observed two populations of MT: a large amount of cold-labile and a small pool of cold-stable MT (Fig. 4). In the cold-stable pool, it does not seem that cold induces a modification of the length of the MT. The possibility of disassembly from inside cannot be definitely discarded, as in some very thin sections some MT appear fragmented. However, our results strongly suggest that cold treatment induces a behavior of MT closely identical to the “dynamic instability” with coldlabile MT, which depolymerize rapidly and completely, and cold-stable MT, which exhibit no modifications in length. Interphasic MT exhibit different reactions when submitted to drugs acting on MT. The most commonly used are colchicine and its derivatives and the Vinca (Carharanthi4s)alkaloids, which are capable of binding specifically to tubulin both in vitro and in vivo and prevent its assembly into MT. Tubulin binds colchicine at a high-afinity site with a stoichiometry of I mol of colchicine per mole of tubulin, although complete inhibition of MT assembly can occur when only a small fraction of tubulin is complexed with colchicine (Olmsted and Borisy, 1973; Wilson and Bryan, 1974; Margolis and Wilson, 1977; Sternlicht and Ringel, 1979). Despite the extreme sensitivity of tubulin polymerization to colchicine, only limited disassembly of M T is generally observed when saturating concentrations

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FIG.4. PEG-embedded 0.5-km semithin sections of the tectal plate of mouse embryo at EIO. Action on interphasic MT:in plate I , of cold treatment; in plate 2, of VBL sulfate 30 pLM. On the left, tubulin irnmunostaining, on the right, phase-contrast micrograph of the corresponding region. Cold treatment leaves few MT intact, and some of them exhibit characteristics suggesting focal depolymerization (arrows and arrowheads). VBL sulfate depolymerizes nearly all MT, and a diffuse fluorescence (large arrowheads) is seen in cells arrested at stage G2 ( x 1200).

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of colchicine are added to the microtubules (see Herzog and Weber, 1971; Wallin and Larsson, 1979; Deery and Weisenberg, 1981). Microtubules resistant to colchicine have also been observed in vivo, even though colchicine is able to prevent the formation of such MT (Mayor et af., 1972; Schnepf and Deichgraber, 1976). At suprastoichiometric levels, the tubulin
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tubulin (Bryan, 1972: Wilson et al., 1978). It is difficult to conclude from these experiments. but it seems probable that the mechanism of action of drugs like VBL is dependent on numerous parameters such as the concentration of drug, the concentration of tubulin, and also the concentration and probably the type of MAP. Using VBL sulfate, Karfunkel (1972)described in Xenopus neurala an inhibition of neurulation and a rounding up of the neuroepithelial cells that have already elongated. We examined the effects of different Vinca alkaloids, VBL, and VCR in the mouse embryo at the earliest stage of neuronal differentiation using whole-culture embryos, immunofluorescence after PEG embedding, and electron microscopy (Fig. 4). The effects on interphasic MT of the BNC are dose and drug dependent. After 90 minutes of culture, a time which corresponds at this stage to a complete cell cycle, VBL and VCR (whatever their concentration) never induce a depolymerization of the whole population of MT, suggesting the existence of a small pool of drug-resistant MT. When the concentration of the drug is increased, the number of stable MT decrease and the appearance of the ventricular processes of the BNC is modified, these processes becoming thinner and thinner, leading to a disruption of the ventricular surface as described with colchicine by Herken (1985). An increased number of mitotic cells near the ventricular surface was observed at very low concentration (0.5 kikl). Our results suggest that the effects of the drugs are different from the effect of cold, giving rise to the hypothesis that stability and lability of MT to cold and to drugs is not a common phenomenon and probably arises via different mechanisms. It seems highly probable that these mechanisms depend not only on tubulin in state of dimer and/or of polymers, but also of the MAP. Cells in which no MT were observed-suggesting that most if not all MT were totally depolymerized-had a typical bipolar shape. The same phenomenon was also observed in cold-treated embryos, suggesting that the depolymerization of MT of the BNC does not induce a modification of the cell shape at least after the closure of the neural tube. However, in classic experiments in cell culture it has been shown that exposing asymmetric cells to MT-depolymerizing drugs such as colchicine or nocodazole causes them to revert to a round morphology again. This phenomenon has been demonstrated in several cell types (Solomon and Magendantz, 1981). but they can be visualized with particular clarity in neuroblastoma cells. Using cytochalasin, Solomon and Magendantz ( I98 I) demonstrated that in neuroblastoma cells depolymerization of MT is not sufficient to cause loss of cell shape. and that some other cellular process, probably ATP dependent, is also involved. Our results obtained in viw after the closure of the neural tube fit these observations well but conflict

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with results obtained by Karfunkel (1972), who observed a rounding up of cells with VBL in Xenopus neurula and with colchicine in chick embryo before the closure of the neural tube. This discrepancy suggests two hypotheses: first, MT are no longer involved in maintenance of cell shape after the closure of the neural tube; second, the rounding up observed is a misinterpretation and is related to the progressive storage of round mitotic cells blocked at this phase by the depolymerization of the mitotic MT induced by these drugs.

B. MICROTUBULES AND THE MITOTIC CELLS 1. Morphology of the Mitotic Cells

At the earliest stages of neurogenesis, mitotic figures are seen in the wall of the neuroepithelium. Most of them are observed close to the ventricle, despite the possibility of the presence of aberrant mitotic cells in the apical part of the neuroepithelium. The cell rounds up during the mitosis. This rounding up is associated with a disappearance of the junctional complexes seen at the contact with the ventricular surface (Cohen, 1987). The mechanism(s) by which the rounded-up mitotic cells is maintained at the contact of the ventricular surface, and the mechanisms of reconstitution of the junctional complexes of the ventricular feet of the daughter cells remain totally obscure. The orientation of the mitotic spindle may be important, not only in determining whether a daughter cell is released or remains attached to the ventricular surface, but also in determining the spatial arrangement of the cells (Jacobson, 1978). Smart (1976) demonstrated that in the ventricular zone, and when the wall of the neuroepithelium is relatively thin, in more than 90% of mitotic cells the spindle axis is tangential to the ventricular surface and the plane of cleavage radial to this surface, so that the daughter cells lie side by side and retain their attachment to the ventricular surface when they reenter the cell cycle. This arrangement of the mitotic spindle seems important in the spatial orientation of the BNC. We observed a high proportion of mitotic cells with a plane of cleavage perpendicular to the ventricular surface. Various methods have been used to determine the kinetics of the BNC cell cycle (Jacobson, 1978). It seems that the duration of mitosis is very short when compared to the duration of the cell cycle (1.5 : 10 of the total cell cycle at stage El0 in mouse neural tube). This is reflected in the scarcity of mitotic figures seen at all stages of development in the CNS. There are also indications that the duration of the cell cycle gradually increases during the development due to the progressive lengthening of the G, phase, whereas the S, G2, and M phases remain relatively constant.

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2 . Microtubules of the Mitotic Spindle Using immunofluorescence, it has been demonstrated in cell culture (Brinkley et al., 1976; Weber and Osborn, 1979) that when cells progress from interphase to mitosis, few (if any) cytoplasmic MT are found outside the spindle, and that the different types of MT of the mitotic spindle are seen-particularly the chromosome-to-pole, or kinetochore MT. and the pole-to-pole, or interpolar MT. We analyzed the appearance of M T in mitosis in situ using immunofluorescencein PEG-embedded semithin sections of the embryonic mouse tectal plate at the earliest stage of neuronal differentiation (i.e., at 10 days postmating; stage E10) (Fig. 5). During prophase, nuclear envelope remains intact and the fluorescent filaments of the interphasic MT disappear. The centrioles derive from the centrioles of the ciliary apparatus, centriologenesis occurring during the cell cycle concomitant with the disappearance of the primary cilium (Cohen et al., 1988). Most cells observed in our material displayed aspects of prometaphase. During this stage, despite the disruption of the CMTC, cells remain more or less elongated. This result, like our observations on the lack of modification of the shape of the BNC after a drug or cold-induced disruption of the interphasic MT favors our hypothesis that the CMTC play no role in maintenance of an elongated cell shape. I n situ, rounding up of BNC was seen only during late prometaphase with the appearance of a complete mitotic spindle and concomitant with the elongation of the spindle. This result differs slightly from the observations in cell culture of Brinkley et al. (19761, who observed a rounding up as the cells entered mitosis. Differences between cell culture and in situ suggest that in the neural tube, the interrelations between cells may play a major roie in the maintenance of the cell shape. Mitotic spindle during prometaphase can assume two forms (Brinkley er al., 1976). Sometimes, chromosomes are grouped around a single bright spot, and the monopolar prometaphase is explained by the failure of centrioles to separate at prophase. Most often, two centrioles can be distinguished and an elongated bipolar fluorescent spindle can be observed. The bipolar prometaphase is explained by the separation of the centriole pairs, which occurs generally prior to nuclear envelope disruption. As described in other cell types, it seems probable that centrosomes are the major site of MT nucleation in the spindle (Mitchison and Kirschner, 1985; Soltys and Borisy, 1985; Mitchison et a / . , 1986).The possibility for cells to regulate the initiation and extent of MT assembly during mitosis has been extensively discussed elsewhere (Vandre er a l . , 1984; Kirschner and Mitchison, 1986), and several hypotheses have been postulated. It is possible that a widespread phosphorylation, triggered by a calcium ions release, signals the transitions between interphase and mi-

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FIG. 5 . PEG-embedded 0.5-pm semithin sections of the tectal plate of mouse embryo at EIO. On the left, tubulin immunostaining; on the right, phase-contrast micrograph of the corresponding region. Different stages of mitosis are shown: (plate I) large arrowheads point to prometaphase, and arrow to metaphase; (plate 2) anaphase; (plate 3) telophase, with a staining of the MT of cytoplasmic bridge, except at midbody (arrow); on the right, prometaphase (large arrowhead) ( x 1200).

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tosis. Phosphorylation of centrosome components at the entry of mitosis may result in an amplification of the nucleation capacity of centrosomes responsible for the decrease in average MT length and increase in frequency of catastrophe according to the dynamic-instability hypothesis (Kirschner and Mitchison, 1986). Another hypothesis is that the binding affinity of one or more species MAP, possibly MAPI, which is present in mitotic cells and in the centrosomes (Sato et al., 1984; de Mey el a / . , 1984), may be reduced by phosphorylation, leading to an increase in the frequency of catastrophe or a decrease in the frequency of rescue as phosphorylation of MAP inhibit the rate and extent of MAP-stimulated MT assembly (Jameson and Caplow, 1981). However, it is also possible that the binding of some species of MAP, particular MAPI b, in the C-terminal (region 1V)subdomain of tubulin, interacts with the assembly capabilities of tubulin and increases the frequency of catastrophes (Maccioni er a / . , 1987: Serrano et a l . , 1984). Using an anti-MAP1 monoclonal antibody (kindly provided by J. de Mey), we were able to observe fluorescent spots in the mitotic cells of BNC, suggesting the existence of this protein in these cells during mitosis. The mechanism(s) that trigger the depolymerization of MT at prophase remains unknown, as does the mechanism that signals a cell to reentry or not in the cell cycle. It is possible that the location of the nucleus plays a role, since there is a close correlation between the location of the nucleus, near the ventricular surface, and the beginning of mitosis. During metccphase the spindle is well stained by fluorescence, and bundles of fluorescent MT are seen extending from the chromosomes, aligned on the metaphase plate, to the poles. The poles are characterized by a brightly fluorescent spot. Interpolar, or pole-to-pole, MT are usually difficult to distinguish at this stage, probably due to their thinness. Very few cells were seen in anuphuse. During this stage, kinetochore MT are short, and bundles of pole-to-pole, or interpolar, MT are seen extending across the interzone. We were unable to observe the diffuse fluorescence of the cytoplasm surrounding the anaphase spindle reported by Brinkley er al. (1976). who suggested that this was due to the release of free tubulin in the cytoplasm during the spindle MT disassembly. It has been demonstrated (Mitchison et a / . , 1986; Wadsworth and Salmon, 1986; Gorbsky et ul., 1987) that kinetochore MT depoiymerize at the kinetochore as the chromosomes move poleward during anaphase (anaphase A of Inoue, 1981). This “Pac Man” model seems to explain the movements of chromosomes toward the poles, because it furnishes a poleward force which pulls the chromosomes, whereas the opposite force-the poleward ejection force, created by the dynamic instability of polar MT-diminishes due to a decrease in number of the polar MT. A concomitant elongation

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of the spindle is observed due to the elongation of the interpolar MT (anaphase B of Inoue, 1981). The site of incorporation of tubulin in anaphase B remains a matter of controversy. Margolis et al. (1978), Mclntosh et at. (1983, and Masuda and Cande (1987) suggests that tubulin adds on to the plus ends of MT, which are in the zone of MT overlap, and which then slide through the midzone as the spindle elongates. Pickett-Heaps et al. (1986) argued that new subunits are added on to preexisting MT at their minus ends, which are clustered round the spindle poles. InouC and Sat0 (1967), in contrast, suggested that new subunits would be added throughout the spindle during anaphase. At telophase two daughter cells start to separate, and during cytokinesis, or cytodieresis, the interpolar MT progressively elongate, leading to a bridge of MT between the two daughter cells. These MT extend from a particular region rich in MT, the midbody. As described by Brinkley et al. (1976) and Weber (1976), the midbody seems to remain unstained, probably due to the electron-dense matrix surrounding MT of the midbody at the ultrastructural level, which may interfere with the antibody binding. 3. Action of Drugs and Cold Action of low temperature on MT of the mitotic spindle has been analyzed in cell culture (Brinkley et al., 1976) and in crane fly spermatocytes (Scarcello et al., 1986). These authors described a differential loss of spindle MT with a high susceptibility of pole-to-pole interpolar MT to cold treatment, whereas the chromosome-to-pole or kinetochore MT are resistant to this treatment. Using cold-treated embryos (Fig. 6), we observed the existence of one or two brightly fluorescent spots from which radiate fine fibers directed toward the chromosomes. Usually chromosomes seem to be located at the periphery of the cell. These aspects suggest that in the mitotic cells of the neuroepithelium, cold treatment has the same effect as in cell culture with a disruption of the pole-to-pole MT and a preservation of the kinetochore MT. The exact significance of the cold stability of kinetochore MT remains unknown. This behavior may be related to the fact that these MT appear more stable in relation to their attachment both to the centriole and to the kinetochore, whereas interpolar MT appear more unstable, one of their extremities being more or less free (Kirschner and Mitchison, 1986). It is also possible that interpolar MT may be more accessible to depolymerization from inside than kinetochore MT. Some cells were seen at telophase with persistent staining of MT extending from the midbody to the daughter cells. These MT derive from the elongation of the interpolar MT, which are cold labile during other stages of mitosis, suggesting that during telophase there is a modification of the behavior of the elongating interpolar MT, probably due to a modification of their

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FIG.6 . PEG-embedded 0.5-pm semithin sections of the tectal plate of mouseembryo at ElO. On the left, tubulin immunostaining to show the effects of cold treatment (plate I ) , of VBL sulfate 10 pM (plate 2), and 30 pM (plate 3) on mitotic MT. On the right, phasecontrast micrographs. Cold treatment (plate I ) depolymerizes the interpolar MT, but leaves intact the kinetochorial fibers (arrowheads) directed toward the chromosomes (small arrows). In plate 2 is a pseudometaphase (arrow). and in plate 3 only kinetochores (arrows) are stained ( X 1200).

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structural components which may precede the entry in G, phase marked by the appearance of the primaril cilium. Antimitotic drugs have been extensively used to disrupt mitotic spindle (Dustin, 1984). Colchicine remains the most frequently used antimitotic drug, probably because it is the most specific poison, combining electively with tubulin and acting at very low concentrations. This drug does not interfere with the rate of mitosis, but induces a progressive accumulation of mitotic cells which seem to be arrested at metaphase stage (pseudometaphase). This effect is reversible after the removal of the drug. Vinca alkaloids are also powerful mitotic inhibitors, comparable to colchicine. Their effect seems less reversible and mitoses display the same aspects as those described with colchicine. Other effects of Vinca alkaloids may interfere with mitosis (Creasey, 1968; Wilson et al., 1974), such as the binding to microfilaments and the inhibition of protein synthesis. Studies using colchicine (Watterson et al., 1956; Herken, 1985) or VCR (Kallen, 1962; Langman ef al., 1966) demonstrated that in the neural tube there are several layers of cells arrested in metaphase at the contact with the ventricular surface, and that after about 8 hours almost all c e h are arrested at this stage. Using two types of Vinca alkaloids (VBL and VCR), we observed a particular dose effect (Fig. 6). In rotatory whole culture of embryos at stage El0 (I0 days postconception)-that is, a stage of high rate of cell production4rug.s were added to the milieu for the same length of time (90 minutes), at increasing doses from 0.1 to 200 pM. At low doses, cells were blocked at metaphase with the appearance of pseudometaphase. Increasing the doses led to (1) a progressive increase of the mitotic index; (2) a progressive depolymerization of the mitotic MT, initially of interpolar MT and then of kinetochore MT; (3) a progressive storage of round mitotic cells arrested at metaphase; (4) a progressive increase of the number of BNC at G , phase with a disappearance of cells at G, phase. These aspects are correlated with the progressive depolymerization of the interphasic MT. Our results suggest different conclusions. First, the interphasic MT seem to play no role in the to-and-fro movements of the nucleus. Second, the accumulation of the cells at G, and M phases is related to the depolymerization of the mitotic MT. Third, it seems that in the neural tube there is a correlation between the duration of the cell cycle and the dose of Vinca alkaloids. It is generally assumed that these drugs have no effects on the rate of mitoses and on the duration of the other phases of the cell cycle (Dustin, 1984). A possible explanation of our own results is that these drugs do not interfere with the mitosis itself but an intracellular signal of the beginning of mitosis. It is well known that depolymerization of CMTC is a prerequisite for mitosis as previously discussed. It is generally assumed that this phenomenon is a consequence

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of the initiation of mitosis. As the antimitotic drugs actively depolymerize the CMTC of the BNC, it is possible that depolymerization of the CMTC is an intracellular signal of the beginning of mitosis, as suggested by the correlation between the intensity of interphasic MT depolymerization and the doses of Vincct alkaloids.

c. MiCRoTUBULES LN MIGRATINGPOSTMITOTIC Y O U N G NEURONS 1. Mechrinisms of Cell Migrution during Neurogenesis

For most cells in the CNS, the withdrawal of a cell from the mitotic cycle and/or the detachment of the ventricular surface seems to be the signal for its migration out of the ventricular zone to its definitive location (Sidman, 1979; Hinds, 1979). The mode of migration resembles closely that of other motile cells. Despite variations, basically the migrating cells extend a leading process containing large numbers of actinlike microfilaments, followed by a flowing into this process of the cytoplasmic organelles, and then by the cell nucleus. As the nucleus is translocated, a relatively organelle-free trailing process is left behind. The migrating cell is generally fusiform in shape, with its two processes radially directed, and can usually be distinguished from the tangentially directed postmigratory neurons and from the BNC, which have a ventricular process still attached to the ventricular surface. Direction of migration is always apicalward at a speed which seems to depend on the region considered. The process of migration starts at different stages of development, depending on the region considered, with a caudocranial gradient, and the rate of migration decreases with development, probably because at late stages of neurogenesis, cells have to migrate a longer distance and to traverse a more complex terrain (Sidman and Rakic, 1973). Migration may use different mechanisms: free ameboid movements (Hinds, 1979; RepettoAntoine and Meininger, 1982): translocation of the nucleus in a leading process still attached to the external surface (Morest, 1970; Repetto-Antoine and Meininger, 1982); or guidance of the migrating cells by a particular cell, the radial glial cell, of which the fiber extending from the ventricular to the apical surfaces is a guide for the migrating neuron (Rakic, 1972). These different mechanisms seem to depend on the regions of the CNS and the distance to be covered. 2 . Microtuhicles in the Migruting Cells Numerous studies have been devoted to study the role of MT in cell motility and in cell migration. Most studies suggest that the mechanical work of cell motility is devoted to microfilaments, while MT would play the role of vector of motion. It has also been suggested that the position

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of the MTOC, generally the centriole, and its associated MT may be important in determining the direction of cell movement (Gotlieb et af., 1981; Kupfer et af., 1982), and that the reorientation of the centriole requires intact MT (Gotlieb et al., 1983). Few data are available on the organization and role of MT during cell migration in the embryonic nervous system. Using serial section reconstruction in various regions of mouse embryonic nervous system, Hinds (1979) demonstrated that the pair of centriole and the primary cilium move progressively toward the perikaryon but remain at the basal pole of the cell, opposite to the direction of migration. As the cell reaches the intermediate layer, the centriole and the cilium adopt a juxtanuclear position, usually basal, but in some cells apical to or on the side of the nucleus. The discrepancy between these results and the data obtained in cell culture leave open the question of the role of MT and MTOC in the cell migration of the young neurons and in specifying the mode of cell migration. Another important question which remains open is the mechanism(s) triggering the initiation of the migratory process. Numerous arguments suggest that the blockade of the reentry in the cell cycle could be quite important. It could be that the modification of the phosphorylation of centrosomes, and/or MAP and/or the expression of new types of MAP, may be the important signal. One possible candidate could be the MAP2, as Izant and McIntosh (1980) demonstrated that this protein is expressed in differentiating neurons in culture and is specific for this cell line in the CNS. Our own results (Binet et al., 1987) also suggest that this protein is specifically expressed in migrating young neurons, although the site of synthesis does not seem to be closely related to the site of initiation of migration.

D. MICROTUBULES IN THE POSTMIGRATORY YOUNGNEURON Factors involved in the process of cell shaping during neurogenesis have been extensively studied (Hillman, 1979; Solomon, 1984). Major factors operating during development to give rise to the specific form of the neuron fall into two classes: intrinsic factors are those controlled directly by the genome, and extrinsic factors are those arising from interactions between cells (Rakic, 1979; Berry er al., 1980; Meininger and Baudrimont, 1981; Dardennes et ai., 1984). We will focus our attention only on the morphology and possible role of MT in this process. In this chapter, we shall summarize the large amount of work devoted to the growth of neurites, axons, and dendrites. However, we will include the extension of neurites in general, since most of the data have been obtained in cell culture in which the clear distinction between axon and dendrites is difficult. It remains to be demonstrated in vivo that the extension mechanisms of these two kinds of neurites and the role of MT are similar or different in light

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of the morphological and functional differences of the two types of expansions, and in view of the differences in the biochemical composition of the MT in the two types of cell processes (see below).

I . Elongoiion of Neiuiies: The Growth Cone Appearance of neurites usually starts when the migrating young neuron reaches the intermediate zone (Shoukimas and Hinds, 1978; Repetto-Antoine and Meininger, 1982), despite conflicting results (Berry and Rogers, 196.5; Stensaas, 1967; Morest, 1970). The first neurite to appear is the axon, followed later by the dendritic differentiation (Ramon y Cajal, 191 I ; Shoukimas and Hinds, 1978). Tissue-culture experiments (Bray, 1973) and analysis of the branching pattern (Berry et al., 1980; Meininger and Baudrimont, 1981) indicate that nerve fibers (axons and dendrites) grow and branch at their terminal both in vifroand in vivo. However, the possibility that growth and branching can also occur at sites along the shafts of growing dendrites (and axons?) is supported by numerous studies (Berry ei a/., 1980). Whatever is the site of elongation and branching, the major structural element responsible for the process of neurite elongation is the growth cone. It was originally described by Ramon y Cajal(191 I ) as "flattened expansions whose edges bristled with short processes or lamellar appendages." Electron-microscopic analyses in vifro (Yamada et al., 1971) and in vivo (Vaughn and Sims, 1978) describe the growth cones as bulbous or club-shaped enlargements of the neurite ending, 1-5 p m in length, from which several types of expansions extend. The filopodia, approximately 0.1-0.2 Fm in diameter, are long and thin cellular projections, also called microspikes; IameUipodia are broad and thin expansions of the cell surface which may extend independently or in association with existing filopodia. Goldberg and Burmeister (1986) proposed to reserve the term of veil exclusively to refer to lamellipodial regions initially devoid of visible organelles in video-enhanced microscopy. Time-lapse microscopy demonstrated that all these projections may play an active role in the elongation of neurites (Bray, 1987). Microtubules and neurofdaments splay out within the cone, being restricted to its central portion in general (Bunge, 1973; Luduena and Wessells, 1973). A variety of membranous organelles are observed in the growth cones and in neurites. The major components of these organelles are elements of the smooth endoplasmic reticulum, often arrayed as a branched system which may appear continuous with the plasma membrane in place. Using phase-contrast or dark-field light microscopy, Hollenbeck and Bray ( 1987) described phase-dense varicosities containing membrane and cytoskeletal components. among them tubulin. probably produced at or near the growth cone and transported toward the cell body using retrograde axonal transport. Although it is difficult to

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identify dendrites and axons in cell culture unequivocally, the existence of distinct morphological features in vivo permitted demonstration that axonal and dendritic growth cones are similar in structure, despite several differences in their detailed arrangement. Since Yamada et al. (1971) described the effects on the growth cones of cytochalasin and of colchicine, it is generally agreed that both microfilaments and MT are required for axonal extension (Bray, 1987). Marsh and Letourneau (1984) also demonstrated the ability of cells in culture to extend axons even in the presence of cytochalasin. These results suggest that membrane vesicles and MT have an innate capacity to “self-assemble” into long, narrow extensions despite the absence of microfilaments. In the presence of colchicine, filopodial activity persists if MT are disrupted, but the axon begins to shorten. Bamburg er al. (1986) demonstrated that the local application of colcemid to the growth cone halts the advance rapidly or causes rapid retraction depending on the dose. Taxol, a drug which enhances polymerization (Kumar, 198 I), also arrests extension, suggesting that neurite extension involves net MT assembly (Olmsted, 1981; Drubin ef a/., 1985; Gard and Kirschner, 1983, and not simply a rearrangement of existing MT in the growing process. This assembly requires the recruitment of tubulin from preexisting monomer pools into tubulin polymers and seems driven by non-tubulin-promotingfactors that accumulate during the process of extension. 2. Mechanism of MT Assembly in the Growing Neurites This mechanism is critical for the elongation of MT in the growing process and for understanding the relations between the perikaryon and the tip of the neurites. The hypothesis that the assembly of MT in the growing neurites may be self-contained and independent of the cell body was supported by the observations that if neurites are cut and disconnected from the cell body, cone formation, movement, and translocation can continue for several hours (Bray, 1987). The existence of initiation sites for MT in the peripheral part of the neurites, and probably in the growth cones proper, was essential to explain the possibility of branching and that the number of M T in a neurites need not limit this branching process. A number of observations demonstrated that, both in vivo (Lyser, 1%8; Shoukimas and Hinds, 1978; Cohen et al., 1987) and in v i m (Sharp et al., 1982), MT in the growing neurites are independent of a perikaryal MTOC. Similar conclusions were drawn from studies performed in adult axons (Chalfie and Thompson, 1979; Bray and Bunge, 1981; Burton, 1987), demonstrating that the number of MT varies along the length of these expansions and that the mean length of the MT does not exceed 100-200 pm. A polarity has also been dem-

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onstrated in adult axonal MT (Burton and Paige. 1981; Heidemann el a / . , 1981) and in amputated neuntes of cultured sensory neurons (Baas and Heidemann, 1986) with an assembly ( + ) pole of the MT directed distalw a d +toward the growth cone. Axonal MT recover this polarity after coldOr drw-induced depolymerization (Heidemann er al., 1984). In adult axOnS (Heidemann et d.,1984; Jones cf ul., 1980). a particular pool of MT exhibits a resistance to cold depolymerization. These cold-stable MT exist in the form of short regions of otherwise labile MT. Due to the lack of precision of the term stable MT as previously discussed, we prefer the term “cold-stable” instead of “stable” to define this pool, to avoid the possible confusion between the resistance to cold (cold stability) and the slow rate of depolymerization (stable MT of Schulze and Kirschner, 1987). Baas and Heidemann (1986) confirmed that the cold-stable MT are not merely “mass-action fragments” maintained because of a high tubulin concentration, but that they are intrinsically cold-stable regions of longer MT. Various authors (Heidemann et a / . , 1981; Morris and Lasek, 1982; Brady ct ul., 1984) proposed that the cold-stable fragments may play a role in MT assembly in axons by acting as nucleating “seeds.” Such seeds are essential for two reasons: first, the absence of relation with perikaryal MTOC; second, the probable existence in axons of factors inhibiting the polymerization of tubulin, which may function in part by inhibiting de novo initiation (Morris and Lasek, 1982; Baas and Heidemann, 1986). These factors may explain the selective degradation of M T in axon terminals (Lasek, 1981; Baas and Heidemann, 1986; Burton, 1987). We demonstrated (Cohen ef al., 1987) the existence of cold-stable fragments of MT in the growing axons of the mouse tectal plate anlage. These fragments did not seem to be concentrated at the distal tip of these axons, near the growth cone. suggesting that the nucleating sites of axonal MT in sifcc may be located at a distance from the growth cone and that these cold-stable fragments do not provide a physical cap against dynamic instability (Baas and Heidemann, 1986). but may act as nucleating MTOC more or less similar to the centrosome (Fig. 7). The assembly of tubulin at the distal end of the growing neurite requires the transport of tubulin from the cell body to the distal tip and the action of assembly-promoting factors. A large body of data indicates that tubulin is transported in the slowest component of anterograde axonal transport and that their steady-state delivery at the distal end continues in mature, nonelongating axons (Lasek and Hoffman, 1976; Black and Lasek, 1980; Heriot ef c i l . . 1985). It is not clear whether tubulin moves slowly down in the form of an assembled framework of interconnected polymers (Lasek and Brady, t982b; Lasek et ui., 1984), or if the cytoskeleton is in fact stationary, while newly synthesized tubulin travels down the axon in an

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FIG.7. PEG-embedded 0.5-pm semithin sections of the tectal plate of mouse embryo at EIO. Effects on axonal MT: in plate I , of cold treatment; in plate 2, of VBL sulfate 30 pM. On the left, tubulin irnrnunostaining, on the right, phase-contrast micrograph of the corresponding region. Cold treatment depolymerizes most MT in the apical process (arrowhead) and leaves intact a large part of the axonal MT (arrow). VBL sulfate depolymerizes the apically located MT and a large part of the axonal MT ( x 1200).

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unassembled or partially assembled form (Nixon and Logvinenko, 1986; Tashiro and Komiya, 1987). This movement of tubulin may be driven by an interaction with the MT through mechanisms closely similar to those used for fast transport of organelles (Tashiro and Komiya, 1987). A similar mechanism has been postulated in growing neurites (Hollenbeck and Bray, 1987), emphasizing the role and the importance of well-organized MT arrays in the neurite behind the growth cone. Among the factors involved in the formation of MT bundles in the neurites, one of the main factors seems to be the nerve growth factor (NGF) (Varon and Adler, 1980). This role has been clearly demonstrated in the PC12 line of NGF-responsive pheuchromocytoma cells (Black et al., 1986; Jacobs and Stevens, 1986a,b). It seems that the uptake and retrograde transport of NGF is essential in its action (Campenot, 1977), suggesting that this mechanism of transport in vivo may provide neurons with growth factors or other different types of macromolecules (Harper and Thoenen, 1980). It has been shown that NGF induces a stabilization of MT by modifying the tubulin protomer-polymer equilibrium (Black and Greene, 1982), leading to an increase in the relative levels of polymerized tubulin (Drubin et al., 1984). Another possible mechanism of NGF on the formation of MT bundles is its action on MAP, since it increases the amount of MAPl, MAP’?, 7 (Drubin et al., 1985; Greene et al., 1983; Black et al., 1986), and it induces a phosphorylation of other MAP, the chartrins (Black e f al., 1986). As suggested by these studies, it seems probable that NGF plays an important role in the formation of MT bundles in vifro through complex actions both on tubulin and on MT assembly-promoting molecules, the MAP. The presence of a relatively high proportion of highly organized MT bundles in the growing axons observed in situ at the first stages of axonal differentiation correlated with the appearance of coldstable MT (Cohen ef al., 1987) and of MAP;?, which is transiently expressed in situ (Binet e f al., 1987), suggest that in vivo a similar mechanism induced by trophic factors may intervene in the spatial organization of the MT. The formation of MT bundles is essential to the mechanism of transport and also to the maintenance of the shape of the neurites.

3 . Determination of Neurite Caliber The most stable parameter of the neuronal processes is the volume (Hillman. 1979).As has been demonstrated in PC12 cells (Jacobs and Stevens, 1986b), four intracellular variables seem essential in the control of the neurite volume: the number and placement of organelles within the neurite; the volume surrounding these organelles; the number and distribution of M T within the neurite; and the M T exclusion zone, which probably corresponds to the “coat” of MAP. These results emphasize the role of MT in the determination and control of axon and dendrites caliber. In

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culture, the density of MT rises during neurite differentiation, this phenomenon being correlated with their progressive organization of the orientation. This modification of the orientation of MT seems to induce a progressive organization of the membranous organelles and a change in the shape of the neurite, which becomes round and smooth (Jacob and Stevens, 1986b). The role of MT in the shaping process of neurites is suggested by the modifications of forms induced by the depolymerization of MT with nocodazole (Jacobs and Stevens, 1986b). These authors and others (Black and Greene, 1982; Black et al., 1986) also described a progressive (over a 4- to Sday period) “maturation” of the MT, which become more resistant to cold- and drug-induced depolymerization. They suggested that MAP may be involved in this maturation and in the development of the “exclusion cylinder” progressively surrounding the MT. However, our own results performed in siru at the earliest stages of axonal maturation suggest that in early growing axons, MT acquire stability to cold and drugs and are organized in dense bundles at the time of their appearance, without any noticeable delay (Cohen et al., 1988). This stability and this organization are correlated with the transient expression of MAP2 in the axons (Binet et al., 1987). The discrepancy between our results and those obtained in cell culture may be explained by the modifications of the environment induced by the cell culture, or by a particularity of MT in the PC12 cell line compared to the neurons in the CNS. Another explanation may be that neurites in PC 12 cells are dendritic and not axonal in nature. 4. Shaping of the Dendritic Tree

We have previously analyzed the growth of neurites and assumed that the actual mechanism of dendritic growth is similar to that of axons. The precise morphology of dendritic networks seems to be subject to extrinsic influences of structural and/or functional nature (Rakic, 1979; Berry et al., 1980).The growth of dendrites-that is, the size, orientation, and topology of the dendritic network-is driven by the formation of contacts between their growth cone filopodia and the appropriate afferent axons (Berry et al., 1980). However, the shape of the dendritic tree is not immediately and definitely established after contacts with afferents but it does undergo some form of remodeling. Functional activity seems to play a major role during this process, and dendrites are able to change their morphology in response to functional influences (Berry et al., 1980; Dardennes et al., 1984). Hormones have been implicated in the remodeling of the dendritic tree, particularly thyroid hormones (Jacobson, 1978; Legrand, 1979). Faivre et al., (1983) observed in throid-deficient rats a correlation between the reduction in the number of MT in dendrites and the modification of the dendrite shape. However, our data on dendritic MT and on their evolution during neurogenesis remain relatively poor. Ap-

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pearance of MAP, and particularly of MAP2, in the dendritic domain will be discussed below, but numerous questions remain open. Are the dendritic MT organized in the same way as axonal MT? What are the relations between dendritic MT and MTOC present in the perikaryon, and do dendritic and perikaryal MT have the same or a different mode of assembly? Do dendritic MT have the same proportion of cold-stable MT, and do they even have cold-stable MT since it appears that this particular pool of MT is localized exclusively in the axonal domain (Brady er al., 1984; Binet and Meininger, 1987, 1988a)?

E. FURTHER EVENTSIN NEURONAL DEVELOPMENT Various events become intermingled in time and in space: migration continues until after birth, guided by radial glial fibers; cell proliferation still occurs-particularly in the subventricular zone-being more devoted to the production of glial cells; neurite growth becomes more important and probably persists throughout life, at least for dendrites. Other events mingle with them: synaptogenesis, and control of neuronal number, of neuronal processes, and of neuronal connectivity. Our data about the morphology of MT at these stages are relatively poor, and we will only summarize the most significant data. 1. Svnciptogenesis

A general rule does not seem to exist about the order of development of the presynaptic and postsynaptic structures, but in each region the order is apparently invariant. The postsynaptic region contains no MT but is usually filled with a meshwork of microfiaments (Landis and Reese, 1983). Besides the presence of actin, other proteins have been observed, particularly MAP2 and probably f3 tubulin (Caceres et al., 1983). We lack information on the progressive appearance and organization of these proteins during synaptogenesis. Microtubules have been observed in the presynaptic regions (Peters er al., 1970), and Westrum el al., (1983) suggested that during maturation in neonates MT may be involved in the formation and maintenance of the presynaptic dense projections and that they may act as channels for the first synaptic vesicles to their site of transmitter release. 2 . Mechanism of Regulation During neurogenesis various mechanisms occur regulating the number of neurons, dendrites, and synapses (Cowan, 1979). In most or probably all regions of the CNS, it has been shown that the initial populations of neurons generated exceeds the number observed at maturity. Spontaneous cell death serves as a regulatory mechanism to permit the adjustment to

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the correct number of cells. This phenomenon seems to be related with factors located in the target region in which the axons of the dying cells terminate. Despite the total lack of information on the role of MT in this phenomenon, it seems possible that they are involved due to the importance of the retrograde transport in the suggested mechanism of cell death. We have seen that dendrites are remodeled during the early postnatal period, usually during a critical period which seems concomitant with the onset of functional activity. Under normal and experimental conditions, it has been shown that the terminal branches of the dendritic tree undergoes a process of reorientation (Berry et al., 1980; Dardennes et al., 1984). It is interesting to compare these results with the modifications of the peripheral part of the dendrites and of the dendritic transport observed in case of reduction of the axonal traffic, implying a balance between the transport mechanisms in dendrites and axons and suggesting the hypothesis of the possible involvement of MT in these phenomena. In probably all regions of the CNS, there is at the end of synaptogenesis a transient period of excessive axonal branching, the “polyinnervation,” which is followed by a phase during which different groups of terminals compete with each other for some as yet unidentified entity. The result of this competition is that unsuccessful axons are withdrawn, although the parent cell and its axon persist. A carefully regulated trophic relationship between the target and the afferents seems necessary to validate or not validate the synaptic connection. It seems also attractive to suggest that MT, via their role in the axonal transport, may be involved in the process of selective stabilization of synapses.

111. Microtubules during Neurogenesis: Biochemical Analysis

Microtubules isolated from the adult brain are composed of various proteins: a structural protein (the tubulin), and MT-associated proteins (MAP), a collection of proteins defined on the basis of their binding and/ or putative interactions with MT. A number of reviews have been devoted to these different proteins (Kirschner, 1978; Luduena, 1979;Dustin, 1984; Vallee et al., 1984; Olmsted, 1986), and at present we will focus on some specific aspects in the developing CNS.

A. TUBULIN 1. Synthesis during Neurogenesis

Immediately after birth in rodents, the synthesis of tubulin rises to reach a maximum until P10 (10 days after birth), and thereafter although the amount of tubulin increases, the proportion decreases as other proteins

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are synthesized preferentially (Fulton and Simpson. 1979). Bamburg et al. (1973) found that the half-life of the colchicine-binding activity of tubulin decreased with increasing age, suggesting that tubulin may undergo some changes during development. Schmitt et al. (1977) showed also that the two pools of tubulin, soluble and particulate, do not exhibit the same behavior during postnatal development, as the soluble pool decreases from 33% of the total soluble fraction at birth to 20% at P20, whereas the rate of increase of the particulate pool parallels that of the total particulate proteins. Our results (Fig. 8) suggest that in the prenatal period in rodents, a dramatic increase of the rate of synthesis of tubulin is correlated with the appearance of the first postmigratory young neurons whose perikarya are diffusely and intensely stained with a tubulin antibody (Fig. 3). Our results also suggest that the most important pool of tubulin at this stage is the cold-stable (CS) fraction, which may represent 70-80% of the total amount of tubulin (Fig. 8). We do not know the mechanisms that trigger and regulate the initiation of synthesis of tubulin, but it seems that the location of the migrating cell is important as it always occurs just below

FIG.8. lsoelectric-focusing pattern of cold-labile (CL) and cold-stable (CS) fractions of tubulin from the tectal plate of mouse embryos at stages E9, El I , E13, and P20. Tubulin purified from 3 mg of tissue is loaded in lanes El I , E13. and €90of the CL fractions. whereas tubulin purified from 7.5 mg of tissue was loaded in lane E9 of CL fractions. Then 4 pI of the supernatant from the lysis of the high-speed pellet were loaded in the lanes of the CS fractions. These 4 pl correspond to 0.4 mg of the homogenate high-speed pellet (for details refer to Binet and Meininger, 1988a). lsotypes are numbered from the basic to acidic poles. lsotypes 1-8 corresponds to the a group, and 9-20 to the p group. Gel is silver stained.

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the intermediate zone (Fig. 3). It seems independent of the presence of axons in this zone, since we were able to observe the perikaryal increase in the intensity of fluorescence in the absence of axons. This increase seems to involve all the MT proteins, since it is correlated with the appearance of a similar increase of fluorescence when staining with a polyclonal antibody raised against MAP2 (Fig. 9). Regulation of tubulin synthesis at the genetic level remains obscure even in cell culture. It has been demonstrated that for most eukaryotes tubulin is encoded by small multigene families (Cleveland and Sullivan, 1985). After the pioneer observations of Ben-Ze’ev et al. (1979), it appeared that levels of tubulin synthesis are apparently established in animal cells by a novel autoregulatory mechanism in which tubulin mRNA stability is closely linked to the pool size of unpolymerized subunits. Alteration in tubulin subunit levels indicated that cells respond to an elevation of exogenous tubulin subunits by a specific and important repression of new tubulin synthesis (Cleveland et al., 1983). Pachter et al. (1987) demonstrated that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes, and they proposed two models of regulation. In the ftrst model, tubulin subunits bind directly to the aminoterminal coding sequences of a ribosome-bound tubulin mRNA. In the second model, tubutin subunits bind to the nascent tubulin polypeptide as it emerges from the ribosome. 2. Biochemical Basis of Cold StabiIity and Cold Lability of Microtubules With regard to the existence of different pools of tubulin in the nervous system, a differential sensitivity of MT to cold treatment has been recognized by numerous studies, but some discrepancies exist in the literature with regard to the definition of CS MT and the nature of this cold stability. As shown by Moms and Lasek (1982), axoplasm from the squid giant axon, and probably from most axons, contains three pools of tubulins: tubulin dimers in form of unpolymerized tubulin, MT in equilibrium with these dimers of tubulin, and MT that are not free to exchange with the dirneric tubulin. Black et al. (1984) and Brady et al. (1984) found polymerized and unpolymerized tubutin and demonstrated that the polymerized tubulin could be separated into a cold-soluble and a cold-insoluble fraction using cold treatment. These studies demonstrated that the cold-insoluble tubulin does not exchange freely with the cold-extractable pool. We have also demonstrated that these three pools of tubulins exist both in the central and peripheral axons (Binet and Meininger, 1988b) and in the developing CNS (Binet and Meininger, 1988a). Webb and Wilson (1980)and Margolis and co-workers (Margolis and Rauch, 1981; Margolis et al., 1986a) use

FIG.9. PEG-embedded 0.5-pm semithin sections of the cerebellar cortex of adult mouse (plate la, b, c) and of the tectal plate of mouse embryo (plate 2a, b, c). (plate la) Tubulin immunostaining; (plate Ibj immunostaining with a specific anti-MAP2 antibody; (plate Ic) phase-contrast micrograph of the corresponding region. It appears clearly that MAP2 antibody stains in adult large (large arrowhead) and medium-sized (small arrowhead) dendrites, and leaves intact the transversely cut parallel fibers, which are intensely stained with an antitubulin antibody (arrows). (plate 2a) immunostaining with an anti-MAP2 antibody; (plate 2b) phasecontrast micrograph of the corresponding region, which is the apical part of the tectal plate. The anti-MAP2 antibody faintly stains the axonal profiles of the intermediate layer (arrows). In plate 2c. immunostaining of the apical ventricular layer of the tectal plate showing the faint staining of a migratory young neuron (arrowheads).

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the term “cold-stable” to designate a CS fraction of MT prepared from whole-brain homogenates by cold extraction and purified by alternating cycles of warm and cold incubations. Margolis and co-workers (1986b) have characterized a complex of proteins which may be involved in cold stability, their STOP proteins. However, as demonstrated by Brady et al. (1984), the CS fraction defined by Webb and Wilson (1980) and Margolis and co-workers does not include the pellet of MT obtained during the purification by the initial cold treatment. They proposed to call this fraction the endogenous cold-stable or cold-insoluble tubulin. It is possible that some proteins responsible for cold stability, or cold insolubility, may be extracted from the cold-insoluble pool during cold extraction. Their incorporation into the cold-labile (CL) fraction may explain the existence of CS MT described by Webb and Wilson (1980) and Margolis and coworkers in this fraction. However, the existence of specific patterns of tubulin using two-dimensional electrophoresis and/or high-resolution isoelectric focusing (HIEF) (Brady ef al., 1984; Binet and Meininger, 1987, 1988a,b) suggest that both in the adult and embryonic nervous system, cold-insoluble tubulin represents a specific pool of tubulin and that cold stability is due at least in part to the assembly of specific components of the tubulin molecule, probably associated with specific companion MAP. As we do not know how to depolymerize the cold-insoluble pool of tubulin at the present time, it seems difficult to reject the possibility that this pool may represent membrane tubulin in totality or in part (Stephens, 1986). This point has been discussed by Brady et al. (1984) and in our own studies, and both of us clearly demonstrated that the cold-insoluble tubulin is not a mernbrane-bound pool of tubulin but a specific pool. We also demonstrated that this pool clearly corresponds to MT morphologically and not to rnernbrane-bound tubulin (Cohen et al., 1987). The existence of different response of MT to cold treatment and the specific localization of this pool in the differentiating neurons suggest that different MT classes might be assembled from different subgroup of tubulin to establish functionally distinct MT. 3 . a- and P-Tubulin Subunits Various techniques have demonstrated that tubulin in the native form exists as a dimer (Luduena, 1979; Dustin, 1984): the a and (3 tubulin, each with a molecular weight of approximately 50,000. These two subunits can be distinguished by their molecular weight, slightly lighter for the p subunit (Marotta et al., 1978); their PI, slightly more acidic for the p subunit (Perry and Wilson, 1982); and their amino acid composition (Luduena, 1979). a. a Subunit. OL tubulin comprises 450 residues plus a COOH-terminal tyrosine. This tyrosine residue can be removed or added by two enzymes

56

VINCENT MElNlNGER AND STEPHANE BlNET

which have been partially purified and characterized. Tubulin : tyrosine ligase (TTLase) is a 35,000 MW protein which catalyzes the ATP-dependent formation of a linkage of a tyrosine residue to the COOH glutamic acid residue of tubulin (Murofushi, 1980). TTLase has been found in all vertebrate tissues (Wehland et al., 1986), but particularly high activity has been found in the embryonic brain where it undergoes a strong developmental regulation apparently correlated with the morphological differentiation of asymmetric cells (Deanin et al., 1977; Rodriguez and Bonsy, 1978). A tyrosyl-tubulin specific carboxypeptidase (‘ITCPase) can remove this tyrosine residue. This enzyme detected in brain tissues has an apparent molecular weight of 90,OOO (Argarana et al., 1980),it shows a high degree of specificity for the carboxy-terminal tyrosine of a tubulin and it seems to act preferentially on polymerized tubulin (Kumar and Flavin, 1981). The importance of the tyrosinated (Tyr-) and detyrosinated (Glu-) MT has been emphasized by the introduction of a monoclonal antibody which recognizes only the tyrosinated form of the a tubulin (YL ‘h, Kilmartin et al., 1982) and of peptide-specific antibodies which distinguish respectively Tyr-a-tubulin and Glu-a-tubulin (Gundersen et al., 1984). It has been demonstrated that MT populations in a single cell are heterogeneous, most MT being stained only with the Tyr antibody and some stained only with the Glu antibody. In addition, some MT are stained with both. Cytoplasmic MT that stain with the Glu antibody are more limited in number and length than Tyr-MT, and they appear as “sinuous” or curly whereas Tyr MT are long and straight (Gundersen ez al., 1984; Gundersen and Bulinski, 1986). Analysis of polymerization in cell culture suggests that Glu- and Tyr-MT exhibit different behaviors with respect to growth and that most, if not all, Glu-MT are not part of the pool of dynamic MT (Webster et al., 1987), as defined by Schulze and Kirschner (1987). GluMT exhibit enhanced stability in comparison with Tyr-MT toward nocodazole treatment and toward dilution, but not toward cold treatment, suggesting that Glu-MT are modified at their ends (Gundersen et al., 1987), since cold treatment is able to allow depolymerization at sites other than at the ends (Mandelkow and Mandelkow, 1985). Gundersen er a/. (1987) proposed that MT become stable in the dynamic sense of Schulze and Kirschner (1987) by a sequence of events which requires, successively, ( 1 ) the binding of a capping protein at the growing end of MT, (2) a subsequent covalent modification (i.e., a detyrosination) and (3) detyrosination might be a signal to some other factors in the cell, which becomes associated to MT and modifies the function of these MT. The level of 01 tubulin that is tyrosinated or able to act as an acceptor for tyrosination decreased during the postnatal development of the brain (Barra et a / ., 1980). Using the specific YL ‘/z antibody, Cumming at al. (1983) dem-

MICROTUBULES IN DIFFERENTIATION AND MATURATION

57

onstrated that tyrosinated a tubulin is absent from or depleted in axons compared to dendrites and cell bodies in adult brain. Burgoyne and Norman (1986) showed that detyrosination in axons occurs close to or in cell bodies and not during its axonal transport, suggesting a segregation mechanism of the components of axonal MT in the cell body, similar to the mechanism demonstrated for T proteins (Tytell et al., 1984). During development of the rat cerebellum, at the postnatal period, the immature axons of the granule cells contain tyrosinated a tubulin, which disappears progressively during the period of axonal maturation and coincident with synapse formation by parallel fibers (Cumming et af., 1984). This switch from Tyr- to non-Tyr tubulin in MT of axons in cerebellum appears to correlate with the conversion of a rapidly growing axon into a mature axon, and it could therefore be related to changes in MT assembly properties or axonal transport in the developing axon (Burgoyne, 1986). This phenomenon does not occur in cell culture, suggesting that the failure of detyrosination may be related to the absence of normal intercellular contacts between granule cells and Purkinje cells in these cultures (Cumming et a[., 1984). Tyrosination is not the only posttranslational modification of a tubulin. Acetylation of the &-aminogroup of lysines of a tubulin has been described (Piperno et al., 1987) with properties of acetylated MT appearing similar to those of Glu-MT. In the nervous system, acetylated MT appear preferentially localized in axons compared with dendrites (Cambray-Deakin and Burgoyne, 1987), despite conflicting results from Black and Keyser (1987) in cell culture. Wandosell et al. (1987) described phosphorylation of a tubulin. This phosphorylation is at the carboxy terminal of a-tubulin subunit and is driven by an insulin receptor kinase in an insulin-dependent fashion with an incorporation of 3.3 mol of phosphate per mole of tubulin. The phosphorylation of a subunit at its COOH-terminal tyrosine precludes the incorporation of other phosphate residues in the a or p subunits. Glu- and Tyr-MT exhibit different behavior when phosphorylated, since upon phosphorylation Glu-MT retain their polymerization capacity, while TyrMT lose it. It has been demonstrated in cell culture (Bockus and Stiles, 1984) that cultured cells in which Tyr-tubulin and insulin receptors are present undergo changes in the organization of their MT network in the presence of exogenous insulin. The presence in axons of various posttranslational modification of tubulin seems related to the presence of stable MT. However, it seems important to raise different questions about stability and development. What is the definition of stability? Is it stability concerning the rate of renewal of MT (Schulze and Kirschner, 1987) and/or a particular behavior of MT

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VINCENT MElNlNGER AND STEPHANE BlNET

toward cold and/or drugs (Brady ef al., 1984)? It seems highly probable that differences exist between dynamically stable, cold-stable, and Vinca alkaloids- or nocodazole-stable MT. These differences raise the problem of the biological and functional significance of these different stability behaviors. Is cold stability, as suggested (Brady et d., 1984; Cohen er d., 1987),a specific and functionally important characteristic of axonal MT, and what are the relations between this cold-stable pool and a pool with a relatively slow rate of renewal? When and where does the stable pool of MT appear? It seems that drug- and cold-stable MT are specific to the axonal domain in the neuron. How is the stable pool of MT modified? Is it a property of the tubulin alone, or of MAP, or of both of them? Are the mechanisms involved in stability the same during the whole process of maturation of the neurons? b. p Subi~nit. The p subunit consists of 445 amino acid residues, and 41% of the molecule appears to be identical to the a subunit. Posttranslational modifications of the p subunit are less well known than those of the a subunit. One such modification shared by p tubulin is phosphorylation by Ca’ +-calmodulin kinase (Eipper, 1974; Luduena, 1979; Wandosell et al., 1986). Gard and Kirschner (1985) reported that in the mouse neuroblastoma cell, phosphorylation occurs at a Ser residue and it increases during differentiation and neurite outgrowth induced by serum withdrawal. This phosphorylation seems coupled to the level of cellular MT and might reflect developmental regulation of MT assembly during neurite outgrowth, rather than developmental regulation of tubulin kinase activity. The probable location of the Ser residue at the carboxy terminal of the p subunit (Eipper, 1974)emphasizes the role of this region (region 1V of tubulin) in polymerization-depolymerization of MT suggested by Serrano ef al. (1985). Indirect arguments in our material suggest a possible preferential localization of p subunits in the dendritic domain of the neuron and their modifications during development. These results favor our critical attitude toward the significance of neurites in cell culture and stand in the way of any generalization between the results obtained in culture and what occurs in vivo. Despite the differences between the cell lines, it seems that neurite outgrowth is correlated with a phosphorylation of a large number of MT proteins. as previously reported neurite outgrowth in PC12 cells induced by NGF is responsible for a phosphorylation of a number of MAP (Black et al., 1986). The similarity of the phosphorylation elicited during neurite outgrowth induced (PC12)or not (Nl15) by NGF suggests that NGF may not be an important factor involved in phosphorylation as proposed by Black et al. (1986).

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59

4. Other Aspects of Tubulin Heterogeneity

Evidence for an extensive heterogeneity of tubulin in brain was obtained by isoelectric focusing. Early reports demonstrated that tubulin from brain tissue, cultured neuroblastoma, and glial cells was resolved into three to nine subspecies, or isotypes (Gozes, 1982). These reports also demonstrated that tubulin heterogeneity increases during brain maturation in rat (newborn to mature) (Gozes and Littauer, 1978), with a critical period during P8-Pl2 (Dahl and Weibel, 1979). They suggested that some of this age-dependent modification was probably controlled at the mRNA level (Gozes et al., 1980). Gozes et al. (1980) suggested that tubulin heterogeneity pattern was different in various regions of the brain, and Gozes and Sweadner (1981) demonstrated that the extensive tubulin heterogeneity was not due simply to the superposition of tubulins derived from the many different cell types of the brain, since the tubulin of a single, isolated sympathetic neuron grown in culture possessed nearly the same number of isotypes as the whole brain. HIEF of tubulin was introduced by George et al. (1981), who demonstrated that the number of peaks and the mass distribution under each peak remained the same when the concentration of protein and/or of ampholytes was altered; that the multiple peaks observed are not a manifestation of tubulin-ampholyte interaction; that purified isoelectric variants yield single bands of the original isoelectric point upon refocusing; that tubulin subunits probably consist of polypeptides with both constant and variable regions in their sequences. They also demonstrated that the number of isotypes obtained by HIEF was independent of the method of extraction of tubulin, a result confirmed by Tran et al. (1987). Since that report, several studies have been devoted to the analysis of tubulin heterogeneity in brain (von Hungen et al., 1981; Denoulet et al., 1982; Wolff et al., 1982; Sullivan and Wilson, 1984; Field et al., 1984; Binet and Meininger, 1987, 1988a,b; Tran et al., 1987). These studies allow the following observations: 1. The tubulin heterogeneity extends between 18 (Field et al., 1984) and 21 (Wolff et al., 1982; Tran et al., 1987), probably depending on the degree of separation of the gels. 2. The extent of tubulin heterogeneity appears extremely similar in various species (rat, calf, pig, chicken, lamb, and human) (Field et al., 1984), even though small differences may be observed when analyzing similar regions in mouse and human. 3. The number of isotypes remains identical, but their relative proportion varies when analyzing different discrete regions of the adult mouse (Tran et al., 1987) and human brain, suggesting a correlation between the function of neuronal subpopulations and the isotubulin pattern.

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VINCENT MEININGER AND STEPHANE BINET

4. The number and relative proportion of isotypes differ in the axonal domain of the neuron compared to the perikaryal and dendritic domain (Binet and Meininger, 1987, 1988b), suggesting a mechanism of selective assembly of tubulin isotypes in the axon similar to the mechanism described for detyrosinated and acetylated tubulin (Burgoyne and Norman, 1986; Cambray-Deakin and Burgoyne, 1987) and for T proteins (Tytell et al., 1984). 5. The number of isotypes and their relative proportion differ when the two pools, cold-stable and cold-labile, of both central (Binet and Meininger, 1987) and peripheral (Binet and Meininger, 1988b) axonal tubulin are analyzed, suggesting that part of cold stability may be related to the arrangement of the different tubulin isotypes within the MT. Tubulin heterogeneity vanes during brain development both in mouse (von Hungen et al., 1981; Wolff et al., 1982) and in chick (Sullivan and Wilson, 1984), with a modulation of both the number and relative proportions of isotypes. These reports suggest that the number of (3 isotypes increases, whereas the relative abundance of a isotypes dramatically changes with an accumulation of the more acidic variants during maturation. They also suggested that some isotypes may disappear (particularly ct6 and &) during postnatal maturation (von Hungen et al., 1981). Despite their importance, the results obtained during development and maturation cannot be interpreted with regard to a correlation between the modifications observed and the events occurring during neurogenesis for three reasons: first, few of these studies have been performed during the earliest stages of development, thus missing events which occur during the initial, and very important, stages (i.e., cell proliferation, migration, axonal and subsequent dendritic growth); second, all these studies have been performed using whole-brain extracts, mixing populations at quite different stages of maturation (the craniocaudal gradient of maturation in Jacobson, 1978); third, all these studies analyze only the cold-extracted pool of MT, avoiding the endogenous cold-stable, which has a specific pattern of isotypes and which represents a large proportion of the total MT population both in neurons and BNC (Brady et al., 1984; Binet et al., 1987; Binet and Meininger, 1988b). We studied the modifications of tubulin heterogeneity during development in a discrete region of the mouse brain, the tectal plate, at various and well-defined stages of neurogenesis (Binet and Meininger, 1988a). As shown in Fig. 8, during axonal growth there is a dramatic increase of the cold-labile pool of MT. Despite the difficulty in obtaining adequate quantitites of CL tubulin at stage E9, it seems that changes both in the multiplicity and in the relative quantity of the tubulin isotypes occur at the end of neuronal migration and during axonal growth. In both a and (3

MICROTUBULES IN DIFFERENTIATION AND MATURATION

61

groups at stage E9 most isotypes corresponding to isotypes seen at El I are faintly stained but still visible-except for isotypes 5, 8, and 9, which probably appear progressively, or their amount largely increases, between these two stages. In the CS pool of tubulin, few modifications occur between stages E9 and El 1. During dendritic growth, which occurs at stage E13, no modifications of the number of isotypes occur in either the CS or the CL fractions. However, the most obvious modification was an increase in the amount of CL tubulin. Dendritic maturation and reorientation, during stages PS-P20, is marked in the CL fraction by the appearance of the most acidic p isotypes and the relative decrease of the’proportion of a group. The CS fraction is characterized by the increase in amount of isotypes 7 and 8 and the decrease of isotype 3. These results suggest that this critical period is marked by profound modifications of MT correlated with a morphogenetic event, the remodeling of the dendritic tree, and a functional validation of the neural network.

5 . Nature and Possible Significance of Tubulin Heterogeneity As previously discussed, different gene products account for some of the heterogeneity of tubulin (Cleveland and Sullivan, 1983, and posttranslational modifications for the remaining heterogeneity. Various arguments favor a close correlation between tubulin heterogeneity and MT function. However, it is probable that no isotype may be responsible per se for a specific function: an argument is the diversity of the patterns of isotypes obtained in the endogenous axonal CS fraction during development and in various regions of the adult brain. Neurons from a discrete region of the brain progressively acquire a specific pattern of MT, in relation to the intercellularcontacts established and to the functional validation. It is also probable that in a single neuron one or more mechanisms exist permitting the selective assembly of these isotypes to obtain specific patterns in the perikaryal and dendritic domain on the one hand and in the axonal domain on the other; in each of these domains, stable and labile MT-both in the sense of dynamics and of resistance to cold and/or drugs-acquire a specific pattern of assembly of the isotypes, which may play a role in the acquisition of this function but not alone and in close relation with MAP. The pattern of isotypes may intervene on the possibility for MT to incorporate one or another type or subtype of MAP.

B. MICROTUBULE-ASSOCIATED PROTEINS Microtubule-associated proteins (MAP) are composed of a group of molecules defined initially on the basis of their binding to MT during polymerization in vitro. Another group of proteins is connected to the MAP.

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VINCENT MEININGER AND STEPHANE BlNET

This class of proteins, less well known in composition than the former one, is defined by its interactions, even transient, with MT (Olmsted, 1986). This class includes proteins associated with MTOC, such as centrosomes and kinetochores, and proteins identified by their binding to MT in immunostaining. Extensive reviews have been devoted to MAP (Vallee, 1984; Vallee et a / . , 1984; Wiche, 1985; Olmsted, 1986), and we shall focus our attention only on the biochemistry, localization, and possible role of MAP during neurogenesis. 1 . Procedure c$ isolation of MAP

Various procedures for isolation of MAP have been used (Olmsted, 1986; Vera et al., 1987). In the procedure of cycled assembly of MT, MAP are the proteins that copurify in constant stoichiometry with tubulin and of which the association with MT depends on the formation of polymer. In this procedure, a MAP is a MT protein that can be sedimented by centrifugation under polymerization conditions in warmth, but not under depolymerization conditions in cold. This procedure allows isolation of the MAP only from the brain MT. Among these MAP, three groups promote the assembly of MT in virro at much lower concentrations than did tubulin, and also stabilize existing polymers: the two high molecular weight proteins (HMW-MAP) and the T proteins. The HMW proteins appear as fine filamentous arms on the surface of the purified MT (Herzog and Weber, 1978; Kim et al., 1979; Voter and Erikson, 1982). On 7.5% polyacrylamide gels, the HMW-MAP appear as two electrophoretic species, the MAPI (300,000-350,000) and the MAP2 (270,000)(Sloboda et al., 1975), each of them being resolved into multiple species when examined on more highly resolving electrophoretic systems. Recently, another HMW protein, the MAP 3 ( 180,000), has been isolated by the same procedure from the brain (Huber et a / . , 1985). The 7 protein isolated from mammalian adult brain consists of four heat-stable polypeptides of different molecular weights (53,000,56.000,59,000, and 63,000) (Cleveland et al., 1977a,b; Drubin and Kirschner, 1986). As judged by peptide mapping (Cleveland et a / . , 1977a, 1979) and amino acid composition, all these proteins are related, and they share epitopes recognized by monoclonal antibodies (Binder et al., 1985). Translation studies in vitro show that the four bovine T proteins are primary translation products; therefore, they are not derived proteolytically from one precursor (Drubin et al., 1984). Further T heterogeneity results from multiple phosphorylation sites on each T polypeptide (Drubin and Kirschner, 1986). Another group of proteins has been described associated with the CS fractions of MT, obtained after the first cycle of cold depolymerization (Margolis and Rauch, 1981). Different proteins have been iso-

MICROTUBULES IN DIFFERENTIATION AND MATURATION

63

lated (50,000, 70,000-82,000, 145,000)and called STOP proteins, claimed to be involved in cold stability (Margolis et al., 1986a,b). Thus all these studies show that MAP, such as tubulin, exhibit an important heterogeneity in brain MT. The procedure of isolation from cycled assembly was largely unsuccessful in purifying MAP from other tissues than brain, and other procedures were developed. The taxol-driven assembly of MT described by Vallee (1982; Vallee and Collins, 1986) permitted the same microtubular components (tubulin and MAP) to be obtained compared with the temperature-driven assembly, and led to the identification of a wide range of closely related proteins isolated from cells of neuronal and nonneuronal origins (Vallee and Collins, 1986). Some disadvantages of these two procedures, particularly the fact that MAP are exposed to undesired enzymatic modifications including proteolysis and phosphorylation during these purification conditions (Sloboda et al., 1975; Sandoval and Weber, 1978; Lindwall and Cole, 1984; Serrano et al., 1986), led Vera et al. (1987) to propose an affinity purification procedure based on heat stability (Herzog and Weber, 1978; Vallee, 1985) and binding to calmodulin (Lee and wolf, 1984; Kumagai et al., 1986). 2 . Microtubule-Associated Protein 2 MAP2, the most abundant MAP of the brain, is a heat-stable protein with an apparent molecular weight of 280,000-300,OoO in SDS-PAGE and which appears hydrodynamically as a monomer of 220,000 MW (Hernandez et al., 1986). The molecule is a flexible and noncompact protein, 90 (Gottlieb and Murphy, 1985) to 185 nm (Voter and Erikson, 1982) long, with a 100-nm axial periodicity (Gottlieb and Murphy, 1983) along the MT. The MAP2 molecule has two structural domains corresponding to two functional activities ascribed to this protein (Vallee, 1980). Upon digestion with trypsin or chymotrypsin, MAP2 can be divided into a small fragment of MW 32,000-39,000 and a large fragment of MW 240,000-270,000. The large fragment is the projection domain of the molecule which extends the wall of the MT and is involved in the interactions between MT and surrounding organelles such as membrane-bound organelles, MT, microfilaments, or intermediate filaments (Vallee et al., 1984). The smaller domain contains the portion of the molecule that binds to the MT surface and to the tubulin. This assembly-promoting domain maintains the MT assembly-promoting activity of intact MAP2 (Vallee, 1980) and binds to the carboxy-terminal region (region IV) of a-and p-tubulin subunits (Serran0 et al., 1984;Littauer et al., 1986) with a probable stronger interaction with the p subunit (Littauer et al., 1986), this binding being prevented by

64

VINCENT MEININGER AND STEPHANE BINET

the phosphorylation of tubulin with Ca”-calmodulin-dependent kinase and casein kinase. MAP2 is subject to phosphorylation both in vitro and in vivo. Various enzymes modulate the phosphorylation in vitro. An endogenous CAMP-dependent protein kinase type 11 or protein kinase A (Sloboda et af., 1975; Vallee, 1980; Murthy and Flavin, 1983) is tightly bound to the projection domain of the molecule (Vallee el ul., 1984) and has a high affinity for M A E . A Ca”-calmodulin-dependent protein kinase also phosphorylates MAP2 and seems more active than the CAMP-dependent kinase (Goldenring er af., 1985; Yamamoto et af., 1985). This enzyme has a widespread distribution in brain and has broad substrate specificity, being active on synapsin I (Nairn and Greengard, 1987), vimentin, MAP2 (Schulman er al., 1985), and tubulin (Wandosell er al., 1986). Other kinases also seem to be active on MAP2 such as a calciumphospholipid-dependent kinase (Tsuyama et a[., 1986) and a serine-threonine kinase activated by insulin (Ray and Sturgill, 1987). These kinases phosphorylate MAP2 in a 42,000 peptide that contains the tubulin-binding domain, but they differ in the level and localization of the sites at which they phosphorylate the projection domain (Hernandez et af., 1987). Different values of phosphate incorporated per mole of MAP2 have been reported, varying from I .7 to 21 (Theurkauf and Vallee, 1983; Yamamoto et ul., 1985). As suggested by Hernandez er af. (l987), the phosphorylation pattern of MAP2 might change and be dependent on the action of different extracellular signals, which may promote such phosphorylation after regulating the intracellular concentration of compounds that activate specific kinase at specific intracellular sites (cytoplasm, inner surface of cell membrane, synapse). The state of phosphorylation of MAP2 seems crucial to the MT assembly-promoting activity of the molecule in vitro (Jameson and Caplow, 1981; Burns et af., 1984), and it has been shown that extensive phosphorylation (24 moUmol) decreases the rate of assembly and increases the loss of tubulin subunits from both MT ends (Murthy and flavin, 1983). A population of endogenous phosphate sites (10 mollmol) is located on the projection domain, but the enzyme(s) responsible for the phosphate turnover and removal has not been isolated. The role of MAP2 in vivo in the dynamics of MT remains obscure. Some results suggest that they play a major role in the structural organization and stability of MT in cells (Farrell er al., 1987). Kumar and Flavin (1985) have determined that the binding activity of MAP2 is lower with detyrosinated MT, suggesting a role of this molecule in the dynamic stability of MT. Besides the MT assembly-promotingactivity of the M A R , this molecule seems to play a major role in the interactions of MT with other cellular components, particularly other MT and intermediate filaments (Bloom and Vallee, 1983). It has been shown that these effects differ from the effect

MICROTUBULES IN DIFFERENTIATION AND MATURATION

65

of T proteins (Black, 19871, a result which might explain the differences in the packing density of MT in dendrites compared to axons. The mechanisms that control this activity are not clearly understood, but it seems probable that phosphorylation of the projection domain may play a role. Different reports (Heimann et al., 1985; Papasozomenos et al., 1985) suggest that there may be specific sites on the MAP2 or its subspecies for the 70,000 polypeptide of the neurofilament triplet, probably on the MTbinding domain of the molecule (Flynn et al., 1987). MAP2 seems also involved in the modulation of the formation of high-viscosity gels of MT and actin filaments (Sattilaro et al., 1981; Pollard et al., 1984), and can induce the reversible formation of actin filament bundles. Phosphorylation modulates this activity, extensive phosphorylation decreases the gelation of actin (Pollard et al., 1984), and calcium
66

VINCENT MEININGER AND STEPHANE BINET

scribed in PC12 cells concomitant with the organization of MT bundles in the neurites (Black et al., 1986).These results, correlated with the wellknown phenomenon of postnatal reorganization of the dendritic tree occurring between P8 and P20 in rodents (Jacobson, 1978; Dardennes et al., 1984). suggest that the modification of MAP2 may be involved not in the assembly of dendritic MT-since MT are observed in dendrites at earlier stages-but in a process of stabilization of the dendritic cytoskeleton possibly triggered by functional and/or hormonal factors. It is also possible that the postnatal modifications of the p tubulin observed during this period may be correlated with the modifications of MAP2, since both molecules seem to have close relationships (Littauer et al., 1986). The localization of MAP2 during development has been carried out in different regions of the brain both in rdtro and in situ and at different stages of development. Earlier observations performed in the cerebellum (Bernhardt and Matus, 1982), in the cerebral cortex (Crandall et al., 1986), hippocampus (Caceres el al., 1984b; Matus et al., 1986), and cerebellum ( Alaimo-Beuret and Matus, 1985) suggested that MAP2 was located initially in dendrites, and sometimes (Bernhardt and Matus, 1982)occurred before the appearance of MT. suggesting that MAP2 acted as a neuronal specifier of dendritic domain and plays a secondary role in the assembly of MT (Burgoyne and Cumming, 1984). The observation of MAP2 immunoreactivity in the axonallike neurites in cell culture during a transient period (Caceres et al., 1986) raised the possibility of the existence of a transient stage in the axonal growth during which MAP2 is present in this process, or the existence in cultured cells of a transient stage of the neurites during which neurites are in an undifferentiated state (Alaimo-Beuretand Matus, 1985; Kosik and Finch, 1987). However, our observations (Binet et al., 1987) (Fig. 9) of an immunoreactivity against MAP2 in situ, in the axonal profile of the postmigratory young neurons in the intermediate zone 2 days before the appearance of the dendrites favors the hypothesis that MAP2 is really present in the growing axons, before the appearance of the dendrites, and disappear later with the differentiation of dendrites. Biochemical analysis confirmed that immunoreactivity against MAP2 was not explained by a cross-reactivity with another MAP subspecies (Fig. 10). This result also favors the hypothesis that the axonal domain of the neuron acquires its specificity progressively during neurogenesis. It seems probable that this phenomenon is correlated with the initiation by the perikaryon of mechanisms which specify the proteins involved in the assembly of axonal MTs. 3 . Microtubule-Associated Protein I

MAPl, though distinguished from MAP2 very early, was not purified until recently (Vallee and Bloom. 1983; Vallee and Davis, 1983) and has

MICROTUBULES IN DIFFERENTIATION AND MATURATION

67

FIG. 10. 7.5% Polyacrylamide gel of a total extract (TE) of the tectal plate of mouse embryo and of microtubules (MT) purified from the tectal plate of the mouse embryo using taxol. Embryos were obtained at stage E l l . Gel is silver stained. Both MAPI and MAP2 are observed at this stage. Tub, tubulin.

been less extensively studied than MAP2. This HMW protein (350,000) also appears as a MT assembly-promoting factor and also exhibits the appearance of a filamentous arm on the MT surface (Vallee and Davis, 1983). It is associated with lower molecular weight components (70,000, 54,000 and 39,000) which are not involved in the binding to MT (Vallee and Davis, 1983). Several proteins seem immunologically related to MAPl , particularly in the nucleus and mitotic apparatus (Sato ef al., 1984; de Mey et al., 1984) and in stress fibers (Asai ef al., 1985). MAP;! and MAPl compete with each other during binding experiments of MAP and tubulin (Kuznetsov ef al., 1981), and they are localized on the same MT (Shiomura

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VINCENT MElNlNGER A N D STEPHANE BlNET

and Hirokawa, 1987). probably both of them involved in the interactions between MT. Recent observations demonstrated that MAPI is also a heterogeneous protein, composed of three polypeptides designated MAPla, MAPlb, and MAPlc in order of increasing electrophoretic mobility (Bloom e f al., 1984). These polypeptides exhibit different sensitivity to proteolysis, are different in one-dimensional peptide mapping, and share specific epitopes (Bloom el ul., 1984, 1985a). Most of MAPlb remains soluble during polymerization, indicating that it polymerizes much less efficiently with MT than MAPIa and MAP2 (Bloom et al., 1985b). The localization of MAP1 in the adult nervous system is still a matter of debate. Bloom ef ul. (1984) observed a wide distribution of MAPla both in neurons and glial cells, particularly oligodendrocytes and within neurons. both in the axonal and perikaryal-dendritic domain. Wiche et ul. (1984),using a polyclonal antibody, did not report a staining of glial ceHs. The axonal localization of MAPIb and MAPla was confirmed by Hirokawa rt ul. (1985). and these authors suggested that these molecules were involved in rapid axonal transport. During postnatal maturation of the nervous system, Riederer and Matus (1985) and Bernhardt and Matus (1984) observed that at birth MAPI is present only in axons and its staining is weak, but the intensity of staining increases progressively to reach its maximum at P20, which is correlated with its appearance in dendrites. These results are too fragmentary to draw any conclusions, but it seems that dendritic reorganization is associated with a progressive modification of MT proteins. Calvert ef al. (1987)reported the existence of an axon-specific MAPI, the MAPlx, which seems to decrease during axonal maturation. 4. Other High Molecular Weight Proteins Huber ef al. (1985, 1986) described another MAP, the 180,000 MAP3 which is present both in neurons and glial cells in sifu and in culture. In neurons, it is detected only in neurofilament-rich axons. Two isotypes have been described, MAP3a and MAP3b, with a progressive sequence of appearance (3a before 3b) and with a dramatic decrease in staining intensity during PI0 and P20 to become restricted to neurofilament-rich axons (Riederer and Matus, 1985). Parysek et al. (1984, 1985) have described a MAP4 (240,000) which is localized exclusively in glial cells.

5 . r Proteins 7 Proteins consist of four closely related polypeptides, with molecular weight between 50,000 and 6O,OOO,which can be phosphorylated (Lindwall and Cole, 1984) and have calmodulin-binding sites (Lee and Wolf, 1984).

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They display the same location on the surface of MT as MAP2 (de la Torre et al., 1986), and binding experiments showed that these two molecules compete with each other (Sandoval and Vandekerckhove, 198l ) and that their tubulin-binding domain is identically located at the anionic C-terminal region (Serrano et al., 1985). They do not project outward from the wall of MT. These differences between T protein and MAP2 suggest that in axons T proteins are not important in specifying MT spacing in situ and that they are probably involved in the regulation of MT dynamic stability (Black, 1987; Drubin and Kirschner, 1986). These proteins can promote tubulin polymerization (Cleveland et d.,1977a), and they are localized exclusively in the neurons in situ and appear to be specific for the axonal domain (Binder et al., 1985). However, it seems that not all isotypes of T proteins are exported in axons, since Tytell et al. (1984) observed only two of the four T proteins synthesized by the cell body in the optic nerve, suggesting that the perikaryon commits to the axonal domain only part of MT proteins synthesized by the cell body and that a mechanism of selection of these proteins exists before the axonal domain. It is not clear whether T proteins intervene in the association between MT and other cytoskeletal elements despite the reports of Seldon and Pollard (1983) and Runge et al. (1979). In cell cultures, Drubin and Kirschner (1986) demonstrated that T proteins increase MT assembly and stability. Earliest reports (Francon et al., 1982) suggested that during postnatal development T proteins were modified with a progressive shift from young forms of T to adult forms. They postulated that these modifications were associated with an increasing MT assembly-promoting activity of the T factors. Ginzburg et al. (1982), using translation assays in vitro, suggested that the regulation occurred at both transcriptional and translational levels. Drubin et al. (1984) also observed a modifcation of the number and amount of T polypeptides during development, but they also determined that embryonic T protein is translated from a 6-kb mRNA that persists throughout development. Other proteins, of 69,000, 72,000, and 80,000, named chartrins have been isolated (Magendantz and Solomon, 1985). These proteins are different from the T proteins and are highly phosphorylated. Their state of phosphorylation seems to depend on the presence of MT in PC12 cells, and they probably intervene in MT stability and neurite elongation (Black e f a / . , 1986; Aletta and Black, 1987). IV. Summary and Conclusions

The developing nervous system has proved to be a very powerful tool to analyze how MT are involved in basic biological processes such as cell

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proliferation. cell migration, cell shaping, and transport. A better knowledge of the basic events occurring during neurogenesis also affords u s the possibility of establishing the basis of experiments and trying to solve unanswered and important questions. Despite the considerable value of cell culture, we need to use more discrete regions of the developing brain in situ in order to analyze the MT and their modifications into cells developing their “natural” environment. One major problem remains the question of the mode of assembly and disassembly, that is, the behavior of MT in Living cells. Dynamic instability andlor treadmilling are accurate interpretations of the dynamics of MT at least in vitro or in cell culture, but we do need more information on what happens in siru and in vitro. One of the main tasks of cell biologists is to devise satisfactory tests to approach this fundamental question. In this view, pharmacological manipulation of embryos treated in whole-embryo culture systems might be a possible way. Microtubules are ubiquitous cell components. However, the extensive heterogeneity of MAP and tubulin in the CNS confers on the neurons a wide range of capabilities of assembly of these proteins and suggests that the neuron has a unique potential of a relation between MT composition and cell function. We have seen that each major event during neurogenesis is related to a specific series of modifications of the MT components. I t remains to be determined if there is a causal or just a correlative relationship between the appearance of specific isotypes and the occurrence of specific events and/or functions. We have also to determine the exact spatial and temporal relations among the different isotypes of MT proteins, tubulin, and MAP. Is there a close correspondence between a tubulin and a MAP isotype? Can the appearance of one isotype of tubulin influence the appearance and the assembly of a specific MAP, or vice versa? Recent results obtained with the Tyr- and Glu-MT shed light on these questions and suggest a whole series of possibilities for cells to modulate the structure, behavior, and function of MT in specific domains of the neuron or in specific regions of the brain, by only a minute modification of the molecule of tubulin. Microtubule protein heterogeneity raises also a number of questions. It is fascinating to observe that neurons are the only cells in organisms that are unable to reenter the mitotic cell cycle. They are also the only cells in an organism which acquire such an important heterogeneity of MT proteins. Is there a causal relationship between these two phenomena? Is there also a causal relationship between MT heterogeneity and the characteristic spatial polarity of neurons? How does the neuron regulate the rate of passage and the type of proteins exported in its two different domains, the axonal and the dendritic-perikaryal domains? Are specific

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intercellular contacts, as synapses, involved in the regulation of the expression of the M T proteins between two neurons, the basis of the specificity of MT observed in functionally related groups of neurons? Observation of the maturation of the nervous system suggests that this process is never arrested and that morphological and molecular modifications continue to occur at various rates in the “mature” and “aging” brain. Microtubules are not sheltered from these modifications. Numerous arguments suggest that they are involved in normally and pathologically aging nervous systems. Studies devoted to the analysis of MT during maturation and aging could shed light on how the different mechanisms regulating MT proteins are modified and what the consequences of these modifications are on the neuronal processes.

ACKNOWLEDGMENTS We are indebted to J. de Mey and M. de Brabander, who kindly introduced us to the world of microtubules and constantly helped us in our research. We also thank them and A. Fellous for kindly providing antitubulin, anti-MAPI, and anti-MAP2, respectively. We also wish to thank J. Wolosewick for perfecting the PEG technique and for helpful discussions. The work described from the authors’ lab was supported by UER Broussais-Hbtel-Dieu, Ministere de I’Education Nationale, and UER Biomedicale des St-Pkres grants. This review covers work published through November of 1987.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 114

Generation of Cell Diversity during Early Embryogenesis in the Nematode Caenorhabditis elegans SUSANSTROME Department of Biology and Institute for Molecular and Cellular Biology, Indiana University, Bloomington, Indiana 47405

I. Introduction

The newly fertilized egg is remarkable in its ability to generate the complex array of diverse cell types seen in adult metazoan organisms. One of the mysteries in this process is when and how early embryonic cells become "determined" to follow different developmental pathways. Two general mechanisms appear to operate: determination by internally transmitted information and determination by external cues. The differences in embryonic programs seen in different organisms largely reflect the timing and relative contributions of these two different mechanisms. The objective of this review is to evaluate when and how cell diversity is generated in embryos of the nematode Caenorhabditis elegans. This organism has become an especially popular developmental system within the last decade, mainly because of its simplicity, invariant development, and genetic manipulability. Its early embryonic program provides a graphic example of the generation of asymmetry and the production of cells that differ visibly as well as developmentally. Embryo manipulation experiments have provided evidence for both internal and external instructing of cells, both of which occur early, and genetic analysis is identifying the genes involved in these early determination events. After introducing the organism, we will consider the experimental embryology in detail. A. LIFECYCLE OF C . elegans The basic body plan, reproduction, and life cycle of C. elegans have been described by Honda (1925), Nigon (1949, 1965), and Nigon et al. (1960), Brenner (1974), and Hirsh et ai. (1976). Caenorhabditis elegans, which naturally grows in the soil, is maintained in the laboratory on agar plates spread with Escherichia coli as a food source. Worms are generally grown at 15"-25"C. Although dioecious nematode species exist, C. elegans 81 Copyright Q 1989 by Academic Press. Inc.

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generally reproduces as a hermaphrodite, which produces both sperm and oocytes and carries out internal self-fertilization. Hermaphrodites have five pairs of autosomes and are XX. Males, which have five pairs of autosomes and are XO, arise at a frequency of about 1 in 700 worms as a result of meiotic X chromosome nondisjunction. Mating of hermaphrodites to males leads to the production of “outcross” progeny, enabling genetic analysis of this organism (Brenner, 1974). It takes approximately 15 hours at 20°C for a newly fertilized embryo to complete embryogenesis, and an additional 3 days for the newly hatched larva to grow into a gravid adult hermaphrodite. During the 3 days of larval growth, the worm increases in length from 0.24 to 1.2 mm and must molt and replace its cuticle four times to accommodate its increasing size. A wild-type adult hermaphrodite makes approximately 300 sperm and, if never mated, produces a similar number of progeny. If mated to males, a hermaphrodite can produce up to 2500 progeny (Hodgkin, 1983). From the time of hatching the average life span is 18 days at 20°C (Johnson and Wood, 1982). However, under conditions of starvation or crowding, worms can enter an alternative longer-lived larval stage termed “dauer” after the second larval molt. Dauer larvae are resistant to desiccation and treatment with harsh chemicals and can survive for months. Upon feeding, dauer larvae molt into fourth-stage larvae and resume development. B. BODY PLANAND CELLTYPES

Adults of C. eiegans are composed of a relatively small number of somatic cells (959 for a hermaphrodite, 1031 for a male; Sulston and Horvitz, 1977; Kimble and Hirsh, 1979; Sulston et a / . , 1983) arranged into a simple tubular body plan (Fig. I): a digestive tract, excretory system, musculature, nervous system, and gonad are surrounded by a cylindrical layer of hypodermal cells and a cuticle. In addition, adult hermaphrodites contain about 2500 germ cells, which includes both oocytes and sperm; males contain about lo00 germ cells, including sperm (Hirsh er a / . , 1976). Detailed descriptions of tissues can be found in Sulston and Horvitz ( 19771, Kimble and Hirsh (1979). Sulston er al. (1983), and in the references below. Briefly, the digestive tract consists mainly of a muscular pharynx (80 cells; AIbertson and Thomson, 1976) attached to the intestine (34 cells). Bacteria are drawn into the frst bulb of the pharynx, crushed in the second bulb. and passed into the lumen of the intestine and eventually out through the rectum. The excretory system (4 cells; Nelson el al., 1983) is an H-shaped duct system that probably functions in the removal of waste material from the body cavity and in osmoregulation. The body wall muscles (95 cells; Zengel and Epstein, 1980) are arranged longitudinally in two

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ovary

-

Y

/

intestine

vulva

intestine pharynx

embryos

sperm

testis deferens copulatory apparatus

FIG.1. Cuenorhabditis &guns hermaphrodite (above) and male (below), showing some of the anatomical features and sexual dimorphisms. Adapted from Sulston and Horvitz (1977).

subdorsal and two subventral rows underlying the hypodermis. Their coordinated contraction and relaxation propel the worm in a sinusoidal pattern either forward or backward. Other muscles are located in the pharynx, the hermaphrodite vulva, and the male tail. The nervous system (350 cells) has been completely reconstructed from serial electron micrographs (Ward et al., 1975; Ware et al., 1975; White et al., 1986). It consists of a nerve ring located between the two bulbs of the pharynx, dorsal and ventral nerve cords, and a variety of ganglia. Most of the sensory receptors are located in the head, enabling worms to sense touch, temperature, and chemicals, and to respond appropriately. The hypodermis and cuticle surround the internal tissues described above. The hypodermis (154 cells, many of which fuse into one large syncytium) secretes the collagenous cuticle that covers the worm. In addition, special hypodermal cells (32 seam cells) secrete cuticular ridges, termed alae, that run along the lateral sides of the worm. The somatic tissues described above are for the most part similarly generated and positioned in hermaphrodites and males. The most dramatic sexual dimorphism is in the ventral hypodermis, which generates a vulva close to the midpoint of the hermaphrodite and a copulatory bursa at the posterior end of the male (Fig. 1; Sulston and Horvitz, 1977). These sexually distinct hypodermal structures are associated with sexually dimorphic musculature and innervation. The other dramatic difference between hermaphrodites and males is in their somatic gonads (Kimble and Hirsh, 1979). The hermaphrodite gonad (143 cells) consists of two reflexed, tubular arms, each of which contains an ovary, oviduct, spermatheca, and uterus

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(Fig. I ) . In contrast, males contain a single reflexed gonad arm (56 cells) with a seminal vesicle, vas deferens, and cloaca (Fig. I ) . Within the somatic gonads, the first germ cells to enter meiosis become sperm in both hermaphrodites and males. Sperm continue to be produced throughout the life of the male, whereas normal hermaphrodites switch from spermatogenesis to oogenesis during early adulthood.

C. CELLLINEAGE AND

AN

OVERVIEW OF DEVELOPMENT

The small size, transparency, and relative simplicity of C. elegnns have enabled the determination of the entire somatic cell lineage of this organism. This tour de force was accomplished by monitoring cell division patterns and fates during embryogenesis (Deppe e? al., 1978; Sulston er n l . , 1983) and larval development (Sulston and Horvitz, 1977; Kimble and Hirsh, 1979) using differential interference contrast microscopy. The analysis has revealed the following:

I . The pattern of somatic cell divisions is essentially invariant between individuals; the few exceptions are found during postembryonic development of the somatic gonad, ventral hypodermis, and male tail. 2. Cell deaths, which are also invariant, contribute to pattern formation. 3. Very few tissues develop clonally; most embryonic “founder cells” contribute to multiple tissues (Fig. 2 ) , and most tissues are derived from the progeny of multiple founder cells. 4. Very few cells undergo long-range migrations; cell division patterns appear to generate cells in approximately correct positions relative to each other. The overall developmental sequence is as follows. Six embryonic founder cells are generated by the 28-cell stage of embryogenesis (Figs. 2, 3). The first half of embryogenesis is marked by cell proliferation, differentiation, and organization of cells into tissues (Figs. 3, 4). During the second phase of embryogenesis the embryo elongates, synthesizes a cuticle, and hatches from the eggshell (Figs. 3, 4). At hatching, the first-stage larva (558 cells) contains a nearly full complement of body wall muscles, and the pharynx and anterior sensory nervous system are nearly complete. The intestine, motor nervous system, and hypodermis are functional, but will increase in cell number during postembryonic development. At hatching, the hermaphrodite and male contain an identical 4-cell gonad primordium and are morphologically identical. During postembryonic development, the gonad primordia develop differently in hermaphrodites and males. In addition, several lineages generate the sexually dimorphic accessory structures required for mating and egg-laying by adult worms.

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Po [zygote) I

AB

I

PI

muscle neurons

Fic. 2. Modified early lineage tree showing the five somatic founder cells (AB, MS, E, C, D)and the germ lineage (Po-P4). A cell anterior to its sister is on the left arm of a lineage branch. The areas of the circles and sectors are proportional to the number of cells generated by each founder cell. Stippling represents ectoderrnal tissue and striping mesodermal tissue. Adapted from Sulston ef al. (1983) and Schierenberg (1987).

D. A MORE DETAILED REVIEWOF EMBRYOGENESIS Development of living embryos within the hermaphrodite and after the embryos are laid (usually at about the 32-cell stage) can be followed using Nomarski differential interference contrast microscopy. Fertilization occurs within the hermaphrodite spermatheca, where the first sperm to contact the advancing end of the oocyte appears to be engulfed (Ward and Carrel, 1979). The end of the oocyte the sperm enters will become the posterior end of the animal. However, it is not known whether the anteroposterior axis is already established in the oocyte prior to fertilization or is determined by the site of sperm entry. Following fertilization, the oocyte nucleus completes meiosis I and 11, extruding two polar bodies at the anterior end of the 1-cell embryo (Fig. 3a), and the embryo becomes surrounded by an impermeable eggshell. The sperm and oocyte pronuclei migrate, meet, and move to the center of the zygote (referred to as Po, Fig. 3b-d). The first mitotic spindle, nucleated from the sperm-contributed centrosomes and oriented along the anteroposterior axis, moves to an asymmetric position (Fig. 3e), leading to an unequal first division (Fig. 30. This is the first of a series of unequal stem cell-like divisions that generate 5 somatic founder cells (AB, E, MS, C, D) and a gerrn-line progenitor cell (P,) by the 28-cell stage of embryogenesis (Figs. 2 and 3f-i). Each founder cell divides and behaves in a

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FIG.3. Embryonic development of C. eleggam. All panels show Nomarski images of living embryos. Anterior is left, posterior is right, dorsal is up, ventral is down. (a) About 60 minutes after fertilization at 16°C. the oocyte ( 0 )and sperm (s) pronuclei appear at opposite poles, and the anterior membrane begins to contract. (b. c) The sperm pronucleus moves away from the posterior cortex and the oocyte pronucleus migrates toward the sperm pronucleus, while a pseudocleavage furrow is formed and resorbed. (d) The pronuclei move to the center and rotate. (e) The mitotic spindle becomes asymmetrically positioned along the anteroposterior axis. The spindle poles are seen as granule-free spheres. (0First cleavage generates a large anterior founder cell AB and a smaller germ-line cell P,. (g) After the division of AB into ABa (anterior) and ABp (posterior), P,divides unequally into a somatic cell EMS and a new germ-line cell P?. (h) After the division of both AB cells, EMS divides unequally into two somatic founder cells, MS and E. Shortly thereafter, P2divides unequally into a somatic founder cell C and a new germ-line cell P,. (i) P3 divides unequally into a somatic founder cell D and the germ-line progenitor cell P4. Soon afterward, gastrulation begins with the migration of the two E cells (intestinal precursors) to the inside of the embryo. +I) The hypodermal cells surrounding the embryo squeeze it circumferentially, resulting in elongation of the embryo into a worm. The approximate stages of elongation shown are "comma" 6 ) . "tadpole" (k). and "pretzel" (I). Bar = 10 pm. From Schierenberg (1986).

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characteristic way and contributes specific cells to specific tissues (Fig. 2; Sulston er al., 1983). The large anterior daughter of the first division, AB, divides proliferatively (symmetrically) and later in embryogenesis generates hypodermal, neural, pharyngeal, and body wall muscle cells. The smaller posterior cell, P,, is a germ-line cell; it undergoes three more unequal stem cell-like divisions, generating the somatic blastomeres EMS, C, and D, and the germ-line cell P,. EMS divides unequally into the two founder cells E and MS. E generates the entire intestine. M S contributes to the pharynx, body wall muscles, somatic gonad, and nervous system. C generates hypodermal, neural, and muscle cells. D generates only body wall muscles. P, gives rise to the entire germ line. Thus, only the intestine and germ line are clonally derived, from E and P,, respectively. Mesodermal and ectodermal tissues are polyclonal in origin, arising from AB, MS,C, and D. Gastrulation begins at the 28-cell stage when the two daughters of E move from the posterior, ventral surface to the interior of the embryo (Fig. 3i), followed by P, and the progeny of MS. Later the progeny of C and D that will contribute to body wall muscle and the progeny of AB that will contribute to the pharynx also move to the interior. During these inward movements, the remaining progeny of AB spread over most of the surface of the embryo, forming a sheet of hypodermis. The overall body plan starts becoming apparent as the precursors of the intestine and pharynx form a cylinder in the center of the embryo and the body muscle precursors become arranged in quadrants between this cylinder and the overlying hypodermis. Cell division and gastrulation are for the most part completed within the first half of embryogenesis (Fig. 4). During the latter half of embryo development, tissues become more highly organized and separated from each other by basement membrane, and the lima bean-shaped embryo elongates into a cylindrical worm. The names of the sequential stages of elongation describe the shape and length of the worm relative to the eggshell: “comma,” “tadpole” or 1 %-fold, “plum” or 2-fold, and “pretzel” or 3-fold (Figs. 3j-I and 4). In an elegant analysis of the effects of cytoskeletal inhibitors and laser ablation of hypodermal cells on embryo elongation and shaping, Priess and Hirsh (1986) demonstrated that microtubules and microfilaments in hypodermal cells participate in squeezing the embryo circumferentially;this squeezing results in embryo elongation. Well after this pressure-driven elongation of the embryo has occurred, the hypodermis secretes the cuticle that surrounds and maintains the shape of the juvenile worm. By the time the embryo hatches as a first-stage larva, approximately 15 hours after fertilization, it has a functional pharynx and intestine, musculature, and nervous system, and it closely resembles the adult worm in overall body plan.

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88

number of live nuclei first cleavage

-(

100200300400500600

0 O

I

'

'

'

,

'

'

founder cells

gastrulation

comma 1 H -fold movement 2-fold

400

500

3-fold

cuticle synthesis begins

-

hatch

-

800 min.

-

FIG. 4. Key events. stages of embryo elongation. and number of living nuclei during embryogenesis at 20°C. Fertilization is normally at - 50 minutes. Adapted from Sulston el ul. (1983).

E.

TISSUE-SPECIFIC MARKERSAND VISIBLE SlGNS OF DIFFERENTIATION DURING EMBRYOGENESIS

The earliest markers of a specific lineage are P granules, which can be visualized both by electron microscopy and by antibody staining of fixed embryos (Fig. 6; Strome and Wood, 1982; Yamaguchi et al., 1983; Wolf et al., 1983). These germ lineage-specific organelles are present in the cytoplasm of the oocyte at the time of fertilization and are progressively segregated to the germ-line cells P,, PI,P3, and finally to the germ-line progenitor cell P,. They persist in the germ line throughout its subsequent development. Similar early markers of the somatic lineages have not yet been identified, and the somatic lineages do not express specific markers of differentiation until considerably later in embryogenesis. The intestine expresses tissue-specific markers as early as the 100- to 150-cell stage at about 3-4 hours after fertilization, when the 4-8 progeny of E can be stained for gut-specific esterase activity (Edgar and McGhee, 1986). At

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about the same stage, refractile autofluorescent “gut granules” (Babu, 1974) appear in the cytoplasm of the E cells (Laufer et al., 1980; Edgar and McGhee, 1986). These granules can be visualized using either fluorescence or polarization microscopy and therefore serve as an excellent marker of intestinal cell differentiation in living embryos. Body wall muscle cell differentiation can be visualized by the 400- to 450-cell stage using antimyosin or antiparamyosin antibodies; although only 2-16 cells are stained at that stage, 81 cells are stained later in embryogenesis (Gossett et al., 1982). The contractile machinery in muscle cells is functional by the late comma stage, when living embryos begin to twitch (Schierenberg et al., 1980). Hypodermal cell differentiation can be monitored by antibody staining of an embryonic sheath antigen that begins to accumulate during the comma stage, approximately 6 hours after fertilization (Cowan and McIntosh, 1985; Priess and Hirsh, 1986). After elongation this sheath is replaced by the larval cuticle, also secreted by the hypodermal cells. Hypodermal cells are also marked by belt desmosomes, which are thought to provide mechanical coupling between cells; belt desmosomes can be visualized using antibodies by at least the comma stage (Priess and Hirsh, 1986). A specific marker for pharyngeal cells, visualized using a monoclonal antibody, appears around the late comma stage (Priess and Thomson, 1987), several hours before these cells express myosin. In addition to the light-microscopic markers listed above, differentiated cells can be distinguished by electron microscopy (Sulston el al., 1983). The appearance of most, perhaps all, of the differentiation markers in the cells described above requires embryonic transcription. Treatment of early embryos with the RNA polymerase I1 inhibitor a-amanitin prevents the appearance of gut granules, gut-specific esterase activity, body wall paramyosin, and hypodermal belt desmosomes (Edgar and McGhee, 1986, 1988). Expression of the intestinal differentiation markers by the appropriate cells becomes insensitive to a-amanitin at about the same stage (75-150 cells) as transcription is detected by the presence of poly(A) RNA in nuclei (Hecht et al., 1981), suggesting that expression is a result of transcription. Expression of muscle and hypodermal markers becomes a-amanitin-insensitive at a later stage. Thus, although the appearance of specific differentiation markers in specific cells may reflect the segregation of maternal information (see next section), it is probably not the case that the differentiation markers themselves are being segregated. +

11. Analysis of Cell Fate Determination by Embryo Manipulation

Cells are considered to be “determined” or “committed” to particular developmental pathways when their differentiation can occur independently of normal cell-cell positioning or contact. Cell fates are thought

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to be determined either by internal cues (e.g., by differentially segregated factors) or by external cues (e.g., by specific cell-cell contacts or diffusible signals). The lineage of C. efegans does not discriminate between these two possibilities because both ancestry and cell-cell patterning are invariant. However, experimental perturbation suggests that embryo development is guided mainly by internal cues; most cells develop autonomously, according to their lineage and independently of normal neighbors. There is, however, recent evidence for induction of certain early blastomeres by external cues. A.

EARLY BLASTOMERESDEVELOP IN CELL-AUTONOMOUS MANNER

“ISOLATED”

A

Analysis of the cleavage pattern and differentiation of blastomeres cultured in isolation offers a means of assessing what role, if any, neighboring cells play in the development of the blastomeres. Laufer et al. (1980) “isolated” blastomeres by exerting gentle pressure on embryos to crack the eggshell and to burst neighboring cells. The results of these blastomere isolation experiments suggest that the characteristic early cleavage patterns of the founder cells are internally specified and that at least one lineage (intestine) differentiates in a cell-autonomous manner. Isolated AB cells underwent many rounds of symmetric, synchronous cleavage, as in intact embryos. Isolated P, cells were observed to undergo the series of unequal cleavages seen in intact embryos, generating EMS (which subsequently divided unequally into E and MS), C, D, and P,. Therefore, AB and its progeny are not required for the early stem cell-like divisions, but are required for normal topogenesis; in the absence of AB, cells do not assume the same positions that they do in intact embryos. Determination of the E lineage can be monitored by assaying for “gut granules” at a stage equivalent to that at which gut granules would appear in intact embryos. Gut granules appeared in P, partial embryos and in clones of cells that probably arose from isolated E cells, demonstrating the autonomy of intestinal cell differentiation. Blastomere isolation has of late been accomplished by different means, such as extrusion of cells through a laser-induced hole in the eggshell (Schierenberg, 1987) and microneedle manipulation (Priess and Thomson, 1987). The results have been generally similar to those of Laufer et al. (1980), with a few exceptions, which are discussed below.

B. CELLABLATIONEXPERIMENTS ALSO SUGGEST CELL-AUTONOMOUS DEVELOPMENT Another classical approach to analyzing the role of cell-cell interactions is destruction of specific cells and evaluation of the developmental fates of neighboring cells. Consistent with the results of blastomere isolation

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experiments, such ablation studies in C. elegans embryos point to autonomous development for the majority of cells tested. Cell ablation has been achieved using a laser microbeam (Sulston and White, 1980; Sulston et al., 1983) or by ultraviolet irradiation of cells treated with psoralen (J. Pries, personal communication). Because it is difficult to kill the large blastomeres in early embryos without killing the embryo, most studies have been carried out on embryos at or beyond the 50-cell stage. In general, after laser ablation of P4and various derivatives of AB, MS, C, and D, embryos developed normally but were missing the progeny cells of the ablated precursor; the surviving cells generated those and only those cells that they generate in unoperated embryos (Sulston et al., 1983). Although the caveat exists that persistence of the ablated cell could conceivably still influence neighboring cells, these results suggest that normal cellcell interactions are not required for many cells to follow their normal developmental pathway. The results also demonstrate that cells generally do not alter their development to compensate for the missing cells or tissues that result from ablation. However, two exceptions to the above generalization provide examples of regulative interactions late in embryogenesis (Sulston et af., 1983); each occurs within the AB lineage and involves a set of homologous cells (GJW and duct/(;,)that meet at the ventral midline of the embryo and apparently compete for a specific primary fate. This is similar to the postembryonic development of the equivalence group that generates the vulva; six hypodermal cells located along the ventral midline follow specific primary, secondary, or tertiary fates depending on their positions (Sulston and White, 1980; Kimble, 1981; Sternberg and Horvitz, 1986).

C. CLEAVAGE-BLOCKED BLASTOMERES EXPRESS DIFFERENTIATION MARKERS Whittaker (1973) demonstrated the usefulness of assaying differentiation markers in cleavage-blocked blastomeres to monitor the segregation of “developmental potential” during the early cleavages of ascidian embryos. The application of this technique to early C. elegans embryos supports the concept of cell-autonomous development and further suggests that “determinants” for different tissue types are being segregated during the early cleavages. The exact nature of such “determinants” has not been elucidated in C. elegans or any other organism. In this review “determinants” are envisioned as being qualitatively distinct macromolecules that confer developmental differences upon the cells to which they are partitioned. However, alternative possibilities exist, such as determination by the quantity instead of the quality of a “determinant” distributed to different cells. This issue will be discussed again in Section IV,C. Laufer et a/. (1980) monitored the appearance of gut granules in cleavageinhibited two-cell, four-cell, and eight-cell embryos. After overnight in-

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cubation of the embryos in culture medium, granules appeared only in the precursors of the intestine (PI in the two-cell, EMS in the four-cell, and E in the eight-cell), indicating that the potential to express intestinespecific markers is segregated during the early cleavages. Gut granules were not observed in cleavage-blocked one-cell embryos. Edgar and McGhee ( 1986) obtained similar results assaying gut-specific esterase activity in cleavage-inhibited embryos. Cowan and McIntosh (1985) extended these studies by assaying muscle-specific and hypodermal-specific markers (pararnyosin and embryonic sheath, respectively). They confirmed the results of Laufer ef al. and demonstrated the segregation of the potential to express muscle and hypodermal markers to the correct early blastomeres (for muscle: P, in the two-cell, P. in the four-cell, and probably MS, P,, and C in the eight-cell; for hypodermis: both PI and AB in the two-cell. and P, and the two progeny of AB in the four-cell). These results also demonstrate that cleavage and correct cell-cell interactions are not required for at least some manifestations of differentiation. In the experiments of Cowan and McIntosh, cleavage-blocked one-cell embryos expressed hypodermal markers in 54% of the cases, but never expressed gut granules or muscle markers. In addition, not all cleavageblocked two- and four-cell stage blastomeres expressed the appropriate tissue-specific marker; for example, 34%, 35%, and 21% of PI cells expressed gut, muscle, and hypodermal markers, respectively. When multiple markers were assayed simultaneously, none of the blocked blastomeres were observed to express markers for more than one tissue, suggesting that cleavage-blocked blastomeres express only one differentiation program even if they contain the potential to express multiple programs. This result must be interpreted with caution; only one marker of each tissue type was used, and in the experiments of Edgar and McGhee (1986) a much higher percentage of PI cells ( 7 5 4 5 % ) expressed gut granules and gut-specific esterase. Simultaneous monitoring of other tissue markers in the experiments of Edgar and McGhee will support or disprove the notion of “exclusivity” suggested by Cowan and Mclntosh. This is an important issue to clarify, since exclusivity implies that differentiation along one pathway prevents or extinguishes the ability of the cell to express genes characteristic of another tissue. If differentiation is controlled by segregated “determinants” and if exclusivity of differentiation holds, then determinants must be segregated to the proper cells and away from each other for full expression of differentiated phenotypes.

D. CELL FUSIONEXPERIMENTS SUGGEST T H E EXISTENCE OF CYTOPLASMIC DETERMINANTS Cell-autonomous development is hypothesized to be controlled by internally segregated factors. Evidence for the existence of such factors and support for their location in the cytoplasm come from experiments in which

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the fate of one cell is changed by exposure to the cytoplasm of a different cell. Using a laser microbeam to perform microsurgery on embryos, Schierenberg and Wood extruded the nucleus from a PI cell at the two-cell stage, fused the resulting cytoplast to an AB progeny cell at the four- to eight-cell stage, and then assayed for the expression of gut granules after overnight incubation in culture medium (Wood et al., 1984; Schierenberg, 1985). P, normally expresses gut granules, but requires a nucleus to do so. AB normally does not express gut granules, but can be induced to do so by fusion to a PI cytoplast. The frequency with which the expression of gut markers can be induced in AB cells and the effect on expression of normal AB markers have not been published. Nonetheless, the results strongly suggest the existence of factors in the cytoplasm of PI that direct nuclear expression of at least one intestine-specific differentiation product; these factors are normally segregated to the E founder cell but can work on the nucleus in an AB blastomere. Edgar and McGhee have exposed nonintestinal nuclei to “gut cytoplasm” by a different method. Cleavageblocked two-cell embryos were allowed to undergo several rounds of nuclear division and were then released from the cleavage block; after incubation, a small percentage of the embryos expressed gut granules in many more than the normal complement of cells, presumably because the intestine-determiningfactors were distributed to multiple cells upon release of the cleavage block (L. Edgar and J. McGhee, personal communication). The results of Schierenberg and Wood and those of Edgar and McGhee support the hypothesis that at least some blastomeres are determined by the segregation of cytoplasmic factors during the early cleavages. Two lines of evidence suggest that cytoplasmic determinants are not freely diffusible. Using a laser microbeam, Laufer and Wood fused EMS to either ABa or ABp at the four-cell stage (Wood et al., 1983). The fused cell subsequently divided directly into four cells, often similar in position and behavior to the four equivalent cells in an undisturbed embryo. Even though non-E-cell nuclei had been exposed to E-cell cytoplasm, in only a very few cases did the non-E cells express gut granules later in development. This result suggests that intestinal determinants are somehow anchored and are therefore not efficiently transferred when cells are fused for short periods (one cell cycle). As described in the preceding paragraph, the determinants apparently can be transferred when cells are fused for longer periods of time. Based on cytoplasm extrusion experiments, Laufer and von Ehrenstein (1981) hypothesized that most, perhaps all, localized determinants are somehow bound in the early blastomeres. Extrusion of up to 20% of the cytoplasm from either end of the zygote or up to 60% of the cytoplasm from various early blastomeres did not prevent the embryos from cleaving and developing into apparently normal, fertile adults. If determinants of cell fates are partitioned to specific regions of the zygote and early blastomeres, they must be bound to a structure that is not readily extruded.

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E. BLASTOMERES ARENOT DETERMINED BY PARENTAL DNA

IMPRINTED

Another hypothetical mechanism for differentially segregating information to daughter blastomeres is via DNA strands; sperm or oocyte chromosomes could be "imprinted" and segregated to specific blastomeres for later activation of lineage-specific genes. Based on monitoring the fate of sperm or oocyte DNA labeled with bromodeoxyuridine, this mechanism does not appear to operate in C. elegans embryos (Ito and McGhee, 1987). Parental DNA strands segregate randomly during the early cleavages, arguing against the existence of nuclear determinants that are stably associated with parental DNA strands.

F. CRITICAL ROUNDS OF DNA SYNTHESIS AREREQUIRED FOR EXPRESSION OF DEVELOPMENTAL POTENTIAL A s discussed earlier, the expression of specific differentiation markers by specific cells appears to be controlled by the segregation of cytoplasmic factors. Because expression of differentiation markers requires transcription from the embryonic genome, at least some of the cytoplasmic factors are thought to be transcriptional activators. Edgar and McGhee (1988) investigated whether DNA synthesis is required for the nuclei in blastomeres to become competent to express differentiation genes. They found that expression of gut granules, gut-specific esterase, a hypodermal marker, and muscle paramyosin was inhibited by exposing early embryos to the DNA synthesis inhibitor aphidicolin. Expression of both esterase and gut granules by intestinal precursor cells became aphidicolin-insensitive during the first cell cycle after clonal establishment of the E (gut) lineage at the eight-cell stage. Hypodermal and muscle cell precursors became aphidicolin-insensitiveat later stages; it is not known whether the transition times for these tissues correspond to clonal establishment of cell types. All three lineages became aphidicolin-insensitivewell before transcriptional activation, as defined by a-amanitin sensitivity. The results of Edgar and McGhee cannot be explained simply by inhibition of cytokinesis by aphidicolin, because cleavage-blocked blastomeres can express all of the differentiation markers mentioned above (see Section 11,C). Furthermore, the timing of conversion of the intestinal precursor to aphidicolin insensitivity was inconsistent with mechanisms of DNA synthesis control of marker expression such as reaching a critical DNA : cytoplasm ratio, counting rounds of DNA synthesis, or lengthening of the cell cycle. Instead the results suggest that C. elegans blastomeres must pass through a critical cell cycle to become competent to express tissue-specific genes later in development. Holtzer et al. (1975, 1983) previously proposed that a critical or "quantal" cell cycle is necessary for

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cells to alter their state of commitment or differentiation. It may be during such a critical cell cycle that cytoplasmic factors-for example, those thought to be partitioned to different blastomeres-enter the nucleus and somehow provide permission or instructions for subsequent gene expression.

G. AN EXAMPLE OF CELLULAR INDUCTION DEVELOPMENT

DURING EARLY

Priess and Thomson (1987) demonstrated that cellular interactions play an important role in determining at least some cell fates during early C. elegans development. The results of their cell ablation, cell extrusion, and cell repositioning experiments suggest that the two daughters of AB are initially equivalent in developmental potential, but that the anterior daughter (ABa) becomes determined to produce pharyngeal muscles by inductive interactions with EMS (or its progeny) between the 4- and 28cell stage of embryogenesis. Anterior and posterior pharyngeal muscles are generated mainly by ABa and MS, respectively. By microneedle extrusion of blastomeres from the eggshell, Priess and Thomson found that extrusion of P, from the 2-cell embryo or of EMS from the 4-cell embryo prevented ABa from expressing pharyngeal muscles. (Reciprocal extrusions showed cell-autonomous expression of pharyngeal muscles by P, and its daughters.) The initial equivalence of ABa and ABp and the inductive effect of EMS (or its progeny) on ABa development were demonstrated by switching the positions of ABa and ABp at the 3- to 4-cell stage; the switched blastomeres developed according to their new positions, and the manipulated embryos hatched into normal-appearing larvae. induction by EMS or its MS progeny cells apparently occurs by the 28cell stage, since laser ablation of the MS-derived pharyngeal precursors at that stage does not affect formation of an anterior half-pharynx by ABa pharyngeal precursors. There is further genetic evidence for the inductive interaction described above. Priess ef al. (1987) isolated and analyzed a maternal-effect lethal mutant, called glp-l (abbreviation for genn-line proliferation), that appears to be defective in induction of ABa cells. In this mutant, P, cells generate an apparently normal posterior half-pharynx, but ABa cells, instead of producing an anterior pharynx, give rise to what appear to be neuronal cells where the anterior pharynx should be. The temperature-sensitive period for anterior pharynx formation is between the 4- and 28-cell stage of embryogenesis, when ABa cells are thought to be induced by EMS or its progeny. A second temperature-sensitive period occurs during larval development and affects another cell signaling event, that in which the somatic gonad distal tip cell signals proximal germ cells to remain mitotic

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(Kimble and White, 1981). In the glp-l mutant, germ cells enter meiosis precociously and differentiate into sperm (Austin and Kimble, 1987). Mosaic analysis has demonstrated that the glp-l defect is in the germ cells and not in the somatic gonad (Austin and Kimble, 1987), suggesting that the germ cells are defective in receiving or processing the signal from the distal tip cell. By analogy, the defect during embryogenesis may be in reception or processing by ABa cells of an EMS or MS inductive signal.

H . OTHERINDICATIONS

OF

EARLY CELL-CELLINTERACTIONS

In addition to the influence of EMS that is needed for proper development of ABa, interactions between EMS and P, and later between P, and the E-cell descendants of EMS may be required for proper development of the intestine and germ line. A striking affinity between germline cells and intestinal precursors is seen as early as the four-cell stage, when P, and EMS display a broad zone of contact and the nuclei in Pz and EMS become eccentrically positioned at the PI-EMS cell boundary (Schierenberg, 1987). This association is maintained during subsequent divisions. In fact, there is a reversal of the pattern with which the germ lineage generates somatic daughters: during the divisions of Poand PI,the somatic daughter is generated to the anterior side of the new germ-line cell, whereas during the divisions of P, and P,, the somatic daughter is generated to the posterior side of the germ-line daughter (Laufer et al., 1980; Schierenberg, 1987). As a result of this reversal of polarity, P, and E and then P, and Ep are generated next to each other. The requirement for interaction between EMS and P, for correct intestinal cell determination has been investigated by monitoring development of EMS after extrusion of PI from the eggshell. Schierenberg (1987) observed that extrusion of P2 during early interphase altered the development of EMS; after an unequal division of EMS, E divided into more than the normal number of 20 descendants, which did not migrate to the interior of the embryo and did not express gut differentiation markers. The development of MS also appeared to be perturbed. Extrusion of P, later in interphase does not perturb the ability of EMS to generate gut differentiation markers (E. Schierenberg, J. Priess, and L. Edgar, personal communications). The results may indicate that P,-EMS interaction is required during a limited period of time for EMS to divide correctly into two different founder cells. However, an alternative possibility is that EMS is damaged by extrusion of its sister cell too soon after the division of P, into EMS and P?. This issue needs to be resolved. Interestingly, the division of EMS apparently must occur longitudinally with respect to the anteroposterior axis for the correct segregation of developmental potential

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to E and MS. Transverse division* of EMS, infrequently induced by removal of AB at the two-cell stage, generates similar daughter cells, neither of which appear to generate gut cells later (Schierenberg, 1988). During normal development, P4follows the two E cells into the interior of the embryo during gastrulation, and later in embryogenesis the two daughters of P4 (Z, and Z,) insert large lobes into two of the intestinal cells (Sulston et al., 1983). It is thought that the intestinal cells may “nurse” the germ cells or otherwise be required for germ cell development. Consistent with this is the observation that in embryos of the nematode Bradynema rigidum, in which contact between the germ-line and intestinal precursor is not maintained during the early cleavages, P4 migrates around other blastomeres to reestablish contact with the E cells (zur Strassen, 1959). I. DISCUSSION Both the ancestry and positions of cells within C. elegans embryos are invariant. Therefore, the cell lineage itself does not tell us whether cells become determined to follow their particular developmental fates by internally transmitted factors and/or by external cues. It has been necessary to manipulate embryos to distinguish between these two possibilities, and the results of most manipulation experiments suggest a high degree of cell autonomy. Cells generally cleave and develop normally in the absence of their normal neighbors. This indicates the importance of internally transmitted factors in determination. Using the operational definition of “determined” as that stage when a cell can develop normally in the absence of its usual neighbors, C. elegans embryonic blastomeres appear to be determined very early. As early as the 2-cell stage, the blastomeres, P, and AB, can cleave normally in isolation (Laufer et al., 1980; Section II,A) and can express appropriate tissuespecific markers, even in the absence of cleavage and normal neighbors (Laufer et al., 1980; Cowan and McIntosh, 1985; Edgar and McGhee, 1986; Section 11,C). Such embryo manipulations reveal that P, contains and is able to express the multiple developmental potentials of its progeny cells. During the series of unequal divisions that P, undergoes in the normal embryo, these developmental potentials are progressively partitioned to *In longitudinal division the mitotic spindle is oriented along the long axis of the embryo. which in C. elegans is the anteroposterior axis. Such a division generates an anterior and a posterior daughter cell. In a transversely dividing cell, the mitotic spindle is oriented perpendicular to the anteroposterior axis, and daughter cells are generated along either the dorsoventral axis or the left-right axis.

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the different founder cells and away from the P lineage. This partitioning is complete and the cells are thought to be “determined” by the 16- to 28-cell stage. The internally segregated factors are probably cytoplasmic, not nuclear. It should be emphasized that “cytoplasmic” as used in this discussion is meant to include cortical and membrane-bound components. The evidence in favor of cytoplasmic factors is that of Schierenberg and Wood (Wood el al., 1984; Schierenberg, 1985; Section 11,D);cytoplasm from an intestinal precursor cell can cause a nonintestinal cell to express intestine-specific markers. The factors are probably not free ions or other very small molecules, because the blastomeres are dye-coupled up until at least the 24cell stage, as judged by intercellular transfer of injected lucifer yellow (D. Knauber and M . Cappechi, personal communication). The evidence against DNA-bound nuclear factors is that of Ito and McGhee (1987; Section 11,E); marked parental DNA strands segregate randomly during embryogenesis. The experiments of Ito and McGhee do not rule out nuclear factors that are not bound to DNA. Although the early blastomeres contain the potential to express multiple differentiation markers, when cleavage-blocked they appear to be restricted to expression of only one differentiated cell type (Cowan and McIntosh, 1985). As discussed in Section KC, because such “exclusivity” has important implications for mechanisms of gene activation, this phenomenon needs to be carefully investigated under optimal blastomere culturing conditions and using other differentiation markers. Cleavageblocked ascidian embryos are able simultaneously to express multiple differentiation markers (Crowther and Whittaker, 1986), demonstrating that exclusivity is not observed in embryos of all organisms. Analysis of the intestinal lineage provides the strongest evidence for partitioning of cytoplasmic determinants directing the development of a blastomere (Sections II,A,C,D). However, this lineage is unusual in being clonal; the founder cell E generates exclusively intestinal cells. In contrast, the founder cell AB has a much more complicated lineage and gives rise to multiple tissue types, and for this lineage both cytoplasmic determinants and cell-cell interactions appear to be required for normal development. In the case of ABa, which generates hypodermis, neurons, and pharyngeal and body wall muscle, the potential to express hypodermal differentiation markers appears to be programmed by internally segregated factors (Cowan and McIntosh, 1985; Section II,C), while extracellular cues appear to be required for the production of both types of muscle (Priess and Thomson. 1987; Section 11,G). In fact, the finding that ABa and ABp appear to switch developmental fates completely after their positions are switched suggests that external signaling is important for the production

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of many ABa- and ABp-derived tissues. It may be the general case that blastomeres are instructed by internally segregated factors to generate the cell type that constitutes the majority of their lineage (AB-hypodermis, MS-muscle, E-intestine, C-muscle, D-muscle), but require external cues for the determination of minority cell types (AB-muscle, MS-neurons). It is evident that maternal information guides early C. elegans embryo development. The zygotic genome is not expressed at an appreciable rate until about the 90-cell stage, as assayed by detection of poly(A) in nuclei (Hecht el af., 1981), although low levels of [3H]uridineincorporation can be detected as early as the 4-cell stage (L. Edgar, personal communication), and run-on transcription assays reveal limited transcription in pre-30-cell stage embryos (I. Schauer and W. Wood, personal communication). More indicative of the importance of the maternal contribution to the embryo is the collection of maternal-effect embryonic-lethal mutants that affect early events (see Section IV). The objective of the next section is to review and evaluate what is known about the localization and functions of maternal components that can be visualized during early development. 111. The Generation of Zygotic Asymmetry and Partitioning of Maternal Components to the Early Blastomeres

One of the reasons C. elegans embryos are so well suited to analysis of cell fate specification is that in addition to developing differently, the early blastomeres appear and behave differently. Through a combination of Nomarski observation of living embryos and immunofluorescence staining of fixed embryos, it has been possible to document the directed movements and differential localization of a variety of intracellular components. The mechanisms of movement and localization and the participation of localized components in blastomere development have been investigated by analyzing manipulated embryos. These approaches have generated a fairly detailed picture of pattern formation and segregation events during early embryogenesis.

A. THEC. elegans OOCYTE Before discussing postfertilization events, it is worth considering oocyte formation and the possibility that some spatial patterning occurs during oogenesis. Within the distal somatic gonads of hermaphrodites (Fig. 3, mitotic and early meiotic germ cells reside within membranous cubicles that are connected to a common core of cytoplasm (Hirsh el al., 1976;

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Proximal Region

Spermatheca

Uterus

FIG.5 . Central longitudinal section showing the germ cells in one of the two gonad arms in an adult hermaphrodite worm. In the distal region the germ nuclei. located in open membrane cubicles. divide mitotically and then enter meiosis. Oocytes form in the loop region and enlarge as they move through the proximal region toward the spermatheca, where fertilization takes place. A one-cell and two-cell embryo are shown in the uterus. The somatic gonad cells form a very thin layer around the germ nuclei and oocytes, and enclose sperm and embryos in the spermatheca and uterus. respectively. From Strome ( 1986b).

Strome, 1986b). Oocytes complete cellularization in the loop region of the gonad, after which they enlarge considerably as they progress through the oviduct toward the spermatheca. There are no nurse cells or follicle cells that directly supply the oocyte or provide spatial cues. Instead. maternal components are synthesized by the germ nuclei, synthesized by nongonadal tissue and imported (for example, vitellogenins are synthesized in the intestine; Kimble and Sharrock, 1983), and also perhaps synthesized by the somatic gonadal sheath cells (Strome, 1986~). The question of whether C. elegans oocytes become polarized prior to fertilization has not been answered. The oocyte nucleus moves to the end of the oocyte far from the spermatheca just prior to fertilization (Fig. 5 ) . and fertilization occurs at the end of the oocyte to enter the spermatheca first; such reproducible movements and apparent restriction in the site of sperm entry suggest polarity. However, immunofluorescence visualization of microtubules, microfilaments, germ-line-specific granules, and membrane antigens reveals homogeneous distributions of each of these cornponents (Strome, 1986a). Thus, there is little evidence for spatial patterning prior to fertilization.

B. ZYGOTIC ASYMMETRIES AND

UNEQUAL

FIRSTCLEAVAGE

The distributions of all of the components listed above become highly polarized during the first cell cycle. After fertilization the positions of the nuclei indicate the polarity of the embryo; the sperm nucleus resides at the posterior end, and the egg nucleus is located closer to the anterior end (Fig. 3a). The completion of meiosis by the egg nucleus, which occurs during the first 60 minutes after fertilization at 16°C. leads to the extrusion

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of two polar bodies, which serve as anterior markers throughout development. After meiosis the two pronuclei form through chromatin decondensation. The centriolar microtubule-organizing centers contributed by the sperm nucleate an astral array of microtubules in the posterior hemisphere, and the anterior membrane begins to undergo visible contractions (Fig. 3a). As the egg pronucleus begins to migrate toward the sperm pronucleus, the contractions subside and a “pseudocleavage furrow” is formed (Fig. 3b) and then resorbed (Fig. 3c). The sperm aster microtubules presumably cause the movement of the sperm pronucleus away from the posterior cortex and may also be responsible for drawing the egg pronucleus into the posterior hemisphere (Strome and Wood, 1983; Albertson, 1984), where the two pronuclei meet (Fig. 3c). The pronuclei then move to the center of the embryo (Fig. 3d), and the mitotic spindle growing around them becomes visible by exclusion of the granular cytoplasm. Once centered, the spindle rotates to lie along the anteroposterior axis. During metaphase and anaphase the spindle becomes asymmetrically positioned (Fig. 3e); the entire spindle moves slightly posteriorly, and during spindle elongation the posterior pole moves closer to the posterior membrane (Kemphues et al., 1988). The asymmetric position of the spindle leads to an unequal first cleavage, generating a large anterior cell and a smaller posterior cell (approximate area ratio of 57 : 43; Kemphues el al., 1988) (Fig. 30. As just described, the anteroposterior axis is evident in the newly fertilized embryo, and may even be set up in the oocyte prior to fertilization. The dorsoventral axis is not apparent until the four-cell stage, at which time ABp lies dorsally and EMS is located ventrally (Fig. 3g). The leftright axis becomes apparent during the divisions of ABa and ABp into left and right daughters (Fig. 3h). The results of Priess and Thomson (1987; see Section II,G) suggest that the dorsoventral and left-right axes are not determined until the division of AB into ABa and ABp; switching the positions of ABa and ABp switches the polarity of both the dorsoventral axis and the left-right axis. Thus it is unlikely that there are dorsoventral or left-right cytoplasmic asymmetries generated in the zygote.

c. SEGREGATION OF P GRANULES TO THE GERMLINE Germ-line-specific structures, termed P granules, provide a striking demonstration of the phenomenon of cytoplasmic localization; they become localized in the cytoplasm destined for the smaller P-cell daughter at the first and each subsequent unequal division within the P lineage (Fig. 6; Strome and Wood, 1982; Yamaguchi ef al., 1983; Wolf et al., 1983).

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The P granules visualized using antibodies are almost certainly the same structures as the germ granules seen by electron microscopy. By electron microscopy they are non-membrane-bound granular masses of electrondense material (Wolf et al., 1983). They are small, numerous (several dozen granules of -0.5 pm diameter), and dispersed throughout the cytoplasm of the oocyte and newly fertilized embryo (Strome and Wood, 1982, 1983). During pronuclear migration and pseudocleavage they begin to coalesce and become localized at the posterior cortex, assuring their passage to P,. P granules continue to coalesce and are progressively passed to P2, P,, and finally P4, where they become perinuclear. They persist as perinuclear granules in germ cells at all stages of proliferation and development, with the exception of spermatocytes, from which they disappear (Strome and Wood, 1982). The segregation of P granules during the early cleavages is thought to reflect their migration. However, an alternative possibility is that they are stabilized in certain regions of P cells and destabilized in other regions, resulting in asymmetric localization without actual movement of granules. Although the function of P granules in the germ line is not yet known, they serve as an excellent model for studying the segregation of lineagespecific factors during the early cleavages. The mechanism by which they are segregated has been investigated by analysis of mutant embryos and embryos perturbed with pharmacologic agents (Strome and Wood, 1983). P granules do not become asymmetrically localized in unfertilized oocytes produced by spermatogenesis-defective hermaphrodites. This indicates that fertilization is required either to provide polarity or to activate the segregation machinery in oocytes. The role of the spindle in P-granule segregation was investigated by analyzing P-granule distributions in embryos that display altered spindle orientation and placement. The spindle remains posterior and oriented perpendicular to the anteroposterior axis in zyg-9(b244) embryos and embryos treated with low levels of the microtubule inhibitor nocodazole; nevertheless, P granules are segregated apparently normally to the posterior cortex, indicating that their segregation is independent of spindle orientation. In fact, P-granule segregation

FIG.6 . P-granule segregation during the early divisions. The left panels show Nomarski images of living embryos (a-c) and a fixed embryo (d), and the right panels and bottom panel show immunofluorescence images. Anterior is left, posterior is right. (a) One-cell embryo during pronuclear formation and early pseudocleavage. P granules are dispersed throughout the cytoplasm. (b) One-cell embryo after pronuclear migration. P granules are localized at the posterior cortex. (c, d) P granules are partitioned to P, of the two-cell embryo (c) and Pz of the four-cell embryo (d). (e) In the newly hatched larva, P granules are in the two germ-line progenitor cells (Z, and Z,) derived from P4. Bars = 10 pm.

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appears to occur normally in embryos in which microtubules have been depolymerized o r disrupted by microtubule inhibitors such as colcemid, griseofulvin, or vinblastine; pseudocleavage also occurs but the membrane contractions preceding it are much more pronounced. Microtubule inhibitors inhibit pronuclear migration, consistent with an important role for microtubules in both the movement of the sperm pronucleus away from the posterior end and the movement of the egg pronucleus to meet the sperm pronucleus. Based on the effects of microfilament inhibitors such as cytochalasin D or B, P-granule segregation and the ability of the zygote to manifest other asymmetries appear to depend on microfilaments (Strome and Wood, 1983). In zygotes treated with a microfilament inhibitor, membrane contractions and pseudocleavage are inhibited. the pronuclei migrate but they meet centrally instead of posteriorly, and P granules coalesce in the center instead of at the posterior cortex of the embryo (Fig. 7b). Such embryos behave “symmetricaUy.” Because cells are not permeable to microfilament inhibitors such as DNase 1 and phalloidin, the effects of the cytochalasins have not been verified using unrelated inhibitors. However, the cytochalasin reversal experiments discussed below argue against nonspecific effects of at least cytochalasin D on zygote development. D. MICROFILAMENTS AREREQUIREDDURING A CRlTlCAL MIDDLE PERIOD IN THE FIRST CELL CYCLE

To try to learn when and how microfilaments participate in generating asymmetry. zygotes were “pulsed” with a microfilament inhibitor (cytochalasin D) to perturb microfilament arrays for defined intervals during the first cell cycle (Hill and Strome, 1988). These studies have defined a critical period of 5-10 minutes approximately three-quarters of the way through the cell cycle during which microfilament function is required. As shown in Fig. 7, the three intervals examined were (1) early-before pronuclear migration, during pronuclear formation and the early phase of anterior membrane contraction; (2) middle-during pronuclear migration, pseudocleavage, and P-granule segregation; and (3) late-after pronuclear meeting, until cytokinesis. Brief disruption of microfilaments during the early period did not affect subsequent zygotic events (Fig. 7c), indicating that microfilaments are not required prior to the events and that microfilaments regain structure and function after cytochalasin disruption. Disruption of microfilaments during the middle period resulted in the same inhibition of pseudocleavage and perturbation of pronuclear migration and P-granule segregation as does continuous cytochalasin treatment; in addition, after washing microfilament inhibitor out, the zygotes divided

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Untreated

CD-

Wash

CD-

Wash

FIG.7. Schematic summary of events in zygotes treated or “pulsed” with cytochalasin D (CD). Each row shows four progressively later stages: pronuclear stage after the completion of meiosis, pseudocleavage and pronuclear meeting, formation and asymmetric placement of the spindle, and first cleavage. Anterior is left, posterior is right. The small dots represent P granules, and the symbols are spindles. (a) Normal zygote for reference. (b) Zygote treated with cytochalasin D during or after meiosis and allowed to develop in the presence of inhibitor. (c) Zygote treated briefly with cytochalasin D during the early time interval, from meiosis until the commencement of pronuclear migration. (d) Zygote treated brieflv with cytochalasin D during the middle time interval, during pronuclear migration until pronuclear meeting.(e) Zygote treated briefly with cytochalasin D during the late time interval, after pseudocleavage, pronuclear migration, and P-granule segregation until just before cytokinesis. From Hill and Strome (1988).

symmetrically or with variable asymmetry (Fig. 7d). Brief disruption of microfilaments during the late interval did not affect the localization of P granules or the asymmetric placement of the spindle and cleavage furrow (Fig. 7e). The results suggest that proper microfilament structure is needed only during the middle interval; microfilaments participate in the events occurring during the interval (pseudocleavage, pronuclear migration, and P-granule segregation), as well as the specification of spindle position and the subsequent cleavage plane later in the cell cycle. When P granules are mislocalized during the middle interval, they do not become properly segregated later in the cell cycle (after removal of microfilament inhibitor); conversely, once P granules are segregated posteriorly, their localization does not appear to require intact microfilaments during the late interval. Analysis of development of the two-cell embryos generated after disrupting microfilaments during the one-cell stage indicates that microfilament function during the critical middle interval is also required for the

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correct segregation of factors that control patterns of division and P-granule segregation (D. P. Hill and S. Strome, unpublished). The normal-appearing two-cell embryos generated after disrupting microfilaments during the early or late intervals do, in fact, develop normally; they undergo the correct pattern of cell divisions, gastrulate, give rise to differentiated cell types (intestine. muscle, and hypodermis), and develop as far as control embryos permeabilized to culture medium lacking inhibitor. (Conditions have not been found that support permeabilized embryo development to hatching; they arrest during morphogenesis.) In contrast, the two-cell embryos generated after disrupting microfilaments during the middle interval undergo one of four different patterns of cleavage and P-granule segregation (Fig. 8): ( I ) normal-the posterior cell behaves like a germ cell and the anterior cell like a somatic cell; (2) reverse polarity-the anterior cell behaves like a germ cell and the posterior cell like a somatic cell; (3) mirror-image duplication-both blastomeres behave like germ cells; (4) symmetricboth blastomeres behave like somatic cells. Embryos of all four classes

Untreated

(-JJ-@-J

FIG.8. Schematic summary of the division patterns and P-granule segregation patterns of embryos pulsed with cytochalasin D (CD) during the critical middle interval in the first cell cycle. Anterior is left. posterior is right. The dots represent P granules. The top row shows a normal two-cell embryo for reference: the germ-line (posterior) cell undergoes a longitudinal and unequal division and segregates P granules to the smaller (germ-line) daughter; the somatic (anterior) cell divides transversely and symmetrically. (Divisions are shown a s they occur in the absence of an eggshell.) The symmetric and variably asymmetric two-cell embryos that result from a cytochalasin D pulse divide according to one of the four patterns shown: normal. reverqe polarity. mirror-image duplication, and symmetric. The posterior cell of a normal two-cell. the anterior cell of a reverse polarity two-cell, and both cells of a mirror-image duplication two-cell resemble germ cells in undergoing unequal division and segregating P granules to the smaller daughter. The anterior cell of a normal, the posterior cell of a reverse polarity. and both cells of a symmetric embryo divide symmetrically, like somatic blastomeres. P granules in these somaticlike blastomeres are distributed to both daughters and are fine and faint.

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generally develop into abnormal masses of several hundred cells which rarely express differentiation markers later in embryogenesis. The question of which cells inherit the “developmental potential” to express differentiation markers during the unusual cleavages described above has not yet been addressed. Analysis of the patterns of segregation of developmental potential for different somatic lineages either will confirm the tentative assignments of cell identities or may reveal that the factors that determine different blastomere characteristics (i.e., cleavage pattern, ability to segregate P granules, ability to express differentiation markers) behave independently. Microfilaments clearly play an important role in the generation of zygotic asymmetry, the directed movements of cytoplasmic components, and the production of daughter cells with different identities. However, it is not yet understood how microfilaments participate in this phase of early development. Rhodamine-phalloidin staining of the distribution of filamentous actin has revealed that microfilaments become reorganized during the interval in the first cell cycle when pseudocleavage, pronuclear migration, and P-granule segregation occur (Strome, 1986b). According to the microfilament inhibitor studies described above, it is during this interval that proper microfilament structure is required. The nature of the reorganization is concentration of dots or foci of actin around the anterior periphery of the zygote (Strome, 1986b). Prior to this, microfilaments exist in a homogeneous array of cortical foci and fine fibers. The fibers remain uniformly distributed around the periphery when the foci become concentrated in the anterior half of the zygote. The anterior foci persist as the zygote undergoes first mitosis, at which time microfilaments align circumferentially around the zygote where the cleavage furrow will form. After cytokinesis, fibers and foci are seen in both daughter blastomeres and in later-stage cells as well. It is not known whether the anteriorly concentrated foci of actin are a critical functional element of the microfilament array. After transient early disruption of the microfilament array (discussed above), proper execution of zygotic events is accompanied by the reappearance and anterior concentration of actin foci. This is consistent with but does not prove that the foci participate in the events. On the other hand, the observation that an asymmetric distribution of actin foci is not observed during P-granule segregation in P, , P2,and P, argues against a crucial role for the actin foci in at least P-granule segregation. If the foci do serve a specialized role in the zygote, they may do so by interacting with myosin. Based on staining C. elegans embryos with an antibody to human platelet myosin (Wong et al., 1984), myosin appears to be located in cortical patches that become concentrated anteriorly at the same time that the actin foci do (S. Strome,

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unpublished). It has been hypothesized that anteriorly localized actin, and perhaps myosin, participate in moving internal components by forming a contractile network around the anterior periphery, which induces cytoplasmic streaming (Strome, 1986a). A pronounced anterior-to-posterior flow of cytoplasm is evident (Nigon et al.. 1960), especially on speededup videotapes of living embryos, but its role in zygote development has not been investigated. Analysis of the requirement for cytoplasmic streaming and electron-microscopic analysis of microfilament structure and association with other identifiable components may help resolve when and how microfilaments participate in zygote development.

E. THEPOTENTIAL FOR UNEQUAL CLEAVAGE IS LOCALIZED AT THE POSTERIOR ENDOF THE ZYGOTE The ability to divide unequally is a characteristic of the germ lineage or P cells in the early embryo. Schierenberg investigated when and where the potential for unequal cleavage is localized by extruding portions of the zygote from laser-induced holes in the eggshell (Schierenberg, 1985, 1987, 1988). Posterior localization of the potential for unequal cleavage in the zygote was demonstrated by the following set of extrusion experiments (Fig. 9):

FIG.9. Schematic summary of the cleavage patterns of zygote fragments. Cytoplasm was extruded through a laser-induced hole at either the posterior (a, b) or the anterior (c, d) end of the zygote. (a) After extrusion of posterior cytoplasm, the zygote fragment left inside the eggshell divides symmetrically. (b) An extruded posterior fragment containing a nucleus and centrioles divides unequally. (c) After extrusion of anterior cytoplasm, the zygote fragment left inside the eggshell divides unequally. (d) An extruded anterior fragment containing a nucleus and centrioles divides symmetrically. Adapted from Schierenberg (1985).

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1. Extrusion of 25% or more of the posterior end of the zygote prevents the remaining cell from dividing unequally (Fig. 9a). 2. An extruded posterior fragment, containing 20-50% of the total cytoplasm and a nucleus and centrioles, undergoes the correct series of unequal germ-line-like divisions outside the eggshell (Fig. 9b). 3. Extrusion of up to 50% of the anterior end of the zygote does not affect the ability of the remaining cell to divide unequally (Fig. 9c). 4. An extruded anterior fragment, containing up to 80% of the total cytoplasm and a nucleus and centrioles, undergoes equal divisions outside the eggshell (Fig. 9d).

Since P granules become localized at the posterior end of the zygote, Schierenberg (1988) tested the possibility that P granules confer the ability to undergo unequal germ-line-like divisions. Posterior fragments (like example 2 above) were extruded before P-granule segregation. Such fragments divide unequally, suggesting that P granules do not control early division patterns. The analysis of the cleavage patterns of cytochalasinpulsed zygotes that distribute P granules to both daughter blastomeres support this conclusion (D. P. Hill and S. Strome, unpublished; Section

111,D).

F. DETERMINATION OF CELLDIVISION AXES The correct partitioning of specific factors to specific blastomeres requires not only the correct localization of the factors in the cytoplasm but also proper orientation of the cleavage planes. As discussed in Section II,H (and accompanying footnote), in normal embryos EMS divides longitudinally to generate the founder cells E and MS.When EMS is induced to divide transversely in manipulated embryos, its ability to generate dissimilar daughter cells is impaired, indicating the importance of the division axis (Schierenberg, 1988; Section 11,H). The germ-line cells (Po-P3), like EMS, normally divide longitudinally and unequally and generate daughters with different developmental potentials, whereas AB divides transversely into developmentally equivalent daughters. Hyman and White (1987) have shown that a rotation of the centrosomes around the nucleus shifts the spindle axis from transverse to longitudinal in EMS, Po, P,, and P,, leading to their longitudinal divisions. Such a rotation is not observed in AB, in which the spindle and division axis remain transverse. Rotation of the centrosomes in the P cells aligns the spindle and division axis with the axis of P-granule segregation. Thus the correct determination of cell division axes is an important aspect of the segregation process.

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G . DISCUSSION The studies described above lead to the following picture of early C. elegans development. The oocyte contains all of the components required

for early embryogenesis; these include ( I ) factors that control cleavage patterns, (2) cytoplasmic determinants that instruct different blastomeres to follow different developmental pathways, (3) factors that signal and/or receive signals from neighboring cells to generate specific cell types, and (4) the precursors, enzymes, and machinery needed to support many rounds of D N A synthesis and cell division. None of the maternal components that can be visualized appear to be prelocalized in the unfertilized oocyte. Instead sperm entry appears to initiate a series of directed movements and somehow leads to the localization of maternal factors. The sperm may actually provide polarity to the egg, or if polarity is determined during oogenesis may provide the machinery (e.g., centrosomes) or a trigger (e.g., a spike in Ca”) for reorganization of the contents of the egg. Reorganization occurs mainly during a 10-minute interval within the first 100-minute cell cycle (at 16°C) and requires the participation of microfilaments. After cytoplasmic factors become asymmetrically partitioned to different regions of the zygote, mitosis and cytokinesis complete the partitioning process. The two daughters AB and PI differ in size and content. The differential segregation of maternal factors to A B and PI cause the two cells to undergo characteristic patterns of cleavage, divide with characteristic cell cycle periods, and generate different cell types later in embryogenesis. The localization process is not completed during the onecell stage; P granules must be actively segregated during the divisions of P I , PI, and P,, to ensure their passage to the germ-line cell P4. Presumably somatic-lineage-specificfactors are progressively segregated during these divisions also. Microfilaments participate in many, perhaps all, of the segregation events in C. &gum embryos. Based on the results of transiently disrupting the microfilament array, proper microfilament structure is required only during the “critical interval” in the first cell cycle for the proper segregation of P granules and factors that control the cleavage patterns of the early blastomeres (Strome and Wood, 1983; Hill and Strome, 1988, and unpublished; Section 111,D). These cleavage factors may be identical to the “potential for unequal germ-line-like cleavage” that Schierenberg showed is normally localized at the posterior end of the zygote (Schierenberg, 1985, 1988; Section 111,E).However, unequal cleavage potential is localized posteriorly prior to the microfilament critical period (Schierenberg, 1988). Microfilaments may then participate during the critical period in stabilizing the posterior localization of unequal cleavage potential.

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When microfilaments are perturbed, the potential may be misdistributed and cause either the posterior, the anterior, or both blastomeres to divide unequally (Section 111,D).This hypothesis does not easily explain the cases in which both blastomeres divide symmetrically, unless the potential for unequal cleavage can be destroyed or somehow inactivated after microfilamentdisruption. It is interesting that centrifugationof Smittia embryos results in a spectrum of pattern alterations similar to those seen after perturbation of the microfilament array in C. elegans zygotes. Centrifuged Smittia embryos display one of four patterns (Kalthoff et al., 1982): (1) normal (head anterior, tail posterior), (2) reverse polarity (head posterior, tail anterior), (3) mirror-image duplication of the anterior half (double heads), or (4) mirror-image duplication of the posterior half (double abdomens). These results are often discussed in terms of morphogenetic centers, one posterior and one anterior, that govern pattern formation in insect embryos (Sander, 1976; Frohnhofer et al., 1986). The critical posterior region identified by Schierenberg in C . elegans zygotes may be such a morphogenetic center. The potential for unequal germ-line-like divisions appears to coincide with the potential to segregate P granules; cells that undergo unequal divisions segregate P granules to the smaller daughter, whereas cells that divide symmetrically distribute P granules to both daughters (D. P. Hill and S. Strome, unpublished; Section 111,D).It is not yet known whether this coincidence reflects a mechanistic relationship between unequal division and P-granule segregation. Another question that remains to be answered is whether microfilaments and/or the posterior region required for unequal divisions are involved in the partitioning of somatic determinants. This may be addressed by analyzing the distribution of somatic developmental potential in embryos that undergo unusual patterns of cleavage and P-granule segregation after brief microfilament disruption. Although P granules serve as a valuable model for investigating how lineage-specific components are partitioned during the early cleavages, their function in the germ line is unknown. Germ granules are ubiquitous and are therefore thought to participate in germ-line determination or germcell development (Beams and Kessel, 1974; Eddy, 1975). The transplantation experiments of Illmensee and Mahowald (1974, 1976) and Wakahara (1978) provide evidence that the cytoplasm containing germ granules can cause cells to develop into germ cells in Drosophila and Xenopus. However, whether this is due to the germ granules or other factors in the cytoplasm has not been answered. The experiment of Laufer and von Ehrenstein (198 1) offers one way of asking whether P granules are required for germ-line development in C. elegans. Posterior cytoplasm was extruded at a stage when P granules are known to be localized at the posterior

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cortex, and many of the disrupted embryos developed into fertile adults (see Section ILD). It is not known whether or not P granules were extruded. Monitoring P granules during such an experiment would shed light on their participation in embryogenesis and germ-line development.

IV. Genetic Approaches to Analyzing Early Development

The C. elegans oocyte contributes the majority of the contents of the zygote, and maternal factors appear to direct early embryogenesis. This section describes some of the maternal-effect embryonic-lethal mutants that have contributed to our understanding of the roles of maternally provided products and the maternal cytoarchitecture. For excellent and more thorough reviews of genetic and phenotypic analyses of embryonic-lethal mutants in C . eiegans, readers are referred to Wilkins (1986) and Kemphues (1987). A. EARLYS E A R C H E S FOR TEMPERATURE-SENSITIVE MATERNALEFFECTEMBRYONIC-LETHAL MUTANTS

Early searches for embryonic-lethal mutants focused on mutants that are lethal at 25°C but not at 16°C. Two collections of temperature-sensitive embryonic mutants were generated by ethyl methane sulfonate mutagenesis: the zyg mutants (Hirsh and Vanderslice, 1976; Vanderslice and Hirsh, 1976; Wood er ul., 1980) and the emb mutants (Schierenberg et a / . . 1980; Miwa et al., 1980; Cassada et al., 1981; Isnenghi et d.,1983; Denich et al., 1984). Based on parental effect tests, 46 of the 54 genes identified showed maternal effects; maternal wild-type product is sufficient for the survival of homozygous mutant embryos. Of these maternal mutants, 29 were strict maternal mutants; maternal wild-type product was required for the survival of embryos, even those carrying a wild-type allele contributed by the sperm. Only 4 mutants were identified in which embryonic expression is necessary and sufficient, and 4 mutants in which both maternal and embryonic expression are required for survival. Temperature-shift experiments indicate when during development exposure to restrictive temperature (25°C) results in expression of the mutant phenotype. Such experiments can identify the approximate period of synthesis andor function of the wild-type product. For most of the embryonic mutants, the temperature-sensitive periods occur during the interval in development predicted by the parental-effect tests. For example, the temperature-sensitive periods for most of the strict maternal-effect mutants are early, beginning before or at fertilization and ending before gastrulation.

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However, the temperature-sensitive periods for some of the mutants are confusing. As discussed by Wood et al. (1980), without an understanding of whether mutants are temperature-sensitive for synthesis of the gene product, produce a thermolabile product, or identify a temperature-sensitive process, results of temperature-shift experiments must be interpreted with caution. Phenotypic analysis of the maternal-effect mutants has revealed a myriad of early defects, including anomalies in zygotic events, cleavage arrest, altered cleavage patterns, altered cleavage rates, and defective gastrulation. Many of the mutants show variability in early defects. Even mutants that display extremely aberrant early development generally continue dividing to 100 cells or more before arrest. A fairly large proportion of the mutants also arrest at larval stages or show defects in gonadogenesis when shifted to restrictive temperature after embryogenesis. None of the collection of temperature-sensitive embryonic-lethal mutants identify what appear to be the cytoplasmic determinants or determinants of unequal cleavage that embryo manipulation experiments suggest exist; many of these mutants may instead identify genes involved in essential metabolic or cytoskeletal functions required at multiple stages of development. In recent screens, over 200 additional temperature-sensitive embryoniclethal mutants have been recovered (R. Schnabel and H. Schnabel, personal communication). These are being examined for early patterning and cleavage defects and for defects in the generation of specific cell types. Several maternal-effect mutants have especially interesting phenotypes and are being characterized.

B.

ISOLATION OF STRICT MATERNAL-EFFECT LETHAL MUTANTS THAT IDENTIFYGENESREQUIRED FOR PATTERNING AND CYTOPLASMIC IN EARLYEMBRYOS LOCALIZATION

To identify genes required only during early embryogenesis, a novel screen developed by J. Priess has been used. This screen utilizes hermaphrodites that have a defective vulva and cannot lay their eggs. As a result, viable embryos hatch inside the mother worms and consume them (Trent et al., 1983), while mother worms containing only mutant arrested embryos survive (Kemphues, 1987; Kemphues et al., 1988). After mutagenesis of these vulva-defective hermaphrodites, newly induced mutants that are non-maternal-effect embryonic-lethal or larval-lethal will arrest as embryos and larvae (usually in the F, generation), and mutants defective in gonadogenesis will be sterile F, adults. Therefore, by screening among homozygous mutant (F, generation) adult hermaphrodites for worms full of arrested embryos, this screen yields maternal-effect embryonic-lethal

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mutants whose products are required only during embryogenesis. This screen can be used to recover both temperature-sensitive and nonconditional mutants. Nonconditional mutants must be maintained as heterozygotes, which is greatly facilitated by the availability of crossover-suppressing chromosomal rearrangements and duplications covering certain regions of the genome. Among the maternal-effect mutants isolated using the above screen, a class of mutants called par mutants (for partition-defective) displays especially intriguing phenotypes and promises to provide important insights into pattern formation and cytoplasmic partitioning in early embryos. Five different par loci have been identified to date, with from 1 to 10 alleles of each (Kemphues, 1987; Kemphues ef al., 1988). The general phenotype is symmetric and synchronous divisions of the zygote and aberrant partitioning of P granules, although the five different par loci show interesting differences in the early defects, and the different alleles of each locus show differences in seventy of the defective phenotypes. Four par mutants (par-1,2,3,4) have been well characterized (Kemphues et al., 1988) and are described below. As a result of abnormal spindle positioning during the one-cell stage, all of the mutant zygotes except par4 divide into two approximately equalsized blastomeres. At the next division, these blastomeres divide synchronously, in contrast to normal embryos in which the anterior blastomere (AB) divides before the posterior blastomere (P,). In addition to the synchrony of divisions, the spindles in the blastomeres of the par mutants are misoriented (Fig. lO).Normally the spindle in AB is oriented transversely while the P, spindle is oriented longitudinally. In 25% of pur-/ embryos, nearly all of par-2 embryos, a small fraction of par-3 embryos, and 2Wo of p a r 4 embryos, the spindles in both blastomeres are oriented transversely. In many par-3 embryos, the spindles in both blastomeres are oriented longitudinally. In addition, some par-/, par-3, and p a r 4 embryos have normally oriented spindles, and some par-3 embryos show “reverse-polarity’’ spindle patterns, with the anterior spindle oriented longitudinally and the posterior spindle oriented transversely. The par mutants show variable but defective patterns of P-granule segregation. Ail of the mutants distribute P granules to both blastomeres during the first division. However, at the next division, there appears to be some differential segregation of P granules in some par-2, par-3, and p a r 4 embryos. Interestingly, P granules cannot be visualized in later-stage blastomeres of par-1 and p a r 4 embryos, but persist in par-2 and par-3 embryos. The disappearance of P granules from par-1 and p a r 4 embryos resembles the disappearance of the P granules that are occasionally dis-

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par-l

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= p par-2 -

par-3 par4

par3

par-3

par-3 -

par-4

FIG.10. Schematic summary of the spindle orientations in two-cell par embryos. Anterior is left, posterior is right. The symbols are spindles. Horizontal spindles are longitudinally oriented, vertical spindles are transversely oriented. A wild-type two-cell embryo contains spindles with the orientations shown in the far left embryo. A par mutant is listed below a particular type of two-cell if a significant proportion of the mutant embryos display those spindle orientations. (For most of the par mutants, the two blastomeres at the twocell stage are similar in size, as shown in the diagram. However, p a r 4 embryos resemble wild-type embryos in undergoing an unequal first division, generating a large anterior cell and a smaller posterior cell. This is not shown in the diagram.) Derived from Kemphues et a/. (1988).

tributed to the somatic sisters of the P cells during the early cleavages of wild-type embryos (Strome and Wood, 1982). Given the severe defects in early cleavage patterns and at least one segregation event in the par mutants, it is surprising that they produce differentiated cell types. The embryos divide into masses of several hundred cells (sometimes more than the normal number of cells) and, based on Nomarski examination and immunofluorescence staining for differentiation markers, generate hypodermal, neural, body wall muscle, and pharyngeal muscle cells (Kemphues et al., 1988). In fact, in some cases, the pharyngeal cells are organized into clusters that resemble a normal pharynx. The intestinal lineage appears to be more severely affected in the par mutants. Nearly all par-f and p a r 4 embryos and the majority of par-2 embryos fail to express intestinal differentiation markers. In fact, this absence of gut granules has served as the basis for identifying par mutants among maternal-effect embryonic-lethal mutants. Another lineage that is severely affected by the par mutants is the germ line. The leaky par embryos that hatch and reach adulthood are generally sterile. The adults contain an apparently normal somatic gonad, but lack germ cells and gametes. This phenotype is maternal effect and so is termed grandchildless. This agametic phenotype may result from either incorrect determination or incorrect behavior of the germ-line progenitor cell P,. Indeed, as a result of missegregation during embryogenesis, P granules are

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often missing from the gonad primordium and are instead aberrantly distributed to rows of cells along the body wall in newly hatched par-2 larvae (S. Strome, unpublished). Whether or not this missegregation of P granules leads to defective germ-line development has not yet been determined.

C. DISCUSSION The extrusion experiments of Schierenberg (1985, 1987, 1988) predict the existence of mutants like the par mutants. Schierenberg’s experiments suggest that a structure or factors in the posterior end of the zygote confer the ability to undergo unequal germ-line-like divisions (see Section 111,E). Mutational alteration or destruction of such a structure or factors would be predicted to cause zygotes to undergo symmetric, synchronous divisions, which is the general phenotype of the p a r mutants. Furthermore, many par embryos that show altered spindle orientations during the second division, show the alteration that would be predicted by Schierenberg’s experiments: both spindles are oriented transversely, as in a normal AB blastomere. The par mutants, in addition to showing cleavage defects, fail to localize P granules correctly. Schierenberg has not analyzed P granules in his manipulated embryos, so it is not known whether the hypothetical posterior structure or factors are involved in P-granule segregation. The similarities between the par mutants and embryos from which the posterior end has been extruded suggest that at least some of the par loci may identify gene products in the posterior structure described by Schierenberg. Microfilaments have been shown to be involved in at least two of the processes that the p a r mutants affect, namely P-granule localization and spindle positioning (see Section IILD). Some of the par mutants, especially par-3, resemble microfilament-inhibitor-pulsed embryos in their altered cleavage and P-granule segregation patterns. Thus, another potential par gene function is interaction with or modulation of the actin cytoskeleton during early embryogenesis. None of the par mutants map to the known actin genes and so are unlikely to encode defective actin subunits, but they could identify actin-associated proteins. If cytoplasmic “determinants” of cell fates exist, as suggested by the blastomere manipulation experiments described in Section 11, then it should be possible to generate mutants that lack them. At this point it is important to reconsider the nature of such “determinants.” If qualitatively distinct determinants for each lineage or cell type exist, then mutants in such determinants would be expected to lack a specific lineage or cell type. Thus far such mutants have not been recovered. Yet it is not clear that such mutants have been effectively sought. The defects in intestinal cell differentiation and germ-line development observed in some of the

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par mutants are consistent with defective cytoplasmic localization of gutand germ-line-specific factors during the early cleavages. However, the intestinal and germ lineages are the only lineages known to be affected in the par mutants; other differentiated cell types are present. The intestinal and germ lineages are unusual in being clonal; E generates the entire intestine, and P4 generates the entire germ line. These special lineages may be controlled by the partitioning of “qualitative determinants,” while other more complex lineages may require other determination mechanisms. This issue may be resolved as investigators screen for maternal-effect mutant embryos that are missing specific lineages. The laboratories of K. Kemphues and J. Shaw are screening for multicellular embryos lacking gut granules in hopes of recovering non-par mutants that specifically affect the gut lineage. An alternative to determination by qualitatively different factors is determination by quantitative differences in the level of one or two factors. For example, one or more factors may be distributed in a graded fashion along the anteroposterior axis, such that the series of unequal divisions of the P cells (see Fig. 3) would generate blastomeres containing different concentrations of the factor(s). As a result of these concentration differences, blastomeres would become determined to follow different developmental pathways. A growing body of evidence indicates that early development of insect embryos is governed in this way: two morphogenetic centers, localized at the anterior and posterior poles of the embryo, generate gradients of morphogens that in turn differentially activate genes along the anteroposterior axis of the embryo (Sander, 1976; Frohnhofer and Nusslein-Volhard, 1986; Frohnhofer et al., 1986). The embryo manipulation experiments in C. elegans do not discriminate between the two general types of determinants described in this and the preceding paragraph. Hopefully the relative contributions of these two types of determinants will be revealed through genetic analysis. The predicted phenotypes of mutants defective in qualitative determinants have been discussed already. Mutants defective in quantitative determinants would be predicted to have much more complex phenotypes and may be difficult to recognize. A third determinative mechanism, induction by cell-cell interaction, has been demonstrated in early embryos (Priess et al., 1987; see Section 11,G). It was by screening for maternal-effect lethal mutants missing specific lineages that Priess, Schnabel, and Schnabel recovered the glp-l mutants, which lack anterior pharyngeal muscle as a result of defects in signaling during the early cleavage stages. Thus screens for mutants missing specific lineages may identify many of the products that play critical roles in early development: signals, signal receptors, determinants, and partitioning machinery. The C. elegans genome is far from being saturated for maternal-effect

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lethal mutations. To date only chromosome I1 has been saturated for such mutations, which appear to fall into two general classes (K. Kemphues, personal communication). The par loci belong to a class of purely maternaleffect genes, genes expressed solely by the maternal genome for use during embryogenesis. Mutations in pure maternal-effect genes were recovered with high frequency during saturation mutagenesis of chromosome I1 for maternal-effect mutants. Many of the temperature-sensitive loci characterized thus far belong to a second class of mutants that may identify genes involved in essential metabolic or structural functions. Such genes appear to be required during multiple stages of development, including oogenesis. Nonconditional alleles of this latter class of mutants were recovered with low frequency during saturation mutagenesis of chromosome I1 for maternal-effect mutants. Based on saturation of chromosome I1 for the high-frequency class of maternaleffect lethal mutations, it is estimated that there are between 24 and 60 pure maternal-effect genes in the genome (K. Kemphues. personal communication). This is out of an estimated 20004000 essential genes in the genome (Brenner, 1974; Moerman and Baillie, 1979), of which 200-1000 are thought to be expressed during oogenesis (cf. Wilkins, 1986). Genetic analysis of embryogenesis has thus far focused on maternally expressed genes, partly because mutagenesis of late larval and early adult hermaphrodites with ethyl methane sulfonate produces mainly maternal.effect lethals. Most of the mutants analyzed are recessive. Only recently have screens for dominant temperature-sensitive maternal-effect lethal mutations been carried out. The six loci so far recovered may identify members of multigene families or haplo-insufficient genes, in addition to dominant alleles of genes that can be isolated in screens for recessive mutants (P. Mains and W. Wood, personal communication). It is not known when the zygotic genome is first expressed or what roles the earliest zygotically expressed gene products play in embryo development. Answers to these two questions may come from current efforts to identify genes that are transcribed in early embryos (I. Schauer and W. Wood, personal communication) and efforts to recover mutations in embryonically active genes by using the mutagen trimethyipsoralen, which has been shown to produce mainly zygotic lethal mutants (L. Edgar, personal communication). V. Summary Caenorhabdifis ekgans zygotes undergo a series of four differentiative divisions to generate 5 somatic founder cells and a germ-line progenitor cell by the 16- to 24-cell stage of embryogenesis. The pattern of divisions. cell positions. and development of the embryonic cells are invariant from

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embryo to embryo. Through a combination of embryo manipulation, treatment of embryos with pharmacologic agents, and genetic analysis of maternal-effect embryonic-lethal mutants, researchers in several laboratories have investigated when and how cell differences are generated and cell fates are specified during embryogenesis: 1. Most blastomeres develop in a cell-autonomous manner. They do not need to undergo cell division and they do not require their normal neighbors to express differentiation products characteristic of their lineage. In embryos in which specific cells have been ablated, the fates of neighboring cells do not change to compensate for the missing cells. These observations suggest that most embryonic cells are determined by lineally transmitted internally segregated information. 2. There is at least one clear-cut example of inductive interactions during early development. The anterior daughter of AB gives rise to hypodermis, neurons, pharyngeal muscles, and body wall muscles. Interactions between ABa cells and P,-derived blastomeres are required between the 4- and 28cell stage for ABa to generate pharyngeal and body wall muscles. ABa appears to be directed to generate hypodermis by internally segregated cues and directed to generate muscle by external cues. 3. Certain of the early internal segregation events require the participation of microfilaments. Disruption of the microfilament array leads to the missegregation of germ granules and of the potential of cells to undergo unequal germ-line-like divisions. Microfilaments may be involved in many other segregation events as well. 4. Several maternal-effect lethal mutants also perturb zygotic segregation events. These par mutants, which divide symmetrically and fail to segregate germ granules, may identify genes whose products interact with microfilaments or otherwise participate in cytoplasmic localization during the early divisions.

VI. Future Perspectives Several current and future lines of investigation will help resolve many of the unanswered questions. The genome has not yet been saturated for maternal-effect embryonic-lethal mutations. Our understanding of when and how maternal products function and interact will improve as more mutants are recovered and analyzed. In addition to analyzing mutants that affect multiple localization and segregation events, like the par mutants, it is worthwhile to screen for mutations in genes encoding the determinants that are postulated to exist and be segregated to specific blastomeres to participate in their future development. It also seems likely

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that other examples of cell-cell interactions will be revealed by systematic genetic analysis of the requirements for the generation of specific cells and tissues in the embryo. Of special interest are those cells and tissues that arise via a complex lineage from a founder cell that produces diverse cell types. Mutants in embryonically expressed genes need to be recovered and analyzed in order to determine when, in what lineages, and for what purposes transcription is turned on during early development. Eventually, cloning and analysis of the genes identified by mutational analysis will elucidate the nature and perhaps mechanism of function of important maternal and zygotic products. P granules serve as the only early lineage-specific marker and visible example of cytoplasmic localization in early C. efegans embryos. Analysis of the behavior of other early lineage-specific markers in mutant and manipulated embryos would add considerably to our ability to interpret the effects of the mutations and manipulations. Monoclonal antibody screens for lineage-specific antigens in early embryos have thus far yielded only antibodies to P granules (S. Strome and W. B. Wood, unpublished). Immunosuppression (Matthew and Sandrock, 1987) and immunization of mice with more defined immunogens may enable the generation of antibodies to lineage-specific antigens that are rare or poorly immunogenic. In addition, using cross-reactive antibodies to different cytoskeletal elements in early embryos may indicate when and how the cytoskeleton participates in early events. cDNA probes and antibodies to the gene products of the par and other maternal-effect lethal genes can be generated once the genes have been cloned. Analysis of the distribution and behavior of the RNAs and proteins will help elucidate when and how they function. The introduction of components into the gonads of adult hermaphrodites may provide an extremely useful means of either perturbing normal embryos or rescuing mutant embryos. Transgenic nematodes are currently being generated by the injection of cloned fragments of DNA into gonads (Fire, 1986). Transient rescue of maternal-effect lethal mutant embryos by injection of cloned DNA into the gonad of the mother may provide an easier method of cloning some of the interesting maternal effect genes than tagging the genes with transposons (Moerman et al., 1986). Injection of antisense R N A may be used to prevent translation of maternal RNAs (Izant and Weintraub, 1985; Melton, 1985). Finally injection of antibodies may be used either to inactivate or to tag intracellular components, for analysis of function and behavior of the components during early development. The determination of the cell lineage for C. efegans has provided a detailed picture of when and where different cells arise during development. Our current challenge is to explain mechanisms, to learn how cells in the

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early embryo become instructed to generate the complex patterns and types of cells found in the adult organism.

ACKNOWLEDGMENTS This review attests to the team spirit of C. elegans researchers around the world; a great deal has been learned by a relatively small group of researchers in a short period of time. I am grateful to all who have contributed to the body of knowledge presented here, and to Lois Edgar, David Hill, Rudy Raff, Bill Saxton, and Peter Cherbas for critical reading of the manuscript.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 114

Uracil-DNA Glycosylases and DNA Uracil Repair N. v. TOMILIN AND 0. N . APRELIKOVA Laboratory of Chromosome Stability, Institute of Cytology, Academy of Sciences of the Union of Soviet Socialist Republics, 194064 Leningrad, USSR

I. Introduction Stability of genetic material in living cells is not only a consequence of the chemical stability of DNA, but the result of the action of multiple enzymatic pathways of DNA repair (for reviews, see Hanawalt and Setlow, 1975; Hanawalt et al., 1978). The mechanism of DNA repair, namely excision repair, is based on the enzymatic recognition of the damaged nucleotide in double-stranded DNA (dsDNA) and a breakage of phosphodiester bond in one complementary strand which contains the lesion (Setlow and Carrier, 1964; Boyce and Howard-Flanders, 1964), followed by local nucleolytic DNA degradation and a repair synthesis-ligation step (Pettijohn and Hanawalt, 1964; De Lucia and Cairns, 1969). Most natural DNAs contain thymine and RNAs contain uracil, which has the same coding potential as thymine and forms complementary pairs with adenine. The first indications of the possible mechanisms of appearance of uracil residues in DNA were obtained in Arthur Kornberg’s laboratory (Bessman et al., 1958), where it was found that dUMP might be incorporated into DNA by DNA polymerase, and by Schuster (l960), who found that nitrous acid induces deamination of cytosine in native DNA at pH 4.2. It was shown later that DNA uracil might be also induced by photolysis of 5-bromouracil-substituted DNA (Wacker, 1963) and by the heating of denatured DNA at neutral pH leading to cytosine deamination (Shapiro and Klein, 1966; Lindahl and Nyberg, 1974). Some bacteriophages (PBSI and PBS2 of Bacillus subtilis) were found to contain uracil instead of thymine as a normal constituent of their DNA (Takahashi and Marmur, 1963), but discovery of the presence in bacteria of the enzyme dUTPase, which cleaves dUTP (Bertani et al., 1963), has suggested that bacteria avoid massive incorporation of dUMP into DNA in vivo. The reason for this avoidance was found by Geider (1972), who detected accumulation of short DNA fragments in semipermeable Escherichia coli cells after incorporation of exogenous dUMP: this result in125 English translation copyrighl 0 1989 by Academic Press, Inc.

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dicated that the enzymes selectively cleaving uracil-containing DNA are present in bacteria. Enzymatic activity inducing breaks in photolysed 5bromouracil-substituted DNA and in single-stranded DNA (ssDNA) treated with nitrous acid was also found in partially purified preparations of U V-endonuclease from Micrococcus lirteus (Carrier and Setlow, 1972; Tomilin er al., 1972). Uracil-DNA glycosylase (UDG) was detected in E. coli by Lindahl (1974), who found that degradation of uracil-substituted DNA was initiated by cleavage of the N-glycosylic bond of the dUMP residue in DNA. This was the first discovered repair enzyme able to cleave the N-glycosidic bond at the DNA level, and later other DNA glycosylases were found (Lindahl, 1982). DNA glycosylases generate the abasic sites which are known to be attacked by special nucleases present in bacteria (Strauss and Robbins, 1968; Verly and Paquette, 1972) and in eukaryotic cells (Verly and Paquette, 1973). Isolation of E. coii mutants deficient in dUTPase (Tye et al., 1977) and in UDG (Duncan et al., 1978) was important in elucidation of the biological function of UDG and DNA uracil repair in vivo. Selective enhancement of C -+ T mutagenesis in ung mutants, deficient in UDG (Duncan and Miller, 1980). suggested the role of UDG-driven repair of spontaneously deaminated DNA cytosines in the maintenance of genetic stability in nondamaged cells. UDG was also shown to be involved in excision repair of uracil incorporated into DNA during replication (Tye et al., 1978: Shlomai and Kornberg, 1978; Goulian ef al., 1980). The induction of increased transient incorporation of dUMP in eukaryotic cells was suggested to be the cause of chromosome breakage (Hagerman, 1984; Yunis and Soreng, 1984) and sister chromatid exchanges, or SCE (Pardo et al., 19871, but the biological role of dUMP incorporation and repair in normal cells remains unclear. Replicative incorporation of dUMP is frequently referred to as “misincorporation” (Lindahl, 1974; Goulian er al., 1980), which might have only deleterious and unwanted effects. However, it was suggested by Shlomai and Kornberg (1978) that “a regulated inclusion of uracil leading to periodic breaks in DNA may be a cellular design to facilitate recombination or other metabolic or structural features of advantage to the cell.” In this review the data supporting this point of view will be discussed. The review is specially devoted to DNA uracil repair and uracil-DNA glycosylases. Other relevant review articles have been cited for further reference (Hanawalt et al., 1979, 1981; Lindahl. 1979, 1982; Duncan, 1981; Lehman and Karran, 1981; Loeb and Kunkel, 1982; Miller, 1983: Tomilin, 1983: Taylor, 1984; Friedberg, 1985; Aprelikova, 1986; Gutierrez, 1987).

U R A C I L D N A GLYCOSYLASES AND DNA URACIL REPAIR

I27

11. Sources of DNA Uracil

A. CHEMICAL MODIFICATIONS OF NUCLEOTIDES 1. Spontaneous Deamination of Cytosine

Shapiro and Klein (1966) were the first to propose that deamination of nitrogen bases might play an important role in spontaneous mutagenesis. They found that cytosine and cytidine are relatively rapidly deaminated by heating in weakly acidic buffers, while there is no detectable deamination of adenosine and guanosine under these conditions. The deamination of cytosine results in the formation of uracil residues. Since dUMP residues are paired with adenine during semiconservative replication, such a deamination process should lead to the GC -+ AT transitions. This might be one of the reasons for the exclusion of deoxyuridylate as one of the typical components of DNA. Since cytosine could be readily deaminated, the product of this reaction should be different from other normal bases in DNA. Otherwise it should not be recognized by a special repair enzyme, which will be potentially able to prevent C + T transitions (Holliday, 1979). The mechanism of spontaneous cytosine deamination has not been established (Shapiro and Klein, 1966; Lindahl and Nyberg, 1974). Two mechanisms are under discussion: after protonation of the N-3 position there is either direct attack at the 4 position or buffer anion addition to the 6 position of the ring and ultimately the nucleophilic displacement of the amino group (Fig. I). Hydrolytic cytosine deamination has also been studied. The rate of deamination of single-stranded and double-stranded E. coli DNA, in the poly(dC), poly(dG) : poly(dC), and in dCMP was investigated as a function of temperature, pH, and buffer composition (Lindahl and Nyberg, 1974). It was shown that heat-induced deamination of cytosine residues in ssDNA took place at an easily detectable rate in aqueous buffers at pH 7.4. At 95°C this reaction as well as the reaction with poly(dC) and dCMP proceeds with the rate constant K = 2 x lo-' residues per second, associated with an activation energy of 29 kcaVmo1, and is essentially independent of the buffer composition. The extrapolation of these data to 37°C results in the K = 2 x lo-'' residues per second. dsDNA is well protected against hydrolytic deamination. According to unpublished data of Lindahl and Nyberg, it occurs at 0.3-0.5% of the rate observed with ssDNA. It is difficult to predict the degree of cytosine deamination in DNA in vivo, but it should be noted that there is transient generation of single-stranded regions during DNA replication and transcription (Henson, 1978). Such

I28

N . V. TOMILIN AND 0. N . APRELIKOVA

y 2 N

H N

FIG.I . Mechanisms for hydrolytic deamination of cytosine and cytidine. R, Hydrogen or -D-ribofuranosyl; B -,buffer anion; C, cytosine or cytidine; U, uracil or uridine. From Shapiro and Klein (1966).

regions would be potential targets for hydrolytic deamination of cytosine residues. Heat-induced rnutagenesis was studied using T4 phage (Baltz ef al., 1976). It is known that 5-hydroxymethylcytosine completely replaces cytosine in DNA of bacteriophages T2, T4, and T6, and the product of deamination of such an abnormal nucleotide is not a substrate for the uracil-DNA giycosylase (Friedberg et al., 1975). It was shown that GC -+ AT transitions are induced by heating in T4 DNA, and they arise from

URACIL-DNA GLYCOSYLASES AND DNA URACIL REPAIR

129

deamination of 5-hydroxymethylcytosine. The reaction was proton-catalyzed. The authors suggested that spontaneous mutations may occur at a high level, especially in the organisms with large genomes. They calculated that in the human cell, which contains about 1.4 x lo9 GC base pairs per haploid genome, the rate of heat-induced (spontaneous) mutagenesis would be about 100 per diploid genome per day, provided that none of the mutation were repaired and that rates of deamination of cytosine and 5-hydroxymethylcytosine are similar. However, it should be noted that in cells of higher eukaryotes DNA molecules are bound to polyamines or chromosomal proteins, which would provide a protective effect.

2. Cytosine Deamination by Bisulfite and Nitrous Acid Cytosine may be also deaminated by amino group-specific reagents such as bisulfite and nitrous acid. Sodium bisulfite catalyzes the deamination of cytosine under mild conditions of temperature and pH (Hayatsu, 1976). It selectively deaminates cytosine without simultaneous deamination of purine residues. The reaction is more rapid at weakly acidic (pH 5) than at neutral pH and is much more efficient with single-stranded as compared to double-stranded DNA (Shapiro et al., 1973). Because of the stereochemistry involved in the bisulfite ions’ attack on the cytosine ring, residues embedded within the Watson-Crick helix of dsDNA are essentially unreactive. The rate of cytosine deamination in duplex DNA appears to be
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N . V. TOMILIN AND 0. N. APRELIKOVA

tants of @XI74 were isolated after treatment of the wild-type phage with sodium bisulfite (Linney et al., 1972), and bisulfite-induced yeast mutants were isolated also (Dorange and Dupuy, 1972). De Giovanni-Donnelly (1985) reported that I M bisulfite is mutagenic for Salmonella strains carrying his G mutation. This was in agreement with the studies of Pagano and Zeiger (1985, 1987). But some conflicting results have also appeared. Kunz and Glickman (1983), using the E. coli lac I system (Miller, 19831, did not detect mutagenicity of bisulfite in these repair-proficient cells. No mutation induction was found in E. coli ung-,dcm-, recA- or other repairdeficient strains. Nitrous acid efficiently deaminates cytosine in DNA, but it also deaminates adenine to form hypoxanthine and guanine to form xanthine (Schuster, 1960). It effectively deaminates both single-stranded and doublestranded DNA (Zimmermann, 1977). The denatured DNA is deaminated two times faster than native DNA. The nitrous acid is a strong mutagen, but besides deamination of bases it catalyzes a number of side reactions. Because of the spontaneous cleavage of the labile xanthine-deoxyribose bond, HNOz induces abasic sites, crosslinking (Dublemen and Shapiro, 1977). and formation of several degradation products of guanine (Shapiro and Pohl, 1968). The mutagenic action of nitrite has been demonstrated in many organisms, including phages (Tessman, 1959; Scearce and Masker, 1986) and Saccharomyces ceresisiue (Zimmermann and Schwaier, 1%7), and among mutations induced by HNO,, deletions were found (Tessman, 1962: Schwartz and Beckwith, 1969). It has been shown that addition of sodium nitrite to cell cultures obtained from newborn Syrian hamsters resulted in malignant transformation of cells (Tsuda et al., 1973). All E. coli ungmutants tested are abnormally sensitive to nitrous acid (Da Roza et al., 19771. Since the ung- mutants are defective in UDG, this observation supports the view that the treatment of cells with nitrous acid causes deamination of cytosine and that slowly repaired uracil is deleterious to the cell. 3. Other Reactions Leading to DNA Uracil

Conversion of cytosine into uracil is detected after exposure of DNA to strong alkali (Ulman and McCarthy, 1973). Another possibility for introducing uracil residues into DNA is the incorporation of 5-bromouracil during DNA synthesis in vivo, or in vitro followed by the exposure to 313-nm radiation (Wacker, 1963; Lion, 1968; Carrier and Setlow, 1972). Cysteamine suppresses DNA breakage by the uracil radicals. The minor quantity of uracil is produced in DNA after exposure to ionizing radiation (Schotes, 1976).

URACIL-DNA GLYCOSYLASES AND DNA URACIL REPAIR

B. INCORPORATION

OF

131

dUMP DURING REPLICATIONAND REPAIR SYNTHESIS

Uracil may be incorporated into DNA molecules during replication and repair synthesis. It is known that thymine differs from uracil by the presence of methyl group in the 5' position of the pyrimidine ring, which does not participate in the formation of hydrogen bonds between complementary bases in DNA. dUTP might be used as a substrate by purified E. coli DNA polymerase I instead of dTTP (Bessman et al., 1958). The same data were obtained later for DNA polymerase TI1 holoenzyme (Shlomai and Kornberg, 19781, as well as for DNA polymerases a and p from calf thymus (Yoshida and Masaki, 1979). However, when two types of DNA templates were examined the incorporation of f3H]dATPby homogeneous E. coli DNA polymerase I on uracil-containing template was lower than on a thymine-containingone (Vilpo and Ridell, 1983). It seems, therefore, that the absence of methyl groups in DNA template influences the DNAbinding domain of DNA polymerase I. In contrast to cytosine deamination, the incorporation of uracil instead of thymine during DNA synthesis should not alter the replication fidelity of DNA. The incorporation of uracil into DNA has been observed in a number of studies. From the study of E. coli DNA replication in vitro, using cell lysates on cellophane disks, Olivera (1978) found that half of the nascent DNA was represented by 10s fragments. Addition of dUTP to the incubation mixture converted all the nascent DNA to small fragments. These findings suggested that only one of two strands was synthesized discontinuously, but both strands may undergo excision repair soon after synthesis owing to uracil incorporation. This transient accumulation of short fragments in newly synthesized DNA due to uracil excision was also observed in mammalian cells (Wist el al., 1978; Grafstrom et af.,1978)as well as polyomavirus (Brynolf et al., 1978) and adenovirus (Ariga and Shimojo, 1979). Using isolated nuclei from polyoma-infected cells, Brynolf et al. (1978) observed the accumulation of short nascent fragments of DNA when dTTP was partially replaced by dUTP. However, DNA replication ceased a short time after addition of dUTP. The deleterious effect of dUTP could be completely suppressed by the simultaneous addition of uracil (an inhibitor of UDG) to the system. When a large amount of dUTP was added, all newly synthesized DNA appeared as small fragments and no synthesis of long strands was observed. But when small amounts of dUTP (2.5% from dTTP) was added, these short fragments changed to high molecular weight DNA. These results suggest that at least a portion of Okazaki fragments formed during replication is derived from an excision repair of uracil incorporated into daughter strands.

132

N . V. TOMILIN AND 0. N. APRELIKOVA

Similar results have been observed with lysates of human lymphocytes supplemented with dUTP instead of dTTP (Grafstrom et al., 1978). However, the authors failed to obtain elongation of short fragments, and the synthesis of chains longer than 20s was not detected. This result may be explained either by the large quantity of dUTP added to the incubation mixture or by the loss of some elongation factor during preparation of lymphocyte lysates. Deoxynucleoside triphosphate samples may be contaminated with dUTP. A reconstituted enzyme system which generates ax174 viral circles from the duplex replicative form incorporated a few uracil residues per circle, although four common dNTPs were used as precursors (Shlomai and Kornberg, 1978). These uracil residues become targets for excision by UDG and DNA breakdown by nucleases destroying the viral circular DNA as a template for further replication. Addition of pure dUTPase prevented dUMP incorporation into DNA in vifro. Another puzzling observation is that dCTP increases the percentage of short fragments in the nascent DNA (Lundquist el a / . , 1974). This may be attributed to dUTP contaminating dCTP or derived from it during storage. Thus, the extent of dUMP incorporation into DNA is apparently related to the size of the intracellular dUTP pool. It is well known that dUTP is a normal intracellular metabolite which participates in dTTP biosynthesis pathways (Kornberg. 1981). There are a number of enzymes synthesizing dUTP, which is then cleaved by dUTPase to form dUMP and PP, (Bertani er a / . , 1961, 1963; Greenberg and Sommerville, 1963);dUMP is then converted to dTMP by thymidylate synthase, and the activity of dihydrofolate reductase is necessary for this reaction (Fig. 2). In bacteria dUTP arises also from enzymatic dCTP deamination (O'Donovan et al., 1971; Neuhard and Thomassen. 1976). In contrast to bacteria, in mammalian cells dCTP deaminase is absent and dUMP may be produced from dCMP via dCMP deaminase (Maley and Maley, 1962; Scarano et a / . , 1963). Shlomai and Kornberg ( 1978) calculated that the minimal steady-state level of dUTP required to supply dTTP for DNA synthesis was about 0.5 pM (this calculation assumed that de novo synthesis of thymidylate proceeds only via the dUMP pathway). Assuming that cellular dTTP concentration is 150 pM and affinities of dUTP and dTTP for DNA polymerase 111 are about the same. 1 uracil should be incorporated per 300 thymines or 1200 total nucleotides. The measurements of the dUTP pool in mammalian cells resulted in controversial data. In proliferating cultures of HeLa cells the quantity of dUTP was 1% of the total d7TP (Mahagaokar ef a / . , 1980). Human DNA contains 58% AT pairs, and 1 molecule of dUTP should be incorporated

-

URACILDNA GLYCOSYLASES AND DNA URACIL REPAIR

dUDP

I

I33

dTDP

laylate

-dTMP

DH F

rnethylene T H F

)AllpH DH F-reductase +Hi

glYc!ne\TH

serine

NADP+

FIG.2. The pathways of dUTP synthesis. DHF, Dihydrofolate; THF, tetrahydrofolate. The conversion of CDP and UDP to dCDP and dUDP is catalyzed by ribonucleoside diphosphate reductase. Adapted from Tye et al. (1977).

134

N. V. TOMILIN A N D 0. N . APRELIKOVA

per 350 nucleotide pairs during DNA synthesis. However, in mouse fibroblasts Nilsson et al. (1980) failed to find any dUTP during proliferation, although the authors considered the sensitivity of their method permitted determination of as little as 0.1% dUTP of dTTP content. Extracts of polyomavirus-infected mouse cells during onset of D N A synthesis contained 0.4% of dUTP. Goulian ef al. (1980) did not find any signs of dUTP in cultivated human cells either. It is possible that the size of the intracellular dUTP pool in mammalian cells depends on physiological factors and is closely regulated (Fig. 2). 111. Uracil-DNA Glycosylases and Excision Repair of DNA Containing

Apyrimidinic Sites Uracil-DNA glycosylase (UDG) was the first enzyme of excision repair found to release uracil from DNA by the cleavage of the N-glycosylic bond; several other DNA glycosylases were described (Lindahl, 1976; Kleihues and Margison, 1976; Karran and Lindahl, 1978; Riazuddin and Lindahl, 1978). Using uracil-containing DNA as a substrate, Lindahl (1974, 1976) and J . Duncan et al. (1976) identified UDG activity in E. coli and B . sirbtilis. This activity was first named uracil N-glycosidase (Lindahl, 1974) and then uracil-DNA glycosidase (Lindahl ef al., 1977), but has now been renamed uracil-DNA glycosylase, in accord with current recommendations for carbohydrate nomenclature (Lindahl, 1979). Uracil-DNA glycosylases were detected in all studied prokaryotic and eukaryotic organisms. The single exception is D . melanogaster. Embryos or cultured cells of Drusophih did not contain detectable amounts of UDG. The enzymatic activity cleaving DNA containing uracil was found only at the third-instar larvae stage of Drosophila development, and this enzyme was endonuclease (Deutsch and Spiering, 1982). Uracil-DNA glycosylases isolated from different organisms have some properties in common, and it appears that this is a rather conservative protein. The activity of UDG can be easily determined by incubation of an enzyme sample with DNA containing labeled uracil residues. For this purpose one can use DNA labeled in vitru by nick translation in the presence of dUTP labeled in uracil residue or DNA of the bacteriophages PBSI or PBS2, which normally contain uracil instead of thymine. The incubation of enzyme with uracil-containing substrate is usually followed by acid or alcohol precipitation of polymers and determination of the radioactivity of the soluble fraction. A more stringent method for assay of enzyme activity involves paper chromatography of released material fol-

URACIL-DNA GLYCOSYLASES AND DNA URACIL REPAIR

135

lowed by determination of radioactivity in the form of free uracil (Lindahl, 1974), or passage of the reaction mixture through a small column of Dowex1 in H,O. The eluate from such a column contains free uracil, while Dowex binds mononucleotides and polynucleotides (Duncan et a f . , 1978). The fact that UDG shows full activity in the presence of 5-10 mM EDTA permits measurements of the enzyme activity in crude cell extracts, since the activity of most DNases requires divalent metal ions (Friedberg et al., 1975; Sekiguchi et al., 1976).

A. PROKARYOTIC URACIL-DNAGLYCOSYLASES 1. E . coli

Escherichia coli UDG was purified by Lindahl and Nyberg (1974) and Lindahl et al. (1977). The procedure involved streptomycin sulfate and ammonium sulfate fractionation, gel filtration on Sephadex G-75, hydroxyapatite chromatography, and DNA-agarose chromatography. The enzyme was purified I 1,000-fold and showed better than 95% homogeneity. It was shown that E. coli UDG consists of a single subunit of molecular weight close to 24,500. This is a typical globular protein with a frictional ratio J’f0 of 1.2. The amino acid composition shows no unusual features, but the protein contains a few cysteine and serine residues and a high number of glutamic acid and glutamine residues. UDG has a broad pH optimum around 8; it shows no cofactor requirement, and the activity is resistant to 5 mM N-ethylmaleimide. It withstands heating for 5 minutes at 45°C in several buffers, but is inactivated after 5 minutes at 50°C. The purified E. coli UDG has a turnover number of 800. A rough estimation shows that one cell of E. coli contains about 300 enzyme molecules. Assuming that enzyme has a low K , = 4 x lo-’ M for dUMP residues in DNA, UDG should have the potential to release many more uracil residues than can be produced by the spontaneous cytosine deamination reaction in vitro. It was calculated that the rate of DNA deamination at pH 7.4 and 37°C is about 30 per hour per 6 x 10” Da (Setlow, 1982). Purified E. coli UDG does not release free uracil from dUMP and deoxyuridine. The shortest deoxyribonucleotide attacked by UDG is tetranucleotide. Uracil is not released by UDG from RNA, but is effectively released from both single-stranded and double-stranded DNA. For dsDNA it does not matter whether uracil residues are hydrogen-bonded as in PBSl and PBS2 DNA, or uracil forms a mismatched pair with guanine, as in partially deaminated DNA (Lindahl et al., 1977). UDG seems to have a strict substrate specificity and does not release uracil analogs from DNA such as thymine (5-methyluracil), 5-hydroxymethyluraci1,5-bromouracil,

I36

N . V. TOMILIN AND 0.N. APRELIKOVA

pyrimidine dimers. or deaminated purines (Lindahl, 1974; Lindahl et ul., 1977). All UDG analogs investigated are inhibited by free uracil. The K, for E. coli enzyme is about I x M (Lindahl et al., 1977). Uracil at 2 mM causes >90% inhibition of the E. coli enzyme in vifro. Another type of inhibitor, the specific protein, has been shown to be induced in extracts of bacteriophage T5-infected E. coli (Warner et al., 1980), even though this phage does not contain uracil residues in its DNA. This protein only inhibits UDG from this organism and has no effect on the enzyme from other sources. The biological role of this T5 inhibitor is not clear. 2 . B . sirbtilis UDG from B . subtilis has been purified and characterized and shown to resemble UDG from E. coli in most respects (Cone et al., 1977). The enzyme has a broad pH optimum between 7.3 and 7.8. At pH 8.4 the activity is 78% of that at 7.5. The enzyme has no requirement for divalent cations. It retains full activity in the presence of EDTA and is not stimulated by Ca" , Mg'+, or Mn" . The heavy-metal ions Fe" , Zn", and Co" inhibited the enzyme. In the presence of 30-50 mM NaCl the enzyme activity is stimulated 2-fold, but this effect is not specific for NaCl; similar results were obtained with other anions and cations. Bacillus subtilis UDG is insensitive to inhibition by 1 mM N-ethylmaleimide. It was shown that the enzyme has a molecular weight of approximately 24,000 and has no subunit structure. The substrate specificity of B. s u b tih UDG i s stringent. Uracil is the only base recognized by the enzyme. Thymine, cytosine, adenine, guanine, bromodeoxyuracil, and 5-hydroxymethyluraciIare not recognized as substrates in DNA; nor is uracil recognized in RNA. In addition, the deoxyribose must be on the same strand as the uracil, since the copolymer poly(rU) : poly(dA) is not attacked by the enzyme. Bacillus subtilis UDG does not release uracil from deoxyuridine, deoxyuridine monophosphate, or deoxyuridine triphosphate. The studies indicate that the oligomer (pU), is the smallest single-stranded oligomer attacked by the enzyme. The phage PBSl and several similar phages that can infect B. subtilis form a unique group of phages carrying DNA in which thymidine residues are replaced by uracil residues (Takahashi and Marmur, 1963). Early in the phage infection, several proteins are induced which alter metabolism of DNA precursors. These proteins include deoxythymidylate phosphohydrolase and an inhibitor of host dUTPase (Katz et al., 1976). In addition, the phage induces an inhibitor of UDG before progeny phage DNA syn-

URACIL-DNA GLYCOSYLASES AND DNA URACIL REPAIR

137

thesis commences (Friedberg et al., 1975; Katz et al., 1976; Makino and Munakata, 1977). When induction of inhibitor was prevented by the addition of chloramphenicol or actinomycin D immediately before PBS2 infection, parental phage DNA degraded in 6 minutes after infection to acidsoluble material (Duncan and Warner, 1977). But this antibiotic-dependent degradation does not occur if B. subtilis mutant deficient in UDG is used (Makino and Munakata, 1977). The protein inhibitor of UDG has been purified. It is a heat-stable protein of -20,000 MW (Tomita and Takahashi, 1975; J. Duncan et al., 1976; Friedberg et al., 1978). It was shown that the phage-induced inhibitor binds not only to the B. subtilis UDG, but also is able to interact with UDG from mammalian cells (Lindahl, 1979). 3. M. luteus UDG has been also purified from M. luteus (Tomilin et al., 1978; Leblanc et al., 1982). Like UDGs mentioned earlier, the enzyme from M. luteus has one subunit and a molecular weight of 16,000-19,000, with a broad optimum pH of 5-7. It prefers ssDNA as a substrate, but also releases uracil from dsDNA. It does not act on guanine residues opposite uracil in dsDNA or on xanthine residues in deaminated DNA (Tomilin et al., 1978). It was shown that M. luteus UDG is active toward UV-irradiated PBSl DNA, containing uracil-uracil dimers. Uracil seems to be a noncompetitive inhibitor of M. luteus UDG with a K i = 3.2 x M. Spermine stimulates UDG activity in concentrations of 10-100 mM. At low concentration spermidine shows the same effect, but it decreases the enzyme activity at high concentrations (Leblanc et al., 1982). The effect of intercalating DNA agents such as ethidium bromide and ellipticine resulted in 2- to 2.5-fold activation of UDG. Delort et al. (1985) synthesized different uracil-containing octadeoxynucleotides and have exposed them to the action of E. coli and M. luteus UDGs. It was found that uracil residue situated at the 5' end is excised by M. luteus, but not by E. coli UDG. If dUMP residue was located at the ultimate or penultimate position at the 3' end, its N-glycosylic bond was not cleaved by either enzyme. The initial rate of excision of two neighboring uracil residues or two uracils separated by a single nucleotide was decreased. The different behavior of the two enzymes with respect to the excision of uracil residues located at the 5' end may be due to a different mechanism of action. The UDG from E. coli is considered to be a processive enzyme (Lindahl, 1974), while the M. luteus enzyme is not (Leblanc et al., 1982). The data obtained by Delort et a / . (1985) also suggested that the sequence of the bases surrounding uracil has an influence on the rate of excision.

-

138

N. V. TOMILIN AND 0. N. APRELIKOVA

4. Bacillus srearothermophilus and Other Thermophilic Bacteria

UDG was purified from B. stearothermophilus (Kaboev et al., 1981) and Therrnothrix thiopara (Kaboev et al., 1985). The properties of these enzymes are summarized in Table I. It was shown that UDG from B . stearorhermophilus exhibits extreme thermostability. It could be heated at 60°C (growth temperature) for 60 hours without any loss of activity. The Arrhenius plot for the enzyme is a biphasic curve with transition temperature at 44°C. This transition probably reflects a conformational change of the protein (Kaboev et al., 1981). The enzyme does not require any cofactors and is not inhibited by EDTA and spermidine, but a high concentration of NaCI decreases the UDG activity significantly. Since the rate of spontaneous base deamination is increased with growth temperature of bacteria, one could expect that the activity of UDG in thermophilic strains is higher than that from mesophilic organisms. The specific activities of UDG were compared in nine species of bacteria with different optima of growth temperature (from 10" to 70°C). No correlation between the activity of UDG was found in cell extracts from thermophilic and mesophilic bacteria and the number of expected (Lindahl and Nyberg. 1974) heat-induced lesions in DNA (Andreev et al., 1983). It seems that the level of UDG activity in thermophiles and mesophiles is high enough to repair all potential heat-induced cytosine deaminations, and the dUTP pool and the rate of dUMP incorporation are about the same. Low UDG activity in psychrophiles (Andreev et al., 1983) might be associated with the low rate of cytosine deamination a n d o r with the low dUTP pool.

B. EUKARYOTIC URACIL-DNA GLYCOSYLASES 1 . Yeust and Plunt Cells

The UDG purified from S . cerevisiue does not differ from prokaryotic enzymes mentioned earlier (Crosby et al., 1981). Similar UDGs were isolated from wheat germ (Blaisdell and Warner, 1983) and Zea mays seedlings (Bensen and Warner, 1987). There were two different UDG activities in Zea mays. One of them was localized in the cell nucleus and the other in mitochondria. Both enzymes have a molecular weight of 18,000, but differ in K,, pH optimum, sensitivity to salts, and substrate specificity. Both enzymes are sensitive to N-ethylmaleimide. Uracil and its analogs, such as 6-aminouracil and 6-azauraci1, inhibit both UDGs from nuclei and mitochondria, but in different ways (Bensen and Warner, 1987). Using an original method of selection, Burgess and Klein (1986) have isolated an S . cerevisiae mutant deficient in nuclear UDG.

-

GENERAL PROPERTIES

OF

TABLE I URACIL-DNAGLYCOSYLASE FROM DIFFERENT ORGANISMS ~

MW

KM ( M )

PH optimum

Escherichia coli Bacillus subtilis

24,500 24,000

4.0 x lo-* 1.1 x

8.0 7.3-7.8

Micrococcus luteus

19,400

7.0 x 10.'

5.0-7.0

Bacillus stearothermophilus Thermothrix thiopara Saccharomyces cerevisiae Dictyostelium discoideum Artemia salina

29,000

Calf thymus

28,700

7.0

X

7.2-8.6

Rat liver Nucleus Mitochondria

35,000 20,000

2.0 x

7.5-8.0

Source

Human fibroblasts Nucleus Mitochondria Human HeLa cells Human acute leukemia cells

26,000 27,800

7.5-9.0 7.5-8.0

55,000

6.5-8.5 8.5-9.0

2.3 x 5.3 x

Activators and inhibitors Inhibited by NaCl Inhibited by heavy metals, stimulated by NaCl Activated by spermine and spermidine, inhibited by spermidine at high concentrations Inhibited by NaCl

Lindahl et a / . (1977) Cone et a l . (1977)

Inhibited by N-ethylmaleimide Inhibited by CaCIz, stimulated by NaCl

Kaboev ef al. (1985) Crosby et al. (1981)

8.0

X

lo-'

7.2-8.0

Leblanc et al. (1982)

Kaboev et a / . (1981)

Guyer et al. (1986) KCI inhibits at high concentrations, stimulates at low concentrations Inhibited by NaCI, MgCI,, CaCI,, p-hydroxymercuribenzoate

Birch and McLennan ( 1980) Talpaert-Borle et al. (1979); Talpaert-Borle and Liuzzi (1982)

Inhibited by NaCl NaCl inhibits at high concentrations. stimulates at low concentrations

Golson and Verly (1983); Domena and Mosbaugh (1985)

Inhibited by NaCl Inhibited by NaCl and KCI

Gupta and Sirover (1981)

50,000

30,000

References

Inhibited by NaCI, KCI, MgCI,, MnCIz, CaCI,, EDTA, EGTA

Wist et al. (1978) Caradonna and Cheng (1980)

Zea mays

Nucleus Mitochondria Wheat g e m

18,000 18,000

Bensen and Warner (1987)

27,000

Blaisdell and Warner (1983)

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2. Mammaliun Cells UDGs from rat liver and calf thymus were well characterized (TalpaertBorle et al,, 1979; Talpaert-Borle and Liuzzi, 1982; Golson and Verly, 1983; Domena and Mosbaugh, 1985). The main steps of purification of mammalian UDG are the same: (NH,),SO, precipitation; chromatography on phosphocellulose, DNA-cellulose, and hydroxyapatite; and gel filtration on Sephadex G-75 or (3-100. It was pointed out that there are two different UDG activities in mammalian cells. Rat liver nuclei UDG has a molecular weight of -35,000. The mitochondria1enzyme shows a molecular weight of -20,000, another profile of inhibition with NaCI, and increased thermostability (Domena and Mosbaugh, 1985). The UDGs were also studied in rat hepatocytes (Golson and Verly, 1983). The majority of UDG activity was found to be associated with chromatin, but some enzyme activity was found also in the soluble nuclear fraction and cytoplasm. Nuclei contained 88% of the enzyme activity, while cytoplasm accounted for 12%. Nearly three-fourths (72%)of nuclear activity is associated with chromatin and the remainder (28%) with the soluble nuclear fraction. Chromatin UDG has interesting substrate specificity. Golson and Verly (1983) have shown that the apparent K, for dUMP residues was the same for the dsDNA and ssDNA substrates, but V,,, was slightly higher with ssDNA; the amount of uracil residues in dsDNA affects the kinetic parameters of the enzyme. The greater the frequency of uracil, the greater K , and V,,,. The inhibitory effect of NaCl and uracil was higher for dsDNA than for ssDNA. The authors speculate that NaCl and uracil change the melting point of double helix, which is necessary for the formation of enzyme-substrate complex. The fact that increased density of uracil residues in double-stranded substrate increases the reaction rate is interpreted as the tendency of enzyme to work in a processive manner. An interesting result was obtained with purified UDG from calf thymus. The enzyme activity was strongly impaired when substrates containing apyramidinic/apurinic (AP) sites were examined (Talpaert-Borle and Liuzzi, 1982). The inhibitory effect of AP sites takes place at a concentration range 100-fold lower than that for free uracil. Both uracil and AP sites are produced by UDG during uracil excision. Since excised uracil diffuses into the surrounding media, the inhibition of UDG by uracil could hardly play any physiological role in the cell (for inhibition it is necessary to have about 2-6 mM concentration of uracil), but AP sites could inhibit UDG. Such inhibition of UDG activity in v i w may be important for coordination of the action of UDG with AP endonucleases. Human UDG has been identified in extracts from human placenta (Sekiguchi er al., 19761, fibroblasts (Kuhnlein et al., 1978), HeLa cells (Krokan

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and Witwer, 1981), human lymphocytes (Sirover, 1979), blast cells of patients with acute myelocytic leukemia (Caradonna and Cheng, 1980) and different white blood cells (Koistinen and Vilpo, 1986a). Human UDG is monomeric protein, which has a molecular weight of -30,000. Sirover (1979) observed two species of UDG in either phytohemagglutinin(PHA)stimulated or nonstimulated human lymphocytes with a molecular weight of -40,000. Attempts were made to detect heterogeneity of UDG of other cell types. Crude extracts of white blood cells from acute and chronic myelocytic leukemia patients as well as extracts of cultured HeLa and Molt 4F cells were subjected to polyacrylamide electrophoresis and isoelectric focusing. No heterogeneity was observed in any of these extracts (Caradonna and Cheng, 1980). UDG from all these sources exhibited similar electrophoretic mobilities (R, = 0.37) and PI values of 9.5. Human UDGs are inhibited by monovalent and divalent ions and, in some cases, by EDTA and EGTA (Caradonna and Cheng, 1980), which may indicate that UDG is a metalloenzyme. Fourteen uracil analogs were tested to establish a structure-activity relationship for this enzyme. It appears that UDG is specific for uracil moieties. Uracil is a noncompetitive inhibitor of human UDG with a K i value of 2.2 x M.For the purified UDG from HeLa cells it has shown a 3-fold increase of the rate of uracil excision from a single-stranded substrate as compared to double-stranded DNA, and a 20-fold decrease of activity when dUMP residues occurred at the 3' end of DNA molecules. In addition, the rate of uracil excision was very low when dNTP surrounding dUTP were changed for rNTP (Krokan and Witwer, 1981). It was proposed that there were specific nucleotide sequences containing dUMP residues which could be preferentially recognized by UDG. For human KB cells almost all UDG activity is localized in cell nuclei. Only 5% activity was associated with mitochondria. PBSZinduced inhibitor of UDG suppressed nuclear enzyme more strongly than enzyme from mitochondria (Anderson and Friedberg, 1980). UDG was purified from human placenta, and a series of monoclonal antibodies (mAb) have been prepared (Arenaz and Sirover, 1983). Using immunoprecipitation technique, interaction of the antibodies with different UDGs was studied. Partial cross-reactivity was observed with rat liver glycosylase and with hamster enzyme. In contrast, no cross-reactivity was observed with yeast or E. coli UDG. Immunoprecipitation reaction of placenta UDG with mAb diminished enzyme activity to approximately 50%. The same results were obtained with glycerol gradient analysis. The authors consider that such incomplete immunoprecipitation of UDG activity cannot be due to low antibody affinity, but they suggest that human

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cells may contain two UDGs that are antigenically distinct. Each antibody may recognize one or the other isoenzyme. Further studies of antibody-glycosylase interactions have shown that additional purification of placenta UDG resulted in the preserving of enzyme-antibody binding with one mAb (Seal and Sirover, 1986).This mAb had no effect on homogeneous human UDG but strongly inhibited the activity of the human DNA polymerase a-catalytic subunit. Addition of purified DNA polymerase OL to homogeneous human UDG restored the inhibition of DNA glycosylase activity by this mAb. These findings suggest physical association of UDG with DNA polymerase a-catalytic subunit and the existence of base excision repair multienzyme complex. Using one of the antibodies recognizing antigenic determinants in homogeneous human placental UDG, the in v i m and in vivo biosynthesis of immunoreactive protein from poly(A)' R N A have been followed (Vollberg et a / ., 1987a). Immunoreactive glycosylase protein was synthesized with MW 37,000 from 16s poly(A)+ RNA. This molecular weight is identical to that observed for catalytically active homogeneous placental UDG. I n vivo biosynthesis, as defined by immunoblot analysis of human fibroblast cell extracts and immunoprecipitation of [35S]methionine-labeledcell protein, gave the same results and failed to detect any precursor polypeptides. Using the set of mAb, Vollberg et a / . (1987b) have detected conformational abnormality of UDG in Bloom's syndrome cells. The role of this abnormality in chromosome destabilization in Bloom's syndrome cells is not known, but the cells are also deficient in DNA ligase 1 (Willis and Lindahl, 1987). Electrophoretically homogeneous UDG isolated from human placenta was found to be inhibited by hypermethylation of DNA substrate (Aprelikova et al., 1988). Using the set of DNA substrates containing variable amounts of 5-methylcytosine and a fixed amount of uracil, these authors have shown that the KM of UDG on methylated DNA is about three times lower as compared to nonmethylated substrate. UDG activity was also examined in cultured fibroblasts from patients with the hereditary diseases xeroderma pigmentosum (XP) and ataxia telangiectasia (AT) (Sekiguchi ef a / . , 1976; Kuhnlein er a / . , 1978). These patients are at high risk for developing cancer, and their cultured cells are deficient in repair of DNA lesions caused by U V irradiation (XP) or X rays (AT). The repair deficiency in the case of XP falls into at least nine genetic complementation groups (Hoeijmakers, 1987); in addition, another variant form exists in which subjects have the clinical symptoms of XP, but cultured fibroblasts have normal excision repair. The latter cells are thought to be deficient in postreplication repair (Lehman et a / . ,

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1975). Kuhnlein et al. (1978) have shown that cell lines representative of complementation groups A and D of XP and AT had roughly the same level of activity as did the normal cells. But the cells from two XP variants had roughly half the normal level of activity. In spite of these quantitative differences, no systematic alterations in reaction characteristics, apparent K , for substrate, or purification characteristics were noted.

REPAIROF DNA CONTAINING APYRIMIDINIC/APURINIC C. EXCISION (AP) SITES 1. AP Endonucleases

The first report for the presence in extracts from Micrococcus lysodeikricus (luteus) of endonucleolytic activity inducing breaks in DNA containing AP sites (AP endonuclease) was published by Strauss and Robbins (1968). Similar endonucleolytic activity was found later in E. coli (Friedberg and Goldthwait, 1969; Hadi and Goldthwait, 1971; Verly and Paquette, 1972; Paquette et al., 1972), in mammalian cells (Verly et al., 1973; Ljungquist and Lindahl, 1974; Teebor and Duker, 1975; Linsley et al., 1977), in birds (Risvi and Hadi, 1977), in lower eukaryotes (Chlebowicz and Jachimczyk, 1977; Thibodeau and Verly, 1978), and in plant cells (Thibodeau and Verly, 1977; Veleminsky et al., 1977; Svachulova et al., 1978). In most organisms AP endonucleases are found to be heterogeneous and associated with many different proteins. In M. luteus at least two different AP endonucleases are present (Tomilin, 1974; Tomilin et al., 1976, 1978; Tomilin and Barenfeld, 1977; Laval and Pierre, 1978; Pierre and Laval, 1980a,b). One of these AP endonucleases, which has a lower molecular weight (- 18,000), has an associated activity inducing singlestrand breaks in UV-irradiated DNA (Tomilin, 1974; Tomilin et al., 1976). This associated activity was originally identified as UV-endonuclease (Rorsch et al., 1964; Nakayama et af., 1%7) specific for pyrimidine dimers (Carrier and Setlow, 1970; Kaplan et al., 1971). Specificity of UV-endonuclease having an associated AP-endonuclease activity (Tomilin, 1974; Tomilin et a f . , 1976) toward pyrimidine dimers in UV-irradiated DNA was evident from experiments where this enzyme was found to complement host cell reactivation defect and to suppress increased UV mutagenesis in uvr A, B, and C mutants of E. coli (Tomilin and Mosevitskaya, 1975). These mutants are known to be defective in the excision of pyrimidine dimers from DNA (Howard-Flanders and Boyce, 1966). The associated activity of low molecular weight M . luteus AP endonuclease toward UV-irradiated DNA was later identified as pyrimidine di-

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mer (PD) DNA glycosylase, which cleaves the 5'-glycosyl bond of dimer (Haseltine et a / . . 1980; Gordon and Haseltine, 1981; Grafstrom et al.. 1982). PD-DNA glycosylase of M. luteus was functional in vivo (La Belle and Linn, 1982), but no evidence of involvement of associated AP endonuclease in the repair of apyrimidinic sites in vivo has been presented so far. AP endonucleases with an associated PD-DNA glycosylase activity are not widely distributed in living organisms. Except for M. h t e U S , such enzyme is present only in E . coli infected with the phage T4 (Demple and Linn. 1980) and was found in human cells (La Belle and Linn, 1982). It seems that enzymes of this type play a minor role in the excision repair of AP sites in vivo. The second AP endonuclease of M. luteus which represents major APendonuclease activity and has a molecular weight of -30,000 shows no associated activity toward UV-irradiated DNA or associated exonuclease activity (Tomilin and Barenfeld, 1977; Tomilin et a / . , 1978; Lava1 and Pierre, 1978). Its role in the repair of AP sites in vivo is not proved, but seems possible because: ( 1 ) high activity of similar enzymes was found in many other organisms including human beings (Kuebler and Goldthwait, 1977; Ljungquist, 1977; Thibodeau and Verly, 1977; Bibor and Verly, 1978; Mosbaugh and Linn, 1980; Shaper et al., 1982); (2) when mixed with M. luteirs DNA polymerase and T4 phage DNA ligase, this AP endonuclease is able to repair AP sites in vitro (Tomilin and Barenfeld, 1977);(3) similar enzyme from rat liver is mainly localized in cell nuclei in association with chromatin (Thibodeau and Verly, 1978). Nuclear localization of the main UDG in proliferating human cells is well established (Gupta and Sirover, 1981). A special type of AP endonuclease associated with 3'. 5'-exonuclease, 3'-phosphatase activity is found in E. coli (Hadi and Goldthwait, 1971; Weiss. 1976) and in Haemophilus influenzas (Clements et al., 1978). There exists strong genetic evidence for involvement of this enzyme, which represents major AP-endonuclease activity in E . coli and is encoded by the gene xth A (Yajko and Weiss, 1975; White et a / . . 1976; Weiss, 1976) in the repair of apyrimidinic sites formed after UDG excision of uracil in vivo (Weiss et a / . , 1978). This evidence is that low viability of dut xth A double mutant deficient in dUTPase and AP endonuclease/exonuclease 111 is suppressed by additional ung mutation. On the other hand, AP endonuclease associated with 3'-exonuclease is not an essential enzyme because E. coli cells having deletion of the xth A gene are viable (Yajko and Weiss, 1975). Apparently in these cells excision repair of AP sites is performed by other enzymes with AP-endonuclease activity, such as endonuclease IV (Ljungquist, 1977). Enzymes with AP-endonuclease activity which are clearly different from AP endonucleases described previously were found in E. coli (Gates and

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Linn, 1977; Bonura et al., 1982)and in other organisms (Ihone and Kada, 1978; Svachulova et al., 1978), but their role in the repair of apyrimidinic sites formed by UDG is not clear. It is interesting that at alkaline pH (-9.3, E. coli endonuclease V cleaves DNA containing uracil residues (Gates and Linn, 1977),but also contains other endonucleolyticactivities, including AP endonuclease, and shows little activity at neutral pH. Some tryptophan-containing tripeptides (e.g., Lys-Trp-Lys) are able to promote the cleavage of AP sites in dsDNA in vitro (Behmoaras et al., 1981), but the significance of this reaction for the nicking of depurinated DNA in vitro is not known. Anyway, one important conclusion is that the incision step of excision repair of DNA containing AP sites might be performed by several different enzymes present in bulky amount in prokaryotic and eukaryotic cells, and this step seems not to be the rate-limiting step in the excision repair of uracil-containing DNA. 2 . Other Steps of Excision Repair

After cleavage of AP sites by AP endonuclease, the ssDNA break is formed having a modified 3' or 5' end depending on which specific enzyme cleaves depurinated DNA. Some AP endonucleases cleave DNA 5' to the AP site, forming normal 3'-OH and 5'-P04 group with base-free deoxyribose linked to polynucleotide chain (Ljungquist, 1977); but other AP endonucleases, such as M. luteus AP endonuclease associated with PDDNA glycosylase, cleaved DNA 3' to the AP site (Haseltine et al., 1980; Gordon and Haseltine, 1981; Grafstrom et al., 1982), forming a normal 5'-P04 end and 3'-OH group of base free deoxyribose. Human fibroblast AP endonuclease having a high affinity to AP sites and AP endonuclease isolated from the same cells, and having low affinity to AP sites, induces a break 5' to the AP site (Mosbaugh and Linn, 1980). On the other hand, AP endonuclease purified from human placenta induces single-strand breaks at either the 5' or the 3' side of the AP site in a given molecule, but not at both sides (Grafstrom e f al., 1982). The base free nucleotide at the DNA break created after the action of 5'-AP endonuclease can be excised by 3'-acting AP endonuclease or by 5' + 3' exonuclease and the base free nucleotide after the action of 3'AP endonuclease can be excised by 5'-endonuclease or 3' + 5'-exonuclease. Many other 3' += 5' and 5' + 3'-exonucleases, some of which are associated with DNA polymerases, exist in E . coli (Kornberg, 1981). Escherichia coli DNA polymerase I or M. luteus DNA polymerase can excise a base free nucleotide from DNA nicked by T4 phage 3'-AP endonuclease-PD-DNA glycosylase or by M. luteus 3'-AP endonuclease associated with PD-DNA glycosylase (Mosbaugh and Linn, 1982), and M. luteus DNA polymerase can eliminate a base free nucleotide from de-

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purinated DNA nicked with a high molecular weight AP endonuclease from M . luteus which has no associated PD-DNA glycosylase activity (Tomilin and Barenfeld, 1977; Lava1 and Pierre, 1978). 5' + 3' and 3' -+5'-endonucleases are present in mammalian cells also (Lindahl et al., 1%9a,b; Cook and Friedberg, 1978; Doniger and Grossman, 1976; Hollis and Grossman, 1981), and some of these enzymes are able to excise damaged or mispaired nucleotides from the nicked DNA. DNA polymerase 6, which is present in mammalian cells, has an associated exonuclease activity (Bymess et al., 1976). This activity is able to excise AP sites from the nicked DNA, and DNA polymerase then refills the gaps with normal nucleotides. Resynthesis during excision repair in E. coli is mainly performed by DNA polymerase I and, to a lesser extent, by DNA polymerase I1 and 111 (Kornberg, 1981; Hanawalt et al., 1981). The majority of DNA lesions are repaired by the short-patch mode of resynthesis (20-30 nucleotides long), but some lesions are eliminated by the long-patch mode (1500 nucleotides), and DNA polymerase I is required for both modes of repair synthesis (Cooper, 1982). Exonuclease VII also plays a role in the DNA degradation and determination of the size of patches during the resynthesis step (Masker and Chase, 1978), as well as DNA helicase I1 encoded by the gene uvr D (Kuemmerle et al., 1982). In mammalian cells the resynthesis step of excision repair is performed by DNA polymerases a and p (Miller and Chinault, 1982; Liu et ul., 1983; Dresler and Lieberman, 1983). However, it is difficult to exclude the potential role of DNA polymerase 6. Possibly DNA gyrase (top 11) is also involved in the resynthesis step during DNA excision repair (Mattern and Scudiero, 1981). The final step of excision repair is performed by DNA ligase, present in prokaryotic and eukaryotic cells (SoderhaU and Lindahl, 1973; Lehman, 1974; Kornberg. 1981). 3. Rupuir of A P Sites in Vivo

AP sites produced by UDG and by other agents are effectively repaired in vivo. In E. coli dut mutant, I dUMP residue is incorporated per 100 nucleotides during each replication, and all uracils are repaired via the U D G pathway (Hochhouser and Weiss, 1976; Tye et al., 19781, which involves AP endonuclease associated with exonuclease 111, DNA polymerase 1, and DNA ligase (Tye and Lehman, 1977; Weiss et al., 1978). dut xth A double mutants show decreased viability which is suppressed by additional ung mutation (Weiss et al., 1978). AP sites should be repaired before DNA replication because they block DNA synthesis (Schaaper and Loeb, 1981; Sagher and Strauss, 1983) and induce misincorporation of dAMP (Schaaper et af., 1983). During repair of AP sites, ssDNA breaks and gaps appear which also should be repaired

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before replication. Otherwise double-strand breaks might be formed. The size of patches of DNA synthesis during excision repair of AP sites in vivo is unknown, but there is no reason to think that the size is different from that during repair of other DNA lesions. In mammalian cells the size of patches of repair synthesis is smaller (1-100 nucleotides) as compared to E. coli (2G1500 nucleotides) and depends on the nature of the damaging agent (Regan et al., 1971; Edenberg and Hanawalt, 1972; Painter and Young, 1972; Cleaver, 1974; Regan and Setlow, 1974; Setlow et al., 1976; Hanawalt et al., 1979). In mammalian cells after X or y irradiation, which produce AP sites directly (Ljungquist and Lindahl, 1974; Tomilin and Barenfeld, 1979) or indirectly after elimination of damaged bases by DNA glycosylases (Chetsanga et al., 19811, the size of patches during repair synthesis is 1-3 nucleotides (Regan and Setlow, 1974). Methyl methanesulfonate, which also produces A P sites in mammalian DNA after spontaneous or DNA glycosylase-catalyzed removal of 3-methyladenine or 7-methylguanine (Cathcart and Goldthwait, 198 I ) , induces repair patches of 30-40 nucleotides (Th’ng and Walker, 1983). A special mechanism exists in normal mammalian cells which prevents initiation of DNA replication during extensive repair: even a few DNA breaks in a cluster of replicons might inhibit initiation of DNA synthesis in this domain (Painter and Young, 1980). This mechanism preventing replication of damaged DNA is defective in cells from AT patients (Painter and Young, 1980; Houldsworth and Lavin, 1980), who are abnormally sensitive to y irradiation (Taylor et al., 1975). DNA replication during repair in AT cells leads to the increased production of dsDNA breaks and chromosome aberrations (Taylor, 1978). Maximal time for the repair of incorporated uracils and AP sites, formed by UDG in mammalian cells, should correspond to the time interval necessary for complete rejoining of short DNA fragments after pulse-labeling with [3H]thymidine. The short DNA fragments, some of which are formed because of dUMP incorporation (see Section V), are completely rejoined within 1 hour after pulse with [3H]thymidine (Hyodo et al., 1970; Huberman and Horwitz, 1973; Gautschi and Clarkson, 1975).

IV. Mutants Deficient in Uracil-DNA Glycosylase and dUTPase

A. MUTANTS DEFICIENT IN URACILDNAGLYCOSYLASE cung MUTANTS) 1. E . coli

Escherichia coli strains containing low UDG activity in crude extract were isolated and the mutation was mapped at 55.6 minutes of E. coli chromosome (Duncan et al., 1978). One of the mutants (BD 10) contains no detectable UDG activity in crude extract, but after partial purification

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the residual UDG activity at the level of 0.5% of the wild-type strain was detected (Riazuddin and Lindahi, 1978). Subsequently, E. coli null mutants (residual activity <0.02%) containing deletion of the ung gene (or insertion in this gene) were isolated (Duncan, 1985), suggesting that UDG is not an enzyme essential for viability. This is consistent with the absence of UDG in Drosophila (Deutsch and Spiering, 1982). In E. coli ung mutant (BD lo), 1 uracil residue is incorporated per 20003000 normal DNA nucleotides (Tye et al., 1978; Shlomai and Kornberg, 1978). This value corresponds to a minimal number of dUMP transiently incorporated into DNA of the ung+ cells, because BD 10 has some residual UDG activity. Because nonreplicating E. coli DNA contains no uracil, UDG activity in the ung+ cells is high enough to repair at least 4 X lo3 uracil residues in each replication. The most interesting property of the ung mutants is their mutator phenotype (Duncan et ul., 1978; Duncan and Weiss, 1978; Duncan and Miller, 1980; Fix and Glickman, 1986). The majority (%%) of the amber mutations in the lac I gene of ung mutants are GC + AT transitions (Fix and Glickman, 1986), and there is no increase of the rate of other transitions and transversions (Duncan and Weiss, 1978). This suggests that most mutations in ring- strains arise via hydrolytic deamination of cytosine. This conclusion is also supported by the observation (Fix and Glickman, 1986) that the presence of AT pairs near mutated cytosine greatly increases the rate of spontaneous mutation in the ung strain. Apparently, AT richness facilitates local denaturations of DNA (“breathing”) and promotes deamination of cytosine because the rate of cytosine deamination in denatured DNA is 100-fold higher as compared to that in native DNA (Lindahl and Nyberg, 1974). Another interesting property of the leaky ung mutants is an increased frequency of genetic recombination in these cells (HYPER-REC phenotype), which probably is associated with slow residual repair of uracil and prolonged persistence of single-strand breaks during the repair. Null mutants having
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possible that slow repair of replicative uracil in the presence of a limited amount of UDG does not inhibit the next round of DNA replication, and some DNA breaks induced by residual UDG are converted into doublestrand breaks. Sensitivity of the ung mutants to chemical mutagens which are able to deaminate cytosine indicates that UDG is involved in the repair of not only the spontaneous but also of some induced lesions.

2 . B. subtilis Bacillus subtilis mutants (urg mutant) having
The intracellular pool of dUTP depends on the activity of deoxyuridine triphosphatase (dUTPase), which cleaves dUTP to form dUMP and PP, (Bertani et al., 1963). Escherichia coli mutants deficient in dUTPase (dut mutants) were isolated by Hochhouser and Weiss (1976). dut mutants accumulated shorter fragments of pulse-labeled DNA (4-5s) as compared

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to the wild-type cells (IOS),indicating that additional ssDNA breaks appear after synthesis of each Okazaki fragment (Tye er a / . , 1977). Apparently, these breaks arise because of increased incorporation of dUMP during replication and UDG-driven repair. The rate of rejoining of short fragments in dirt mutants is increased by additional pol A and /ig mutations (Tye and Lehman, 1977). and dur xrh A double mutants show decreased viability (Weiss el d.,1978), suggesting that DNA polymerase I , DNA ligase, and AP endonuclease/exonuclease I11 are involved in the repair of uracil. Shortening of pulse-labeled DNA fragments in the dut mutants and decreased viability of the dirr xrh double mutants are suppressed by ung mutation (Tye et d.,1978; Weiss er a / . , 1978). Despite smaller size of the Okazaki fragments in the dur mutants, the fragments are completely rejoined and DNA isolated from the mutants contains no detectable uracil (Hochhouser and Weiss, 1976). DNA isolated from the dut ung mutant contains up to 1% of dUMP residues (Duncan et al., 1978; Tye cr a / . , 1978). which indicates the capacity of the ring + x f h A + pol A --+ lig pathway to repair DNA uracil. Because the transient incorporation of dUMP in the dur' cells is 0.03-0.05% (Shlomai and Kornberg, 1978), the capacity of the ung pathway is about 20 times higher than that needed for complete repair in wild-type cells, and the pathway should be able to repair I% of DNA uracil appearing in the E. coli genome (-lo7 nucleotides) in each replication (i.e.. -2 x lo5 nucleotides of DNA uracil per hour). The phage T4, deficient in the virus-encoded dCTPase/ dUTPase and grown in the dur ung mutant, contains >7% of their nucleotides replaced for dUMP (Duncan and Warner, 1978). Infection with this phage of dut ' ung+ cells leads to the loss of viability and rapid degradation of phage DNA, and the effect is suppressed by the ung mutation (Duncan and Warner, 1978). UDG cleaves ssDNA containing uracil, and this explains the inactivation of uracil-substituted single-stranded M 13 phage DNA in ung+ host (Kunkel, 1985). But when the uracil-substituted double-stranded phage T4 is introduced 'into ung + cells, excision repair should take place. and the capacity of the ung pathway (- lo5nucleotides per hour) seems in principle enough for complete repair even at a high extent of dUMP substitution. Loss of viability of the dUMP-substituted T4 phage in the ung + host is probably associated with too extensive repair and formation of double-strand breaks because of overlapping of the gaps in complementary strands. The high frequency of incorporation of dUMP into DNA of the dut mutants (-1 per 100 nucleotides) should take place not only during replication but also during repair, which should proceed in these cells more slowly as compared to dur' cells. The increased incorporation of dUMP into DNA of the dur mutant and

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subsequent slow repair probably explain its hyperrecombinational phenotype (Konrad and Lehman, 1975). Therefore, a partial defect in the ung gene and in the dut gene both stimulate genetic recombination. 2. Use of Double dut ung Mutants in Oligonucleotide Mutagenesis

New methods of site-specific oligonucleotide mutagenesis in vitro have been developed (Smith, 1985), but these methods have low efficiency. The standard procedure for directed mutagenesis (Zoller and Smith, 1982) involves several steps: (1) isolation of single-stranded MI3 phage DNA containing a target sequence; (2) annealing of a “mutant” oligonucleotide or “mutant” restriction fragment which forms a mismatch with a target sequence and which serves as a primer for in vitro synthesis of the second MI3 strand by DNA polymerase; (3) joining of the synthesized strand by DNA ligase and isolation of circular covalently closed (ccc) molecules in alkaline sucrose gradients (enrichment step); (4) transfection of mismatched cccDNA into appropriate E. coli host and selection of the mutant phage progeny. Using this procedure, 1045% yield of mutants might be obtained without precautions to prevent mismatch repair (Zoller and Smith, 1982). It should be pointed out that in the absence of ligation and enrichment step the yield of mutants is very low, suggesting that the phage strand containing a single-strand break gives no progeny. Using the dut ung mutant to prepare M13 DNA with a target sequence, Kunkel has developed (Kunkel, 1985; Kunkel et al., 1987) a modification of the standard procedure which eliminates the time-consuming alkaline sucrose gradient enrichment step and increases the yield of mutants up to 90%. In Kunkel’s modification, MI3 phage is grown in the dur ung strain, and ssDNA isolated from this phage contains 20-30 uracil residues (Sagher and Straws, 1983). After priming with “mutant” oligonucleotide or restriction fragment and the synthesis-ligation step, the uracil-substituted nonmutant phage strand is degraded by the treatment with UDG and alkali in vitro, and the in vitro-synthesized “mutant” strand is transfected into ung+ or ung- host, which results in 80-90% yield of “mutant” plaques. The high yield of mutants (>50%) was also observed when mismatchcontaining ligated M 13 DNA with uracil substituted the phage strand and the uracil-free in vitro-synthesized “mutant” strand was introduced (without UDG and alkali treatment in vitro) into ung+ cells (Kunkel, 1985). By analogy with the experiment previously described (i.e., UDG and alkali treatment in vitro), it was suggested that the enrichment of mutant production observed after introduction of the double-stranded mismatched DNA into ung+ host is also associated with degradation of the uracilcontaining nonmutant phage strand in vivo (Kunkel, 1985; Kunkel et al., 1987). This interpretation of the latter experiment seems to be incorrect,

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since under identical conditions the absolute yield of the mutant in ung' host (587) was 2.4 times higher as compared to isogenic ung- host (248) (Kunkel, 1985). This increase of the absolute mutant yield on ung' host cannot be explained by the higher efficiency of transfection in ung+ host, because the same DNA treated with UDG in vitro shows the identical biological activity on ung- and ung+ cells (Kunkel, 1985). It is clear from the data presented in Table 2 of Kunkel's paper (1985) that not only inactivation of uracil-substituted strand, but also mismatch correction is responsible for a high yield of the mutant phage progeny after transfection of heteroduplex DNA into ung' host. This conclusion is supported by the observation that even in the absence of ligation of "mutant" M13 strand synthesized in vitro by DNA polymerase of the phage T4 (which causes no strand displacement). the yield of mutant progeny without selection might be as much as 67% (Kunkel er al., 1987). Escherichiu coli strain dur ung used by Kunkel for growth of M 13 phage and uracil substitution contains no dam or dcm mutations and is not defective in DNA methylation. On the other hand, transfection of heteroduplex M I 3 D N A with one strand substituted with uracil into img+ mi4t H. L, S of E. coli strain only slightly increases the yield of mutants (from SO to 60-70%; see Kunkel et al., 1987), suggesting that methylation-directed mismatch correction driven by mur H, L, S gene products (Radman et al., 1980; Radman and Wagner, 1986) plays a minor role in prevention of transfer of mutations from in virro-synthesized uracil-free strand to uracilsubstituted phage template strand. Kunkel's data therefore indicate that another mismatch correction system is operative in ung+ strains of E. coli which is independent of dam, mur H, S , L genes and depends on U D G , because it is eliminated by the ung mutation: frequency of mutant phage progeny after transfection of a half uracil-substituted heteroduplex DNA into i m g - host is 6.7% (Kunkel, 1985), in agreement with the value ( 10%) expected in the absence of correction at 20% ligation efficiency. It seems that U DG-dependent mismatch correction happens during extensive excision repair of uracil in the phage DNA strand, which takes place after transfection of heteroduplex DNA into ling+ host. This repair actually occurs, since survival of ligated dsDNA in ung- host is only three times higher as compared to that in ung+ host; this is in contrast with ssDNA of the uracil-substituted phage, for which plating efficiency in ung+ host is 10' times lower as compared to ung- host (Kunkel, 1985). Experiments performed recently in our laboratory by V. Golubovskaya using Kunkel's system with phage T4 DNA polymerase and synthetic oligonucleotide inducing frameshift mutations in the lac gene of M13 mp19 indicate that after the high efficiency in vitro synthesis step, even in the absence of DNA ligase (which results in about the same survival of half-

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uracil-substituted DNA on ung + and ung- cells), the absolute yield of lac- frameshift mutations is about five times higher in ungf as compared to ung- host. These data support the idea of involvement of uracil-DNA glycosylase and DNA uracil repair in the directed transfer of mutation from the oligonucleotide-containing uracil-free DNA strand to the uracilsubstituted phage DNA strand in E. coli. Indirect evidence for the existence of a similar system of UDG-dependent mismatch correction in mammalian cells will be discussed later.

c. DEAMINATION OF 5-METHYLCYTOSINE AND SPONTANEOUS MUTACENESIS The rate of spontaneous (hydrolytic) deamination of 5-methylcytosine in vitro is about four times higher as compared to dCMP (Scholes, 1976). Because the deaminated 5-methylcytosine residues formed in DNA (thymines) are not recognized by UDG (Lindahl, 1979), a high mutagenesis rate should be expected in the absence of GT mismatch repair. Analysis of spontaneous mutation in the episomal lac I gene of E. coli (Coulondre et al., 1978) showed that several hot spots of G : C+ A : T transitions were

associated with the second cytosine of the sequence 5'-CCAGG-3', which is known to be methylated at this point by the dcm gene product (Marinus and Morris, 1973). In ung+ strain, 60% of C ---* T transitions are associated with 5-methylcytosine (Fix and Glickman, 1986), and in ung strain other cytosine hot spots appear and contribution of 5-methylcytosine to spontaneous C + T transition becomes lower (Duncan and Miller, 1980; Fix and Glickman, 1986). These data suggest that spontaneous deamination of 5-methylcytosine occurs in bacteria in vivo. However, E. coli contains a short-patch mismatch correction system which preferentially excises thymine from G : T mismatches in the sequence 5 '-CTAGG-3' 3'-GGTCC-5'

(Radman and Wagner, 1986). This system seems to be efficient in E. coli, and no 5-methylcytosine mutation hot spots were detected in chromosomal lac I ,gene (Radman and Wagner, 1986). There is also indirect evidence for 5-methylcytosine deamination in higher eukaryotes. In sea urchin a minor fraction of thymine derives its methyl groups at the 5 position from methionine via S-adenosylmethionine (SAM) and not from tetrahydrofolate (Scarano et al., 1967; Grippo er al., 1970). Because there is no evidence for enzymatic deamination of 5-methylcytosine in DNA or for SAM-mediated transfer of the methyl group to DNA uracil (Taylor, 1984), the results of Scarano et al. suggest that spon-

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taneous deamination of 5-methylcytosine takes place in sea urchin and possibly in other eukaryotes. This is supported by strong evolutionary pressure to eliminate S’-CpG sequence (which could be methylated) from many structural genes and regulatory sequences in vertebrate cells (SubakSharpe, 1967; Russel et a f . , 1976; Bird, 1986). 5-Methylcytosine deamination might be responsible for genome destabilization via point mutations (Selker and Stevens, 1985). It is interesting that the housekeeping genes which are not methylated retain a high frequency of CpG (Bird, 1986), supporting the view that CpG elimination is associated with DNA methylation. Some fractions of mammalian DNA (e.g., bovine satellite I) also retain a high frequency of CpG doublet despite high levels of DNA methylation in somatic cells (Sano and Sager, 1982). The reason why bovine satellite I does not lose CpG sequences during evolution is that satellite I is not methylated in germline cells (Taylor, 1984). The methylation in somatic cells might have some regulatory role in differentiation. High levels of methylation in somatic cells are typical for satellite DNAs and palindromes (Bond et a f . , 1967; Kiryanov et a f . . 1974; Smirnov et al., 1977; Romanov and Vanyushin, 1981).

The regulation of mutation rates might be achieved not only via cytosine methylation but also by modulation of UDG activity: the decrease of relative UDG level should result in the increase of the frequency of C + T transitions. It should be noted, however, that some organisms (e.g., Drosuphifa) lack both DNA methylation and UDG (Taylor, 1984; Deutsch and Spiering, 1982), but nevertheless show a “normal” frequency of spontaneous mutations, most of which are insertional (Sprandling and Rubin, 19811.

V. Physiological Variations of UDG Activity and Intracellular dUTP Pool A. VARIATIONSOF UDG ACTIVITYI N EUKARYOTIC CELLS 1. Z’issue Distribution

UDG activity was studied in different tissues and cells of mammals. If the main function of UDG is the release of uracil incorporated during replication, a correlation between activity of the enzyme and the rate of DNA synthesis should be expected. According to this suggestion, a comparative study of UDG activity and the rate of DNA synthesis in different rat tissues was performed (Aprelikova and Tomilin, 1982). The results show good correlation between these two parameters: the higher the pro-

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liferative activity of tissue, the greater the specific activity of the enzyme in it. In accord with these data is the study of UDG in different types of human peripheral blood cells. The highest activity of glycosylase was found in cells able to perform replicative or repair DNA synthesis, or in cells containing actively transcribed genes (Koistinen and Vilpo, 1986a).These cells include T lymphocytes, B lymphocytes, and monocytes. Peripheral blood cells at the stage of terminal differentiation-namely erythrocytes, plateleis, and granulocytes-contained little or no UDG activity. The expression of UDG was also studied in human normal hematopoietic bone marrow cells and in their malignant counterparts, obtained from patients with chronic granulocytic leukemia (Koistinen and Vilpo, 1986b). The enzyme activity was highest in the proliferating granulocytic compartment (myeloblasts through myelocytes), and it was diminished in more differentiated cells. UDG activity was also higher in immature red cells. The same tendency was demonstrated for human malignant monoblasts, which were induced to differentiate by tetradecanoyl phorbol acetate (TPA).

2. Induction of UDG during Cell Proliferation and afrer Carcinogen Treatment With the beginning of studies of eukaryotic UDG, new aspects of enzyme function have appeared. The regulation of repair capacity of cells was studied as a function of cell growth. As compared to basal levels of base excision repair in quiescent cells, proliferating cells have greater capacity for DNA repair synthesis after exposure to methyl methanesulfonate (Scudiero er al., 1976), sodium bisulfite (Gupta and Sirover, 1980), and ionizing radiation (Lavin and Kidson, 1977). Stimulation of DNA synthesis and cell proliferation also resulted in induction of UDG activity. Desiccated gastrula of brine shrimp (Artemia salina) starts to develop when cysts are incubated in seawater. The period of preemergence development includes cell differentiation without any DNA replication. Only after hatching does DNA replication take place and the number of cells begin to grow. This experiment shows 40-fold stimulation of UDG activity before hatching (Birch and McLennan, 1980). Using regenerating liver as a model system, the specific activities of UDG as well as DNA polymerase activity and DNA synthesis were determined at definite intervals after partial hepatectomy (Gombar er al., 1981; Aprelikova and Tomilin, 1982). Both enzymes and DNA synthesis were stimulated at the same time intervals. No increase in either activity was observed in controls undergoing sham surgery.

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The induction of UDG activity was also found in the liver of rats treated with hepatocarcinogen 4-dimethylaminoazobenzene (Aprelikova and Medvedeva, 1983). After a single administration of carcinogen, proliferation of nondifferentiated cells takes place. The induction of UDG was observed to take place synchronously with the increase in proliferative activity of liver cells. The coordinate stimulation of UDG activity. DNA synthesis, and DNA polymerase activity was observed in different human cells in culture. Treatment of peripheral blood lymphocytes with PHA increased glycosylase activity 10-fold (Sirover, 1979). Gel filtration on Sephadex G-100 has shown the presence of two glycosylase peaks either in PHA-stimulated or unstimulated cells. However, PHA stimulation results in an increase of only the first peak of glycosylase activity. The occurrence of two lymphocyte glycosylase species may be due to heterogeneous culture of human lymphocytes. Since T lymphocytes are stimulated to cell division rather than B lymphocytes, the inducible UDG activity may arise from T lymphocytes, and the uninducible peak from B lymphocytes. Furthermore, treatment with either actinomycin D or cycloheximide at the moment of maximal stimulation (96 hours after PHA addition) diminished enzyme activity after an appreciable interval. This suggests that induction of UDG requires transcription and translation processes. The glycosylase activity started to decrease 5 hours after cycloheximide addition. This is in contrast to the immediate effect of puromycin on DNA polymerase activity during lymphocyte stimulation (Loeb et al., 1970). Thus, the glycosylase is a fairly stable enzyme. The regulation of glycosylase activity was studied in a thermosensitive mutant strain of Chinese hamster (Duker and Grant, 1980). Under nonpermissive temperature, cells of this strain are arrested in G, phase. The stimulation of cell division increases both UDG and dUTPase activities. The level of both enzymes is minimal in Go and is maximal in S phase. Besides these two enzymes the activity of AP endonuclease was examined at different phases of the cell cycle, and the activity was found to be constant. Further evidence for the capacity of normal human cells to regulate UDG activity was obtained using a synchronous population of W1-38 human diploid fibroblasts (Gupta and Sirover, 1980). Cells were synchronized by serum starvation. Fibroblasts were stimulated to proliferate by replating the quiescent cells in fresh media containing 20% calf serum. DNA synthesis, DNA polymerase, and UDG activity, as well as unscheduled DNA synthesis after exposure to sodium bisulfite or methyl methanesulfonate, were measured at time intervals during cell division. This experiment showed that UDG activity increased 5-fold during cell proliferation and unscheduled DNA synthesis was enhanced 4- to 30-fold in a similar fash-

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ion. But the increase of glycosylase activity and the enhancement of DNA repair occurred prior to the induction of DNA replication. Furthermore, at the maximal stimulation of DNA replication and DNA polymerase activity both glycosylase activity and DNA repair had substantially diminished. As cells entered the second cell cycle, the glycosylase activity again increased and then diminished. The authors assumed that this time schedule of activation evolved to ensure the transfer of correct genetic information to daughter cells and the stimulation of DNA repair before DNA replication would result in the correction of preexisting DNA modifications to ensure further the fidelity of DNA synthesis (Gupta and Sirover, 1980). However, it seems difficult to explain the physical association of UDG with DNA polymerase, observed in this laboratory later (Seal and Sirover, 1986), as well as the association of glycosylase activity with replicating rather than mature minichromosomes of SV40 (Krokan, I98 I). Perhaps a more attractive hypothesis to explain such an association is that UDG is necessary for the repair of uracil residues incorporated during replication. The amount of UDG in cells in the absence of DNA synthesis is enough for the prescreening of rare deaminated cytosines. Stimulation of UDG synthesis before DNA replication may be necessary for effective building of repair enzyme complex, working immediately after DNA synthesis. Temporal regulation of UDG during synchronous cell proliferation was examined in Bloom’s syndrome skin fibroblasts. Individuals with Bloom’s syndrome have an increased risk of neoplasia (German et al., 1977);their cells have an abnormally high spontaneous mutation rate (Gupta and Goldstein, 1980; Warren et al., 1981) and a high incidence of chromosomal aberrations (Chaganti et al., 1974; Bryant et al., 1979). In contrast to normal fibroblasts, neither nucleotide excision repair nor base excision repair was enhanced prior to the onset of DNA replication in Bloom’s syndrome cells. Expression of DNA repair pathways as well as the induction of UDG were coordinate with the induction of DNA replication and of DNA polymerase (Gupta and Sirover, 1984). This result suggests that Bloom’s syndrome cells may be characterized by specific alterations in the temporal sequence of gene regulation. The more detailed studies showed that only nuclear UDG was induced in WI-38 cells during proliferation (Gupta and Sirover, 1981). In contrast, the specific activity of mitochondrial glycosylase remained constant during cell proliferation. The previous data of these authors concerning with the existence of two species of UDG in lymphocyte culture (Sirover, 1979) could not be explained by the presence of nuclear and mitochondria1 enzyme, because the nuclear glycosylase represents >90% of total enzyme activity. But in lymphocytes both species were present in equal amounts. The discovery that the repair capacity depends on the proliferative state

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of the cell led to the suggestion that the extent of proliferation of a given cell population will determine the extent to which repair was increased. The enhancement of DNA repair would depend on the mean generation time of a specific cell population. Thus, cells characterized by short generation times would be expected to increase DNA repair to a greater extent than would cells with longer generation time. This question was examined in experiments with asynchronously proliferating hamster fibroblasts (mean generation time 13.79 hours) and normal human skin fibroblasts (mean generation time 27.64 hours) (Vollberg et al., 1984). The experiment shows that the extent of enhancement of UDG was greater in the hamster cells than in normal human cells (9.5-fold versus 4-fold, respectively). The exposure of normal human cells to 20 pM dirnethyl sulfate reduced the rate of cell growth and the proliferation-dependent stimulation of glycosylase. Plant cell UDG activity seems to have a different cell cycle-regulatory mechanism. The enzyme levels were studied in two types of nondividing cells in onion, Allium cepa (presprouting dormant and differentiated cells), and in meristems proliferating at different rates (Hernandez and Gutierrez. 1987). The extracts from both quiescent root cells (isolated from the bulbs before sprouting) and proliferating root meristem cells do not show large differences in UDG activity. Upon entry into the proliferation cycle, the UDG activity is enhanced, being directly proportional to the proliferation rate. However, as cells leave the meristem zone and initiate their differentiation program the UDG activity decreased below 5% of the level found in the meristem zone. Thus, root proliferating cells and quiescent root cells, but not differentiated cells, show a high UDG activity (Gutierrez, 1987). The regulation of UDG synthesis was also investigated in virus-infected cells. When HeLa cells were infected with either the type 1 or the type 2 herpes simplex virus (HSV), an increase of the activities of both UDG and dUTPase was observed (Caradonna and Cheng, 1981). The UDG activity increased 6- or 40-fold in cells infected with HSV type 1 and 2, respectively. At the same time, dUTPase activity increased only 3- to 4fold. Furthermore, the UDG extracted from infected cells exhibits characteristics which differ from that of the enzyme extracted from mockinfected HeLa cells. In addition, the serum from rabbits immunized against cells infected with herpesvirus specifically inhibits UDG activity and dUTPase activity from infected, but not from uninfected cells. It is not known whether viral infection results in a modification of a host enzyme or the virus induces a new enzyme. To determine whether viral transformation alters the proliferationdependent regulation of UDG, the induction of this enzyme in a normal

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cell strain and the corresponding SV40-transformed line was examined (Dehazya et al., 1986). Results obtained also demonstrated that the level of glycosylase activity in SV40-transformed cells is greater than in the nontransformed WI-38 cells. In transformed cells the constitutive and basal levels of UDG were greater than those observed in growing or in confluent nontransformed cells. Furthermore, the extent of UDG induction was also greater in transformed cells. The glycosylase regulation was also examined by immunological analysis using mAb. The data demonstrated an equivalence between the enzymes induced in both cell types, although other repair processes are altered upon SV40 transformation (Teo et al., 1983; Shiloh et al., 1983; Sklar and Strauss, 1983; Gantt et al., 1984). These results suggest that the proliferation-dependent regulation of base excision repair enzymes is retained in SV40-transformed human cells. The localization of UDG in SV40-replicating minichromosomes was studied by sedimentation analysis (Krokan, 1981). Replicating and mature minichromosomes isolated from the extracts of SV40-transformed CV- 1 cells were separated by sedimentation in a sucrose gradient. It was shown that the UDG activity cosediments with replicating, but not with mature chromatin. Only a small part of the enzyme activity was associated with mature chromatin and 34% was in a soluble form on top of the gradient. Such preferential association of the enzyme with replicating material suggests that most of the UDG activity in mammalian cells was used for repair of dUMP incorporated during replication.

B. METHOTREXATE-INDUCED INCREASEOF dUTP POOLAND CHROMOSOME FRAGILE SITES Goulian et a / . (1980) reported that dUMP is incorporated into DNA of animal cells in vivo. The same question was studied with bacterial cells, deficient in dUTPase, which resulted in accumulation of abnormally short Okazaki fragments (Tye et al., 1977). No animal cell mutants are available that lack either dUTPase or UDG, but the drugs that inhibit thymidylate synthase offer the possibility of studying uracil incorporation in vivo. Inhibitors of thymidylate synthase (e.g., the dihydrofolate reductase inhibitor, methotrexate), in addition to lowering the level of thymidylate (Tattersall et al., 1973; Friedland, 1973; Skoog et al., 1976), greatly expand the pool of dUMP (Tattersall et al., 1973; Jackson, 1978). Human lymphoid cells treated with methotrexate and labeled with [3H]deoxyuridinewere found to contain dUMP in DNA in readily detectable amounts (-0.8 pmol of dUMP per micromole of total DNA nucleotide), and this value was further increased when the cells were treated with uracil, an inhibitor of UDG (Goulian et al., 1980). No dUMP could be detected by this method

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in DNA from cells not treated with methotrexate, whether uracil was present or absent. These data were in accord with the report of Sedwick et ul. (1981). concerning the exposure of a human lymphoblastoid cell line to another antifolate, metoprine. The treatment with metoprine was also shown to increase the apparent level of dUMP in DNA in the presence of increasing concentrations of exogeneous deoxyuridine (Sedwick et al., 1978). When newly synthesized DNA was extracted from metoprinetreated cells that had been labeled with deoxyuridine for up to 3 minutes, most of the DNA synthesized was no larger than 4s on an alkaline sucrose gradient. In contrast, the predominant form of newly synthesized alkalistable DNA in cells not treated with the drug was larger than 4s. Abnormal progression of DNA synthesis, degradation of the newly synthesized DNA, or both, occurred as a delayed consequence of the drug treatment in the absence of exogenous deoxyuridine when thymidine was used to label DNA. This problem is closely related to the problem of fragile sites. A few specific chromosome sites tend to be expressed as gaps or breaks during routine human metaphase chromosome preparations. With some exceptions, these sites have appeared infrequently and at low levels of expression. Deprivation of folic acid and thymidine results in the expression of a majority of heritable fragile sites and in the appearance of spontaneous chromosome breaks, termed constitutive fragile sites (Yunis and Soreng, 1984). The incidence of the breaks was enhanced by caffeine, which inhibits DNA repair in cells starved in buffer (Vikchanskaya, 1977). The detailed mechanism by which folic acid deficiency or fluorodeoxyuridine treatment induces the expression of fragile sites is not yet known. It was proposed that their expression was related to partial inhibition of thymidylate synthase, resulting in thymidine deprivation and misincorporation of uracil in place of thymine. Heritable fragile sites may occur at the DNA sequences wherein the methyl groups bind proteins and are involved in chromosomal folding. If the fragile sites remain demethylated after misincorporation of uracil (unmethyiated analog of thymine), or if uracil is excised prior to mitosis, the chromosome structure may collapse at a specific point, yielding gaps and breaks. However, this model met several difficulties which were discussed by Hagerman (1984). For example, in methotrexate-treated human lymphoid cells the quantity of uracil incorporated into DNA (according to Goulian et al., 1980) is < 1 uracil residue per 10' nucleotides. Based on an estimated DNA length per X chromatid, <300 uracil substitutions would be present over the entire X chromosome. This deficit of thymine methyl groups should have no effect on chromatin assembly. It should be noted that Fraser and Pearson (1986) could not detect any uracil residues in DNA from methotrexate-treated cells. Fur-

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thermore, the uracil level in DNA from patients with the fragile-X syndrome do not appear to be elevated (Wang et al., 1983). Moreover, the studies of Simpson and Kunzler (1979) have suggested that the 5-methyl moiety does not play a major role in the nucleosome organization. In spite of this controversial information, Hagerman ( 1984) considers that uracil incorporation may participate in the expression of fragile sites. The transient incorporation of uracil may be quite high during DNA replication. Both prokaryotic and eukaryotic DNA polymerases readily incorporate dUTP in place of dTTP. Due to rapid repair, such abnormal residues are almost quantitatively removed under normal circumstances. Such excision invariably involves a new DNA repair synthesis, and if such a repair process is itself carried out under conditions of dTTP depletion, the resulting DNA may end up with extensive single-strand nicks or gaps as a consequence of such an abortive repair process.

c. EFFECTOF METHYLATIONINHIBITORS AND FREEURACIL IN THE SIZEOF PULSE-LABELED DNA

IN

HUMAN CELLS

The measurements of dUTP pool in nontreated mammalian cells indicate that the dUTP pool size is 0.41% of the dTTP pool (Nilsson et al., 1980; Mahagaokar et al., 19801, although some authors were unable to find any detectable quantity of dUTP (Goulian et al., 1980). The experiments with semipermeable mammalian cells and isolated nuclei suggest that the addition of exogenous dUTP leads to degradation of the newly synthesized DNA, and that the effect is suppressed by free uracil (Brynolf et al., 1978; Grafstrom et al., 1978), an inhibitor of UDG (see earlier). We used a similar approach in the experiments with human cells (primary culture of embryonic fibroblasts). It is known that the pulse-labeled DNA in mammalian cells after alkaline denaturation consists of small fragments of 100-200 nucleotides (Gautschi and Clarkson, 1975; Taylor et a/., 197% which are formed because of asymmetry of DNA strands and the necessity to form primers for Okazaki fragments. If dUMP incorporation and uracil excision also contribute to the small size of pulse-labeled DNA, it should be expected that the inhibition of UDG by free uracil will result in the increase of the size of newly synthesized DNA. This effect is actually observed in our experiments (Aprelikova, 1983). Calculation of molecular weights indicates that at least 1 uracil is incorporated and excised per 1000 nucleotides of nascent DNA (Aprelikova et al., 1988). This is a minimal value, because inhibition of UDG in vivo by 10 mM uracil should not necessarily be 100% and because some apyrimidinic sites may be repaired during 1 minute of pulse-labeling. The estimated number of dUMP residues transiently incorporated during replication in mammalian cells (1 per 1000

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nucleotides) is in agreement with the value for E. coli dut' cells (Tye el al., 1978; Shlomai and Kornberg, 1978). Large variations of the dUTP pool and dUMP incorporation in cultured mammalian cells might be associated with different conditions for cultivation, and suggest that dUMP incorporation could be modulated by physiological factors. The actual dUTP content in different mammalian tissues in vivo is not known, but variations of UDG activity are rather high (Aprelikova and Tomilin, 1982). It is known that mammalian DNA is enzymatically methylated during and after replication (Adams, 1971; Demidkina et al., 1979). Fast methylation of the short newly synthesized fragments takes place just after their synthesis (Kiryanov ef al., 1980; Kautiainen and Jones, 1985), and slow methylation takes place later (Kiryanov et al., 1982). Methylation of DNA in mammalian cells may be inhibited by I-ethionine, which substitutes methionine in S-adenosylmethionine (donor of methyl groups), and by Sazacytidine, which substitutes DNA cytosine (Boehm and Drahovsky, 1979; Groudine ef al., 1981; Jones and Taylor, 1981). Actually, I-ethionine was shown to be capable of inhibiting the enzymatic methylation of newly replicated DNA (Sneider er al., 1975; Cox and Irving, 1977; Boehm and Drahovsky, 1979). Because the fast DNA methylation takes place at the same time as uracil excision, we were interested in determining whether the two processes interfere with one another or occur independently. We have found (Aprelikova, 1983; Aprelikova et al., 1988) that a 24-hour treatment of cultivated human cells with ethionine leads to a decrease in the size of pulse-labeled DNA as compared to control nontreated cells, and the same effect occurs with 5-azacytidine. Uracil incorporation and/or repair seems to be involved in the observed effect, because the addition of free uracil along with ethionine suppressed in part the effect of ethionine, and the combined effects of the two agents were not additive (Aprelikova ef al., 1988). Our data (see Section 111) also suggest that DNA hypermethylation decreases the initial rate of uracil release from DNA catalyzed by UDG from human placenta. It seems likely, therefore, that the lowering of the number of methyl groups in the newly synthesized DNA in vivo induced by methylation inhibitors stimulates excision of uracil by UDG, although the possibility that methylation inhibitors stimulate dUMP incorporation is not excluded. D. POSITIVE CORRELATION BETWEEN LIVER UDG ACTIVITYA N D AVERAGEPLOIDY OF HEPATOCYTES

If the only biological function of UDG is antimutagenic repair of hydrolytic cytosine deamination, then large variations of UDG activity should not be expected in different species of mammals. We have measured (Ku-

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dryavtsev et al., 1984) the liver UDG activity in 13 species of laboratory, farm, and wild mammals which differ in the average ploidy (the average number of genomes per cell) of hepatocytes. It is seen from Fig. 3 that there exists a positive correlation between the average ploidy of hepatocytes and the specific activity of UDG (UDG activity calculated per microgram of liver DNA). The highest average ploidy were found in the laboratory mouse (12), laboratory rat (13), and field mice as follows: Microfusfortis ( 1 l ) and Microfus subarvalis (8). The lowest UDG and neardiploid hepatocytes were found in the horse ( I ) , cow (2), raccoon dog (3), and mink (4). The intermediate levels of UDG and ploidy are shown in other species: polar fox ( 5 ) , pig (6), fox (7), field mouse Microtus sahalinensis (10). Large interspecies variations of the level of UDG activity might reflect the basal level of cell proliferation in the liver of a given species, as was found during studies of tissue distribution of UDG in the laboratory rat (Aprelikova and Tomilin, 1982), but no differences in the basal level of proliferation of liver cells in different mammalian species were reported so far. On the other hand, the liver is the main organ which metabolizes toxic and promutagenic chemical compounds (Alvares, 1982; Bartsch et af., 1982, 1983) which come with food, and different mammalian species differ in their ability to activate promutagens (Schwartz, 1975). The direct mutational test for fibroblasts treated with the mutagen 7,12dimethylbenz[a]anthracene shows the decrease of mutagenesis in the order: laboratory rat > guinea pig > rabbit > horse > elephant > human (Schwartz, 1975). The level of spontaneous chromosome aberrations in hepatocytes revealed in regenerating rat liver decreases in the following order: laboratory mouse > laboratory rat > guinea pig > polar fox (Curtis and Crowley, 1963; Brooks et a f . , 1973). Differences between mammalian species in the average ploidy of hepatocytes were observed earlier by Carriere (1969), who found that the ploidy drops in the following order: mouse > rat > guinea pig > cat. We have extended the data on ploidy to other species (Fig. 3). Developmentally regulated genome multiplication in the liver is considered to be a compensatory mechanism of genetic adaptation to a high level of DNA damage induced by the activated chemical mutagens (Brodsky and Uryvaeva, 1985): the increase of ploidy diminished the deleterious consequences of induced recessive mutations. Correlation of liver UDG activity with ploidy suggests that the increase of UDG might be a similar compensatory mechanism of genetic adaptation designed for antimutagenic repair of DNA damaged by activated chemical compounds. Some mutagens (e.g., nitrous acid and bisulfite) could directly interact with the exocyclic amino group of DNA cytosine, leading to DNA deamination and formation of DNA uracil; other mutagens interact with pool nucleotides and are incorporated into DNA during replication, leading to the formation of mismatched pairs.

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This explanation is consistent with the observation that the chemical carcinogen dimethylarninoazobenzene induces UDC in rat liver (Aprelikova and Medvedeva. 1983).

VI. Biological Role of DNA Uracil Repair-Speculations and Discussion SPONTANEOUS MUTAGENESIS,CHROMOSOME REARRANGEMENTS, RECOMBINATIONS,MISMATCHCORRECTION, EVOLUTION The major biological role of UDG is antimutagenic repair of DNA uracil arising through spontaneous hydrolytic deamination of DNA cytosine ("hydrolytic" uracil). The existence of spontaneous mutation hot spots at 5-methylcytosines in bacteria (Miller, 1983), and a strong evolutionary trend to avoid the CpG sequence from eukaryotic genes which are methylated in the germ line (Subak-Sharpe, 1967; Russel et ul., 1976; Taylor, 1984), suggest that deamination of 5-methylcytosine takes place at a biologically significant rate. Cytosine deaminates at about the same rate as 5-methylcytosine (Lindahl and Nyberg, 1974). Analysis of spontaneous mutagenesis in E. coli u n g mutants (Duncan and Miller, 1980; Fix and Glickman, 1986),which shows selective enhancement of GC -+ AT transitions, supports the view that cytosine deamination is important in mutagenesis. This also indicates that a significant proportion of DNA in vivo is present in the single-stranded state which promotes deamination, since denatured DNA deaminates 100fold more easily than dsDNA (Lindahl and Nyberg, 1974). The enhanced "breathing" of DNA in regions of base sequence rich in AT pairs leads to the increased rate of spontaneous cytosine deamination and 20- to 30fold enhancement of the rate of C += T transitions in ung- strains; when cytosine is situated in GC-rich sequence, only I .5- to 3-fold enhancement of mutagenesis is observed (Fix and Glickman. 1986). In normal cells conT rnutagenesis is suppressed, but when cytosine is taining UDG, C methylated no UDG-driven repair takes place, which leads to genome destabilization in germ-line or somatic cells (Taylor, 1984). In E. 'coli a short-patch mismatch correction system driven by mut S and mirt L gene products eliminates thymine from G : T mismatch (Radman and Wagner, 1986), but the significance of this mismatch correction system for other organisms is unclear. Even in the absence of functional mur S, L systemdirected correction of T : G mismatches takes place in E. coli. It is not immediately apparent why mutagenesis via cytosine deamination is enhanced in E. coli ung- mutants, because most of these mutants (e.g., BD 10) are leaky and contain residual UDG at the level of >0.5% of the

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wild-type strain (Lindahl, 1979), which is quite enough to repair all “hydrolytic” uracil. However, the residual UDG in ung- mutants is mainly used for slow repair of more frequent “replicative” uracil, and “hydrolytic” uracil is repaired with reduced efficiency. It seems, therefore, that when the cellular UDG repair pathway is saturated with an excess of “replicative” uracil, one should expect an increase in the rate of spontaneous C -+ T transitions because of nonrepaired “hydrolytic” uracil. Normal cells contain enough UDG to repair all “hydrolytic” and “replicative” uracil, and the contribution of C -+ T transitions to the overall rate of mutagenesis is apparently small. It is interesting that the majority of mutations induced by thymine deprivation in B. subtilis might be reversed by 5-bromouracil (Bresler et al., 1973), suggesting that thymine starvation which increases dUMP incorporation (Gouliam ef a f . , 1980) induces mainly C -+ T transitions, since 5-bromouracil induces AT + GC transitions (Skopec and Hutchinson, 1982). It seems possible that the saturation of the UDG pathway by enhanced incorporation of dUMP increases the possibility of C -+ T transition via cytosine deamination. Although the antimutagenic role of UDG in the repair of “hydrolytic” uracil is well established, it seems that the enzyme is also involved in other pathways of antimutagenesis. The rate of dUMP incorporation in proliferating cells is one for each few kilobases, which is several orders of magnitude higher than the maximal expected rate of cytosine deamination (Lindahl, 1979), and it is important that during replication uracil is transiently incorporated only into the daughter strand of DNA. This incorporation and UDG-driven repair might serve for strand discrimination during mismatch correction (Tomilin, 1983). Incorporation of incorrect nucleotides during replication is the major source of spontaneous mutagenesis in E. cofi, and many mutations leading to mutator phenotype (mut H, S, L, U) inactivate the genes involved in mismatch correction (Radman and Wagner, 1986). Two general mismatch elimination systems are postulated in E. cofi: GATC-dependent mismatch repair directed by transient undermethylation of daughter DNA strands (Marinus and Morris. 1973; Wildenberg and Meselson, 1975; Glickman et al., 1978; Radman et a f . , 1980; Laengle-Rouault et a f . , 1986), and nondirected recombinational repair of dsDNA gaps produced in one sister nonmethylated duplex at mismatched base pair (Hastings, 1984; Radman and Wagner, 1986). The GATC-dependent system is apparently absent from eukaryotic organisms (Taylor, 1984; Radman and Wagner, 1986), but some data suggest the importance of CG methylation in strand discrimination during mismatch correction in mammalian cells (Hare and Taylor, 1985). However, many eukaryotic structural genes are free of CG sequences, and in housekeeping

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genes CG-rich promoters are nonmethylated (Bird, 1986). In E. coli,elimination of GATC methylation by the dam mutation leads to partial loss of strand discrimination during correction and enhanced mutagenesis by the “replicative” mutagen ethyl methanesulfonate (Gtickman et al., 1978). In mammalian cells the methylation inhibitor I-ethionine suppresses mutagenesis by the “replicative” mutagen 6-hydroxyaminopurine (Knaap et ul., 19811, suggesting that undermethylation stimulates strand discrimination and directed mismatch repair or recombinational repair. Recombinational repair of dsDNA gaps which could potentially eliminate mismatch (Hastings. 1984; Radman and Wagner, 1986) probably exists in eukaryotic cells (Szostak et a / . , 1983; Natarajan et al., 1985; Brenner et d.,1986). For this repair the dsDNA breakage at mismatch pair is required and no strand discrimination is necessary. Recombinational repair might explain mismatch elimination in the absence of methylation, but it is not clear how this repair would eliminate mismatch without strand discrimination from hemimethylated DNA when the double-strand breakage is not expected. Evidence for uracil-directed UDG-dependent mismatch correction in E. coli was obtained by Kunkel (1985; Kunkel et al., 1987) and was discussed in Section 1V,B,2. Kunkel used MI3 phage for mutagenesis studies, but if this system is important in the repair of misincorporated nucleotides one could expect that the ung mutants will be mutators. This was actually observed (Duncan and Miller, 1980; Fix and Glickman, 1986), but mutator effect was rather weak and specificity of observed mutations (C + T transitions) was not in accord with the idea of replicative errors. It should be noted. however, that the irng mutation used in these studies (BD 10) is leaky mutation, and slow UDG-driven repair undoubtedly takes place in this mutant because ( 1 ) uracil-substituted phage T4 DNA is degraded in BD 10 (Duncan and Warner, 1978); and (2) E. coli cells containing this mutation exhibit a hyperrecombinational phenotype (Duncan, 1985), explained by slow DNA breakage during UDGdriven repair. This slow repair might be effective in directed correction of mismatches in chromosomal DNA, but inefficient for correction of heteroduplex M 13 DNA (Kunkel, 1985), which should start replication immediately after entering the cell. Evidence of DNA strand targeting by single-strand breaks during mismatch correction in Streptococcus pneitmoniar was obtained by Lacks (Claverys and Lacks, 1986), who also suggested that discrimination of newly replicated DNA strands by virtue of its single-strand breaks provides a basis for antimutator action of the Hex system. DNA strand targeting by single-strand breaks or gaps in newly synthesized DNA was also postulated for eukaryotic cells (Taylor, 1984; Hare and Taylor, 1985). ssDNA breaks are known to be intermediates in the UDG-driven repair (Section 1ll.C).

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The results presented in Section V,C and D are consistent with the idea that UDG-driven repair of uracil incorporated during replication is involved in targeting of daughter DNA strands during mismatch correction in human cells (Tomilin, 1983). Inhibitors of DNA methylation I-ethionine and 5azacytidine were found to stimulate DNA uracil excision from newly synthesized DNA, resulting in smaller DNA fragments (Aprelikova, 1983; Aprelikova et af.,1988), and I-ethionine was found to suppress mutagenesis induced by “replicative” mutagen 6-hydroxyaminopurine (Knaap et al., 1981). The increased number of breaks in newly synthesized DNA possibly leads to more efficient discrimination of DNA strands containing misincorporated 6hydroxyaminopurine during mismatch repair and suppressed mutagenesis. Alternatively, the extensive repair of DNA uracil in undermethylated DNA might stimulate formation and repair of dsDNA gaps, which would eliminate mismatches (Hastings, 1984; Radman and Wagner, 1986). Actually, the inhibitor of DNA methylation 5-azacytidine increases the frequency of SCE in hypermethylated chromosome bands in human cells (Hori, 1983). UDG-dependent targeting of mismatch correction helps to explain the possible role of UDG in the repair of DNA lesions induced in liver cells by activated chemical mutagens (see Section V,D). Many chemical mutagens are able to interact not only with DNA but also with pool nucleotides, and some modified nucleotides are incorporated into DNA during replication forming mutational heteroduplexes which might serve as substrates for mismatch correction systems. High UDG activity in polyploid rodent hepatocytes (Fig. 3) could stimulate strand discrimination during the repair of mutational mismatches and could suppress mutagenesis. This explanation is in accord with the view that a high level of ploidy and a high UDG activity in rodent hepatocytes is a developmentally regulated mechanism of genetic adaptation to the deleterious genetic effects of activated chemical mutagens (Brodsky and Uryvaeva, 1985). Uracil incorporation during replication and UDG-driven repair seems not only to be important in the control of point mutations, but also to play a role in chromosome destabilization in eukaryotic cells. Deprivation of folic acid and thymidine from media for cultivation of human cells leads to the expression of chromosome gaps and breaks at specific sites named as fragile sites (Sutherland, 1983), and the expression of these genomic weak points is enhanced when the cells are exposed to caffeine (Yunis and Soreng, 1984). A hypothesis on the nature of folic acid-sensitive fragile sites was proposed (Krumdieck and Howard-Peebles, 1983; Hagerman, 1984), which postulates that dUMP incorporation and repair play a role in chromosome destabilization. Transient extensive incorporation of dUMP during replication and UDGdriven excision repair under conditions of a high dUTP : dTTP ratio induced by depletion of dTTP pool (Goulian

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IOiQ

2

1-0-112 I

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3

1-0-1

j 11

4

5

6

ploldy n FIG.3. Correlation between average ploidy of hepatocytes and specific activity of uracilDNA glycosylase in the liver of 13 species of mammals. Abscissa: average ploidy of hepatocytes ( n . average number of haploid genomes per cell) measured by cytophotometry (Kudryavtsev et a / . . 1984). Ordinate: specific activity of uracil-DNA glycosylase (UDG activity calculated per gram of liver DNA). I . Horse: 2, cow: 3, raccoon dog: 4, mink: 5, polar fox: 6, pig: 7, fox; 8, field mouse Microtus subarvalis; 9, guinea pig: 10, field mouse Microtus sahalincnsis; II,field mouse Microtusfortis; 12, laboratory mouse: 13, laboratory rat. Modified from Kudryavtsev ct a / . (1984).

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er al., 1980) may result in repeated rounds of abortive repair, ultimately leading to chromosome breaks and gaps (Hagerman, 1984). Inhibition of folate-dependent thymidylate synthase in animal cells is known to be inducing DNA fragmentation (Perlman and Huberman, 1977). One of the consequences of abortive repair could be underreplication of some chromosome regions (Laird et al., 1987). Specific localization of fragile sites might be associated with an evolutionarily conserved class of thymine-rich sequences which incorporate more dUMP as compared to other sequences (Yunis and Soreng, 1984). The caffeine enhancement of expression of fragde sites might be explained by the inhibition of rejoining of DNA breaks during UDG-driven repair. Caffeine is a potent inhibitor of rejoining of y-ray-induced ssDNA breaks in human cells starved in buffer (Vikchanskaya, 1977). It is interesting that some fragile sites are expressed when cells are treated with 5-azacytidine (Sutnerland et al., 19851, which could stimulate excision of “replicative” uracil andor formation of double-strand breaks. The localization of 5-azacytidine-dependent fragile sites in human chromosomes (Sutherland et al., 1985) is different from that of folic acid-dependent sites (Yunis and Soreng, 1984)-consistent with the view that the maximal effect of methylation inhibitors should be expected in chromosome regions which are hypermethylated in the absence of inhibitors. Fragile sites have appeared infrequently and at low levels of expression in normal cells not deprived of folic acid and not treated with inhibitors, and specific chromosome aberrations detected in human neoplastic cells frequently coincide with fragile sites (Yunis and Soreng, 1984). This indicates that uracil incorporation into DNA occurs in mammalian cells under normal physiological conditions and that karyotype evolution in tumor cells might be accelerated by antifolates used for therapy. The increased level of chromosome aberrations in the liver of the laboratory mouse as compared to other species (Curtis and Crowley, 1963; Brooks el ul., 1973) might be a consequence of more extensive DNA uracil repair (see Section V,D). Chromosome rearrangements in the evolution of primates frequently occur at bands containing fragile sites (Miro el al., 1987). It is possible, therefore, that dUMP incorporation and UDG-driven repair are involved in reorganization of chromosomes in somatic and germ-line cells. UDG seems to play a role in genetic recombination. Escherichia coli mutants partially deficient in UDG show a hyperrecombinational phenotype, and the deletion of the ung gene restores the frequency of recombination typical for wild-type strains (Duncan, 1985). Apparently, recombination is stimulated by a slow DNA breakage initiated by UDG. Escherichiu coli dur mutants deficient in dUTPase (Tye er al., 1977), which incorporates into DNA up to 1% of dUMP (Tye er al., 1978), also show the hyperrecombinational phenotype. In dur mutants the long-range per-

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sistence of single-strand breaks is probably associated with saturation of the UDG pathway. The folic acid-dependent fragile site at human Xq27q28, associated with X-linked mental retardation, occurs in a region prone to a high frequency of meiotic recombination (Szabo et al., 1984; Brookwell and Turner, 1983; Davies et a / . , 1985). As was noted earlier, inhibitors of DNA methylation which stimulate uracil excision and DNA breakage at chromosome replication forks (Section V.C) also induce SCE in heavily methylated chromosome bands (Hori. 1983). SCE may be also induced in plant cells by dUMP residues persisting during replication (Gutierrez, 1987). It seems possible that not dUMP itself, but secondary DNA damage (e.g., single-strand break in parental DNA strand), is responsible for SCE formation (Gutierrez, 1987). It is interesting that the level of UDG in different proliferating plant cells appears to be finely regulated (Gutierrez, 1987), suggesting that UDG may play a role in regulation of SCE frequency and the rate of spontaneous mutagenesis in plants. Intrachromosomal DNA recombination at replicative forks is postulated to be the first step of gene amplification in mammalian cells (Schimke, 1984). Inhibitors of thymidylate synthase greatly expand the dUMP pool (Jackson. 1978), inducing a dramatic increase of dUMP incorporation (Goulian et al., 1980). UDG is not an essential enzyme for E. coli, since the deletion of the ung gene does not lead to the loss of viability (Duncan, 1985). UDG seems also not to be essential for eukaryotic organisms, since the enzyme is absent from Drosophila (Friedberg et al., 1978; Deutsch and Spiering, 1982). However, extracts of the third-instar larvae contain the nuclease degrading uracil-containing DNA, which is thought to initiate the histolization process in Drosophila pupae (Deutsch, 1987). Incorporation of dUMP into D N A of the third-instar larvae is induced by the protein inhibitor of dUTPase (Giroir and Deutsch, 1987). The synthesis of UDG seems to be turned off as plant cells enter their differentiated state (Gutierrez, 19871, but differentiated hepatocytes in nonregenerating rat liver still contain a high UDG activity (Aprelikova and Tomilin, 1982). Further studies of regulation of UDG synthesis and of corresponding eukaryotic genes, as well studies of the dUTP pool in differentiated cells, will be helpful in understanding the ways by which cells modulate their genetic stability.

VII. Conclusions The major source of DNA uracil in prokaryotic and eukaryotic cells is transient incorporation of dUMP during replication. This “replicative”

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uracil is quickly repaired by UDG, AP endonucleases, and other enzymes of excision repair. The rate of dUMP incorporation depends mainly on the size of the intracellular dUTP pool, which vanes from 0.001 to 1% of the dTTP pool and seems to be closely regulated by physiological and genetic factors. The minor source of DNA uracil is spontaneous hydrolytic deamination of DNA cytosine, and the rate of cytosine deamination is several orders of magnitude lower than the rate of dUMP incorporation in proliferating cells. Normal capacity of UDG-driven repair is enough to repair both “replicative” and “hydrolytic” uracil, but saturation of UDG with “replicative” uracil leads to inefficient repair of “hydrolytic” uracil and to increase of C +. T mutagenesis. Inefficient or slow repair of “replicative” uracil caused by the increased incorporation of dUMP or by the deficit of UDG results in a prolonged persistence of repair-dependent DNA breaks which induce genetic recombination and chromosome destabilization. Uracil-DNA glycosylasedependent excision of “replicative” uracil is possibly used in DNA strand targeting during mismatch correction andor for elimination of mismatched base pairs by recombinational repair of dsDNA breaks. Therefore, the rate of mutagenesis might be modulated by the size of the dUTP pool and by the capacity of the UDG-driven repair pathway.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 114

Age-Related Changes in DNA Methylation: Do They Represent Continued Developmental Changes? LAURAL. MAYS-HOOPES Department of Biology, Occidental College, Los Angeles, California 90041

I. Introduction The intent of this review is to examine a single system that undergoes extensive changes in development, namely DNA methylation, to see whether or not age-related changes in this system can be plausibly explained as an extension (or pleiotropic effect) of developmental programs. As a guide to those not familiar with some of the fields covered, brief overviews of the roles currently envisioned for DNA methylation and of the theories presently dominant in gerontology will precede the review proper. In the review per se, sections will be focused on particular sequences (overall genome, proviral sequences, specific genes) and will describe the known developmental and age-related changes so that trends can be more easily recognized. This is a useful way of organizing the material to address the specific issue at hand, but of necessity, it obligates the review to deal almost exclusively with the few genes that have been examined in senescence, which may not be a typical or representative set of all genes. Nevertheless, it is possible to make some tentative generalizations at present, which could be useful as a guide to fruitful areas of investigation in molecular gerontology. A. OVERVIEW OF ROLES OF DNA METHYLATION DNA methylation has been widely studied as a means of silencing transcription of mRNA from genes (Jones and Taylor, 1980; Razin and Riggs, 1980; Ehrlich and Wang, 1981; Doerfler, 1983; Cooper, 1983; Riggs et al., 1985; Bird, 1987; Cedar, 1988). The evidence in these and other reviews can be summarized in three main conclusions: ( I ) Some sites, usually 5’ to genes, are unmethylated in all known cases where transcription can occur. This correlation is particularly strong for genes of proviruses (Cooper, 1983; Doerfler, 1983). (2) The lack of methylation at a possible 181 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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5’-regulatorysite is not the sole and sufficient control of any system known. (3) In the most clear-cut cases, the unmethylated status of a site or group of sites is necessary but not sufficient to start transcription. This could be because methylation interferes with binding of some trans-acting factors (Becker et al., 1987), but evidently not SPI (Harrington et al., 1988). It is important to realize that eukaryotic regulatory systems are just beginning to be deciphered in all of their complexities, and that the role(s) DNA methylation may play in binding of trans-acting factors and regulating transcription remain clouded in uncertainty. Nevertheless, in some cases it does seem that programmed changes in DNA methylation occur in concert with changes in gene expression. Since, as mentioned earlier, most of the methylation sites are evidently not of major regulatory importance, in that their methylation status does not correlate with the transcription of the genes in which they occur, a brief mention of several other roles that have been postulated for DNA methylation is in order. Razin et al. (1985) have proposed a nucleosomelocking model for DNA methylation. This model includes the proposal that genome-wide transient demethylation allows nucleosomes to slide along the DNA. Specific proteins called determinators (possibly transacting factors) can gain access to particular regulatory sites during this period of sliding. At the conclusion of the transient demethylation, a process of remethylation locks the nucleosomes into position wherever no determinators are protecting the DNA. At subsequent cell replications, the methylation of the DNA is catalyzed by methyltransferases that prefer to methylate hemimethylated sites (Woodcock et al., 1983; Bolden el al., 1984; Zucker et al., 19851, so that the locations of the nucleosomes can be passed on to future cell generations without the necessity to reinduce the determinator proteins. Evidence in favor of this model includes the preferential location of the 5-methylcytosine (5mC) in DNA that is tightly bound in the nucleosomes (Razin and Cedar, 1977), a transient demethylation of DNA over the whole genome that occurs during differentiation of Friend erythroleukemia cells (Razin et al., 1983, and the site-specific effects of methylation on gene expression in contrast to the broad specificity of known DNA methyltransferases (Gruenbaum et al., 1982; Zucker et a / . . 1985; Bolden et al., 1985; Woodcock et al., 1983). Another possible role for some of the methylation sites is in stabilizing condensation of chromosomes for cell division. For example, Schmid et 01. f 1985) found that there is a distinct small group of fragile sites that can be induced in chromosomes using 5-azacytidine (SazaC), a potent inhibitor of DNA methylation. This drug clearly inhibits DNA methylation (Jones and Taylor, 1980), evidently by covalent linkage to the DNA methyltransferases traversing the DNA (Santi et al., 1984). However at high concen-

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trations it inhibits synthesis of DNA, RNA, and protein (Cihak et af., 1974). It interferes with the methylation of tRNA (Lu et a / . , 1976) and with the processing of RNA in general (Reichman et a / . , 1973; Cihak et al., 1974). Therefore, it is by no means clear-cut that all effects described for this drug should be attributed to inhibition of DNA methylation. For example, the inhibition of DNA synthesis has been strongly considered as an inducer of differentiation (reviewed by Ley and Nienhuis, 1985). However, it is true that the concentrations used in many experiments reported in this review were such as to minimize cell toxicity while still affecting methylation, and it is likely that the effects seen were due to DNA methylation. One must always view such studies with caution, however, and seek evidence that the DNA is in fact demethylated in the regulatory sites for the genes of interest in the experiment, that toxicity is low, and that the deoxy5azaC is effective in lower concentrations than the ribose analog. In this fragile-site experiment, it is not clear that the effects of the drug were on DNA methylation, for example. Several groups have presented evidence that specific methylation patterns constitute paternal versus maternal imprinting. McGrath and Solter (1984) had shown that both parental genomes were essential for development. Sapienza el a / . (1987), Reik et al. (1987), and Swain et al. (1987) all found gamete-specific methylation patterns of genes they had introduced into transgenic mice. Swain et al. (1987) also found that their transgene, autosomal c-myc with a Rous sarcoma virus (RSV) long terminal repeat (LTR), was expressed and unmethylated only when inherited from the male parent; when inherited from the female parent, it was unexpressed and methylated. Another possible role of DNA methylation is in preventing chromosome rearrangements, as suggested by Feinberg (1985). One might visualize an endonuclease that recognized unmethylated sites. If cuts occurred, free ends might invade other duplexes to generate legitimate or illegitimate recombinations. The idea is one that arises naturally from consideration of the prokaryotic restriction-modification systems, in which methylation protects self-DNA from attack by self-endonucleases. B. OVERVIEW OF INTRINSIC AND EXTRINSIC THEORIES OF AGING There are a number of theories of aging, but the major cause of the loss of adaptation and viability as a function of age is usually attributed to extrinsic causes (i.e., environmental attack) or to intrinsic causes (i.e., genetic programs of the organism). The extrinsic theories can be illustrated by the free-radical theory, suggesting that oxygen-containing radicals incidental to respiration can attack and damage cellular macromolecules,

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and that aging is secondary to the accumulated damage (Harman, 1981; Sohal, 1981; Ames et al., 1985). The intrinsic theories are again subdivided into those which attribute aging to the accumulated effects of the neuroendocrine system (Finch et al., 1984; Nelson and Felicio, 1985) or the immune system (Weindruch et al.. 1982), and those which attribute aging to individual cellular programs (Phillips and Cristofalo, 1985). An attractive version of the intrinsic theory suggests that aging results from the pleiotropic effects of genes regulated to suit the needs of development (Shmookler-Reisand Goldstein, 1984). From such a theory, one can predict that changes occurring during development would continue into senescence. Alternatively, one might view senescence as a breakdown of normal cellular differentiation processes of development (Cutler, 1982a,b, 1985; Shmookler-Reisand Goldstein, 1984; Holliday, 1985, 1987b).This version of the theory would imply that tissue specificity might break down during senescence, allowing events normally occurring in one tissue to proceed in others. This idea has been named dysdifferentiation (Cutler, 1985). The intent of this review, as mentioned before, is to see whether or not DNA methylation changes that occur in programmed fashion in development appear to proceed continuously into aging, as well as whether or not tissue specificity is reduced in senescence.

11. Methylation of the Overall Genome

In assessing the overall methylation of the genome, the contributions of repetitive sequences and genes are not distinguished from each other: neither are the contributions of the regulatory and nonregulatory sites seen separately. This type of analysis can reveal some global mechanisms at work. however. For example, Ehrlich et al. (1982) found distinct, reproducible differences between human tissues in the percentage of methylated cytosines. It is probably through developmental regulation that such differences arise. A . CHANGES I N OVERALL GENOME METHYLATIONIN DEVELOPMENT 1. Gametogenrsis

In spermatogenesis, methylation decreases overall; in oocytes, specific sequences have been examined, but not overall methylation. In rooster spermatogenesis, for example, 4.4% of the cytosines are methylated in premeiotic gonial cells, falling to 3. I% by the stages of round and elongated spermatids (Rocamora and Mezquita, 1983). It is possible that the methylation is largely depleted in heterochromatic repeated DNA, since this

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timing coincides with an extreme reduction in the heterochromatin blocks visible in the nucleus (Dressler and Schmid, 1976). Mice also appear to have low methylation of the overall genome in sperm (Adams et al., 1983; Razin et al., 1984). And in human sperm DNA, Ehrlich et al. (1982)found 0.84 mol % 5C compared to 0.98 rnol % 5mC in brain and 0.88 rnol % in liver, using high-performance liquid chromatography (HPLC) analysis.

2. Early Development after Fertilization In preimplantation embryos, the bulk of the embryo is devoted to extraembryonic membrane, with only a small inner cell mass reserved for the future animal itself. Singer et al. (1979) found that 20-30% of the CpG dinucleotides recognized by MspYHpaII endonucleases were cut by HpaII endonuclease (sensitive to methylation), in either preimplantation mouse embryos or adult tissues, implying little overall change in methylation. Manes and Menzel (1981) found that in rabbit blastocyst stage, the extraembryonic trophoblasts’ DNA was only 48% methylated at HpaII sites but that the DNA of the embryo itself was 71% methylated. This study implied a decreased methylation in extraembryonic cells compared to those of the embryo. Kroger et al. (1983) found that embryos of BALB/c and NMRI mice had only 0.73 and 0.7 mol % 5mC in their DNA, while the adult organs tested (liver and kidney) had 0.82 mol % 5mC. It is not clear how much extracellular membrane was associated with these embryos, nor what their gestational age was. 3. Tissue-Specijic Differentiation First, we will examine differentiation of embryonic cell lines in vitro. The F9 embryonal carcinoma, derived from the 129/SV mouse line, is induced to differentiate by retinoic acid. During this differentiation, the cells lose their ability to synthesize the stage-specific embryonic antigen unique to early embryos (SSEA-I) and begin to synthesize collagen type IV, receptors for epidermal growth factor (EGF), and transplantation antigens (Tanaka et al., 1983). This differentiation is thought to involve specialization into cells like the parietal endoderm and visceral endoderm of embryos, and involves genome-wide demethylation of genes (Young and Tilghman, 1984; Tanaka et al., 1983). Bestor et al. (1984) estimated that the DNA lost 9% of its 5mC, corresponding to 2.2 million methylated sites during retinoic acid-induced differentiation. Razin et al. (1984) noted an even more dramatic 30% decrease in methylation upon differentiation. These authors related this finding to the 77% methylation of CpG sequences examined in the mouse embryo compared to 49% in placenta and 53% of yolk sac, showing that this differentiation in vitro may mimic that of extraembryonic tissues in vivo.

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The C3H 10T 1/2 line of mouse embryo cells is not transformed, and appears to differentiate into several mesodermal cell types upon exposure to the DNA methylation inhibitor 5azaC (Constantinides et al., 1977; Taylor and Jones, 1979, 1982). The largest group forms myotubes, but some also differentiate into adipocytes and chondrocytes. Single clones from this line were found to undergo multiple differentiation, ruling out selection of preexisting variant lines. It was also found that the treatment worked best during early S phase of the cell cycle, and that differentiation required cell division after SazaC treatment (Taylor and Jones, 1982). One probable target gene for control of myoblastic development has been cloned by Davis ef af. (1987), by preparing subtracted, myocytespecific cDNA libraries from C3H IOT 1/2 cells treated with SazaC and differentiating into myotubes. This gene, called MyoDI, was able to convert IOT 1/2 cells into myoblasts when transfected into them on an expression vector. It also appeared to cause the cells to withdraw from the cell division cycle. There is no direct evidence yet that MyoDl is activated by 5azaC treatment, presumably by demethylation, but this possibility is under active investigation. It has been found by Hsiao ef al. (1984) that IOT 112 cell line, when exposed to SazaC, does appear to demethylate several genes and perhaps a large part of the genome. An interesting‘feature of that study was that c-mos and p major and minor globin genes did not begin transcription, although they became less methylated. Yisraeli et ali (1986) found that myoblasts, but not fibroblasts, could specifically demethylate the regulatory regions of a-actin gene, which was then able to direct gene expression. Demethylation has also been postulated to play a role in adipocyte differentiation of the CHEF118 line of embryonic fibroblasts from Chinese hamster (Sager and Kovac, 1982; Harrison et af., 1983). In vivo, there have not been studies tracing the development of the tissue-specific differences in methylation, shown for humans (Ehrlich et af., 1982) and for other mammals (Gama-Sosa et al., 1983; Vanyushin et al., 1973; Kroger et al., 1983). However, studies have demonstrated genomic DNA demethylation associated with differentiation, in cell lines that are more determined for specific differentiation than the embryonic lines described earlier. For example, the cell line HL-60, a line established from peripheral blood of a patient with acute promyelocytic leukemia, undergoes extensive DNA demethylation when treated with 5azaC (Christman et al., 1983) or with 5-aza-2’-deoxycytidine (Saza-dC), which goes more selectively into DNA (Momparler et al., 1985). This demethylation occurs as the cells differentiate into granulocytoid appearance. To summarize findings concerning overall genomic methylation in development, it appears that there is a strong move to lower overall meth-

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ylation during spermatogenesis; following fertilization, there is a period of stable methylation in development but a loss of methylation in extraembryonic membranes. The tissue-specific differences in overall methylation imply regulation of methylation during differentiation, and studies of methylation during differentiation of cultured cells, induced by 5azaC or retinoic acid, have implicated at least a transient demethylation in the establishment of differentiated functions.

B. CHANGES I N GENOMIC METHYLATIONI N AGING 1 . Mammalian Aging in Vivo A long series of early studies by Vanyushin and collaborators established a general trend toward demethylation of DNA in various tissues during mammalian aging (for examples, see Vanyushin et al., 1973, and additional references cited in Mays-Hoopes, 1985a). These studies were extensive, but involved determination of the percentage of 5mC via thin-layer chromatography (TLC), which is by no means so sensitive or accurate as HPLC. Ehrlich et al. (1982) studied samples from seven male humans ranging in age from 8 months to 82 years and two female humans, aged 8 and 12 months, along with six placentas, via a very precise and accurate HPLC technique. The authors of this study stated that they detected no age-related changes in human DNA methylation. In liver, they found 0.88 mean mol % 5mC, with a standard deviation of 0.02% in analysis of 18 samples including all nine subjects. In the analysis of lymphocytes, however, only two individuals were tested, whereas for heart, only three were tested (males, 2 6 4 0 years old), and the variation was significant at p < 0.05, although the trend, if any, was not described. In any case, the authors found much larger differences between the tissues than between individual samples of any particular tissue. Although small decreases or increases in DNA methylation in human tissues during aging cannot be ruled out from these results, there does not appear to be the kind of age-related decrease in methylation reported by Vanyushin et al. (1973) for rat tissues. This raises the question of whether the rodent findings can be confirmed via more accurate techniques. The age-related demethylation of total genomic DNA has been confirmed for male C57BL/6 mouse liver DNA using HPLC (Singhal et al., 1987) and via postlabeling and TLC (Wilson et al., 1987). In Singhal et al. (1987), the mole percent of deoxy-5-methylcytidine (d5mC) in male mouse liver DNA was found to decrease from 1.67 ? 0.2 to 1.02 ? 0.3 between 6 and 24 months, followed by a plateau with a slightly increasing slope. In parallel, the mole percent of deoxycytidine (dC) increased from 18.4 ? 0.3 to 19.1 k 0.5 mol %-the deoxythymidine

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(dT) remaining constant at 29.7. The total of d5mC + dC appeared to remain constant during aging, suggesting that the replacement of d5mC sites with dC sites was occurring rather than deamination of thymidines to form d5mC. In Wilson et al. (1987), the Mus rnusculus DNA was compared with DNA from Peromyscus leucopus, a rodent having a life span approximately twice as long as that of Mus. The data from the postlabeling and TLC analysis were presented as percentage of cytosines methylated, and this value for M . musculus male liver decreased from just over 3.00% to -2.60% between 1 and 31 months, appearing to be a linear decrease, but with a noticeably larger standard deviation at ages >20 months. This change was thus a 14% decrease over the life span, compared to a 40% drop seen by Singhal er al. (1987)at the lowest point. 24 months. However, by 28 months (the oldest males in that study), only a 28% decrease over the value at 6 months was seen. A further discrepancy between the two studies is that methylation of 3% of the cytosines (Wilson et al., 1987) gives a value of 0.6 mol % (total of dC + d5mC is 20.05%), which is considerably lower than the value found by Singhal et al. (1987). The difference cannot be attributed to the strain or the sex, since these were identical. Literature values for mouse liver are not in perfect agreement with either study. For example, Kroger er al. (1983) found 0.82 0.015 mol % 5mC for BALB/c and NMRl mouse liver, whereas Feinstein e f al. (1985)found Swiss mice to have 0.69 mol % 5mC in liver DNA main band (separated from the more highly methylated satellite DNA). Romanov and Vanyushin (1981) reported 0.9-1.1 mol % 5mC in mouse DNA. The Peromvscus-Mus comparison in Wilson et al. (1987) is particularly interesting in that the rate of methylation loss observed in the longer-lived animal’s tissue was much slower, going from 2.6% methylated to 2.3% methylated over a time span from 3 to 60 months (a 12% decrease over a much longer period). The same techniques were used to examine the genomic level of d5mC in cultured human bronchial epithelial cells obtained from autopsy donors of ages from 18 to 58 years. In this case, the methylation decreased from 3.08% of the cytosines to 2.95% over this age span, a 5% decrease over decades. Without the final two points, at 56 and 58 years in this data set, one could easily conclude that no change had occurred, as Ehrlich et al. (1982) decided for other human tissues.

*

2 . I n Vitro Replicative L$e Span In cultured cells undergoing replicative life spans, Wilson and Jones ( 1983) found that a progressive loss in overall genomic methylation occurred except in immortal cell lines. In their study, the progressive loss in methylation was much more rapid in rodent lines than in human lines.

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In a more extensive study using human fibroblasts from six donors ranging in age from 11 to 76 years, no correlation of percentage of CCGG sites methylated with donor age could be found (Shmookler-Reisand Goldstein, 1982a).A number of these lines lost methylation during culturing, but not all of them. Holliday (1986)found that culturing normal human fibroblasts with SazaC or Saza-dC shortened the culture’s replicative life span and resulted in cells that resemble those usually seen at the end of the replicative life span. In summary, overall genomic methylation may decrease during in vivo aging and in vitro replicative life span, and it appears that the rate of such loss is correlated inversely with the life span, in the limited number of cases so far examined. This change appears to be the opposite of the trend in development, which includes an overall increase in methylation, at least by comparison with the DNA of male gametes. 111. Methylation of Highly Repetitive Sequences

The highly repetitive sequences do not contain genes, but consist of groups of tandemly repeated simple-sequence DNA, occurring at centromeres and in other locations in heterochromatin (Pardue and Gall, 1969; Radic e f al., 1987). The prototype such sequence is mouse satellite DNA, a 234-bp sequence (Manuelides, 1981) repeated about a million times, and located at the centromeres (Pardue and Gall, 1969). The repeats are not perfectly identical; some endonucleases have recognition sites in virtually every copy of the repeated sequence, while others cut in some but not all copies. There is an MspIIHpaII site that is not present in all repeats, but that occurs tandemly in some sets of repeats (Reilly et al., 1982). In addition to the major mouse satellite, there is a minor mouse satellite with a different sequence, established by cloning mouse repeated DNAs and sequencing the clones (Pietras et al., 1983). It also occurs at centromeres.

A. CHANGESI N HIGHLYREPETITIVE DNA METHYLATION IN DEVELOPMENT 1. Gametogenesis There is a very interesting pattern of demethylation of repeated sequences in mouse germ cells. Sanford et al. (1984) and Ponzetto-Zimmerman and Wolgemuth (1984) both found a striking demethylation of the highly repetitive mouse centromeric satellite in spermatogenesis. At the earliest premeiotic stages that could be purified in quantities sufficient

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for analysis, the major satellite probe detected that a large number of the tandem repeats were sensitive to digestion by HpuI1. This demethylation persisted during all of spermatogenesis. Dilution analysis indicated that about 30-50% of the copies of major satellite were unmethylated in sperm (Sanford er ul., 1984). In addition, the minor satellite sequence was undermethylated in all of these stages of spermatogenesis. The study of Sanford et d . also included oocytes that were isolated from the ovaries of newborn mice, and the DNA isolated from these was similarly undermethylated in both major and minor satellite sequences. The results have been confirmed for sperm by Feinstein et al. (1985), who also detected the low methylation using the endonuclease MnlI, which cuts in every repeat of the major satellite, as well as by Maxam-Gilbert sequencing. There are some studies concerned with other mammalian gametes. For example, the bovine sperm satellite DNA (Sturm and Taylor, 1981; Sano and Sager, 1982) has been shown to be demethylated in sperm. In humans, the EcoRI family of repeats has been shown to be undermethylated in sperm (Gama-Sosa et al., 1983). The evidence to date suggests that extensive demethylation of the highly repetitive DNA is characteristic of mammalian gametes. The time of establishment of the methylation difference is unknown, and it is possible that the germ line is permanently unmethylated in some highly repetitive sequences. Alternatively, the sequences could lose methyl groups before they are recognizably pregametes, as a component of determination.

2 . Development after Fertilization There is no evidence concerning the timing with which the essentially complete de novo methylation of satellite DNA is achieved, but control adult tissues in Sanford et al. (1984), Ponzetto-Zimmerman and Wolgemuth (1984). Sano and Sager (1982),as well as sequencing studies such as Manuelides (1981) and Feinstein er a / . (1985), show that such methylation is essentially complete at seven of the eight CpG sequences of mouse satellite DNA and -50% methylated at the eighth CpG. Thus the evidence with regard to highly repetitive sequences suggests that they change in development from 30-50% unmethylated in gametes to essentially fully methylated. The timing and mechanism($ of this change remain to be elucidated. B. CHANGES I N METHYLATIONOF HIGHLY REPETITIVEDNA AGING

IN

During the replicative life span of human fibroblast cell lines, highly repetitive DNAs were found to maintain their high levels of methylation even though other sequences in the same cells had lost methylation

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(Shmookler-Reis and Goldstein, 1982a). The major and minor mouse satellite DNA sequences can be examined for methylation via methylationsensitive endonucleases. Mnn cuts at unmethylated sites in the major satellite (Manuelides, 1981), while HpaII cuts at unmethylated sites in the minor satellite, although the cloned sequence lacks such a site (Pietras et al., 1983; Sanford e f ai., 1984). MspI can cut sites that resist HpaII due to methylation of CpG sequence, but will not cut if the 5’C of the CCGG site is methylated (Sneider, 1980). A study of such sites in aging mouse liver samples showed that methylation remained constant from 3 to 31 months as assessed by these endonucleases (S. Dalrymple and L. L. MaysHoopes, unpublished data). However, banding the mouse satellite in Hoechst dye-CsCI gradients and isolating BstNI monomer and dimer fragments, followed by HPLC, showed that there was a loss in methyl groups overall that paralleled that of the whole genome (D. Howlett and L. L. Mays-Hoopes, unpublished data). So it is probably the case that in vivo aging may similarly affect overall methylation and satellite methylation, at least in liver DNA. Although much more testing of different tissues and life span times is needed, it could be that highly repetitive DNAs in vivo lose methylation during aging, while those in cells undergoing their replicative life span in vitro do not. In vivo, this trend would be the opposite of that in early development. IV. Methylation of Proviral and Interspersed Repeated Sequences

Proviral and interspersed repeated sequences are examined together because in the case of two interspersed repeats ( L l M d and intracisternal A particle), that have been examined in aging, it is strongly suspected that they originated from proviruses (Kuff et al., 1978; Fanning and Singer, 1987). There is no intention to imply that this is the case for all such interspersed repeats.

A. CHANGES I N METHYLATIONIN DEVELOPMENT 1. Gametogenesis

Sanford et al. (1984) studied methylation of the major mouse repeatedsequence family, Ll Md, during spermatogenesis and oogenesis. The LlMd sequence was found to be strongly methylated in sperm and their progenitor cells, but the oocytes were undermethylated in L l M d as well as in the highly repetitive sequences. This was one of the first indications of methylation differences between sperms and eggs in the same sequences of DNA. The findings with regard to sperm and their progenitors were independently confirmed by Ponzetto-Zimmerman and Wolgemuth ( 1984).

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2. Earlv Development afier Fertilization The loo0 genes for intracisternal A particle (IAP), a defective retrovirus, are expressed early in mouse development (Piko et al., 1984; Calarco and Szollosi, 1973; Wive1 and Smith, 1971). Morgan and Huang (1984) found that methylation of two HhaI sites in the 5'LTR of IAP resulted in its inactivity. Feenstra er a/. (1986) confirmed this and also found methylation of a nearly HpaIl site in transcriptionally silent IAP, using in v i m methylation and transfection into (2057 cells. A very productive approach in analysis of proviral methylation changes has been the study of methylation of exogenous Moloney murine leukemia retroviruses (MoMLV) that are transcribed by reverse transcriptase and inserted into the genome at different times during development. If the preimplantation embryo of mouse is infected, the genes are fully methylated and not expressed. This result is typical of infective viruses added to the embryo up to the blastocyst stage, but by days 12 and 17 of development, infection with the same retroviruses results in no methylation of the reverse-transcribed genomes, and the genes are expressed (reviewed by Jahner and Jaenisch. 1985a). Studies of mouse strains in which only one copy of MoMLV provirus has been integrated at a random location in the mouse genome (Mov strains) have highlighted the role of enhancer methylation and demethylation in regulatory events occumng during early development. For example, Jahner and Jaenisch (1985a) examined the methylation patterns of viruses and flanking sequences in six Mov strains: Mov 1. 5. 7 , 9, 10, and 13, each having a different insertion site in the mouse genome. These results are summarized in Fig. I . In Mov 7 , 9 , and 13, the 3' LTR enhancer sequences were not fully methylated in sperm DNA, but in the other strains. all of the methylation sites were fully methylated in sperm. By day 12 of development, all of the sites in all of the viruses were fully methylated, regardless of their insertion site. By day 17, the 39 methylation sites in the coding sequence of each virus remained methylated, but tissue-specific patterns of demethylation, affecting just the enhancer sequences in the LTR of the integrated proviruses, had become evident. These persisted and intensified in the adult tissues of the Mov strains. This study showed that the enhancer sequence demethylation was dependent on site of integration of the provirus, but that the methylation status of the surrounding DNA did not spread into the provirus; instead, the enhancers seemed to respond independently to tissue-specific signals. In another study by the same authors, the effect of provirus insertion in Mov 7, 9. and 13 upon the methylation of the surrounding sequences was studied. The adjacent mouse sequences were highly methylated in Mov 9, less methylated in Mov 7, and completely unmethylated in Mov

AGE-RELATED CHANGES IN DNA METHYLATION Mov 5

Mov 7

DNA from sperm

mm-mm mm-mm

mm-00

liver

mm-um mm-om am-em -am

Mov 1

d17 [brain embryo kidney

Mov 9

Mov 10

mm-0~1 m-mm ~.IM

193 Mov 13

mm-00

mm-om

mm-mm mm-om mm-om mm-om mm-om mm-om mm-mm m m - m ~

region site

5 0-a

0

H p l Hp3 Hp4-36 Hp37 H13+14 + 2 H1-3 H4-12 +38 A12+13 A1-3 A4-11

FIG.1. Developmental changes in methylation of the enhancer sequences of the LTR of MoMLV proviruses located at different chromosomal positions. The structure of a single provirus is diagrammed at the bottom, with the enhancer regions symbolized by the striped boxes. The top of the figure shows the methylation patterns of the single provirus of each Mov strain, determined by cutting with PstI, SstI, or KpnI depending on the probe used, and by secondary digestion with the methylation-sensitive endonucleases HhaI (B), Aval (A), or HpaII (Hp). Filled boxes indicate virtually complete methylation, half-filled boxes indicate 2040% cleavage by the methylation-sensitive enzyme, and open boxes indicate >80% cleavage (i.e., essentially unmethylated). Reprinted, with permission, from Jahner and Jaenisch (1985b) Molecular and Cellular Biology.

13, which is known to have the provirus integrated between the upstream regulatory sequences and the coding sequences of the a21-collagen gene. In both Mov 7 and Mov 13, a strong increase in methylation of adjacent sequences within I kb of the inserted provirus was noted (Jahner and Jaenisch, 1985b). In the case of another type of virus, mouse mammary tumor virus (MMTV), early development has also been shown to include d e novo methylation of newly introduced proviruses (Cohen, 1980; Breznick and Cohen, 1982). With regard to the extraembryonic tissue, there is a very low level of methylation of the LZMd interspersed repeated sequence of mice (V. Chapman et al., 1984) and of at least one other interspersed repeat (Tolberg and Smith, 1984), as well as other sequences in the DNA of this extraembryonic tissue (reviewed by Sanford et al., 1985).

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3. Cirltirrcd Embryonic Cells Let us now turn to a consideration of the findings concerning proviral and interspersed repetitive DNA methylation in cultured multipotential embryonic cells, before and after they are induced to differentiate. In 1984, Hojman-Montes de Oca et al. studied three lines of cells derived from 129 mouse teratocarcinoma: PCC4, PCC3, and PCDI, as well as the 129 fibroblast Line WME. Treatment of all of these cell lines with 5azaC resulted in the induction of (IAP). The induction was particularly striking in the case of the PCC4 cell line, which originally produced fewer particles than the control line (<2% of cells with particles increased to 92% of cells with particles). In this cell line, the authors showed that the genes for IAP became demethylated upon 5azaC treatment, and that the transcription of the IAP R N A increased 20-fold and 30-fold, for two different-sized transcripts. Gautsch and Wilson (1983) also studied DNA methylation in a line derived from PCC4, called PCC4aza1 EC cells, or EC-A cells. They found that infection of these cells with MoMLV results in integration of the provirus but no transcription of the integrated virus. The study showed that the provirus is methylated at SmaI restriction sites by 16 days after infection, but is not methylated detectably after 8 days. However, the DNA of the provirus is inactive in transcription and transfection from the beginning of the infection. This study suggests that differentiating EC cells have an inactivation process that is not mediated by DNA methylation, and that de novo methylation progressively increases with time after infection. An alternative suggestion might be that the sites important for transcriptional and transfection activity were not those sensitive to Smal. However, Niwa er al. (19831, working with EC-A1 cells derived from PCC4 cells, confirmed the findings of the other authors and, in addition, showed that 5azaC was able to reverse the block to transcription of the MoMLV in the differentiated cells, but not in the undifferentiated cells. In undifferentiated cells, methylation of the provirus increased with time. Even so, treatment of these cells with 5azaC did not reverse the block to transcription. even though the DNA became demethylated. These two groups have shown that de novo methylation is characteristic of this line of cells in culture, and that in the earliest stages, before differentiation, one or more other regulatory signals other than methylation must inactivate gene transcription. A later study (Niwa, 1985) showed that the LTR of MoMLV, controlling the neo gene that can confer resistance to G418 upon cultured cells, was expressed very inefficiently in undifferentiated cells, even when it was not methylated. The efficiency of the construct was checked in mouse LTk cells, and it was expressed perfectly well, making the cells resistant

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to the antimetabolite. Another experiment reported in the same paper by Niwa (1985) showed that lines of EC induced to differentiate by retinoic acid varied in their spontaneous rate of production of infectious virions. Three such lines were studied, and the two that produced virus were hypomethylated with regard to the line which did not. This finding implies that DNA methylation may have an important regulatory function in the expression of the retrovirus genes in more differentiated cells. B. CHANGESDURING AGING The endogenous copies of the MMTV in C3Hf mice were studied by Etkind and Sarkar (1983). They found that the Mtv-I provirus, implicated in spontaneous formation of mammary carcinomas in this strain, underwent a progressive demethylation in spleens of non-tumor-bearing mice at ages 3, 6,9, and 12 months. The spontaneous tumors examined in this study resulted from integration of an additional copy of the provirus from Mtv-1 (identified by specific restriction fragments) into a new location in the DNA of mammary gland. It is possible that the demethylation of the Mtv-1 provirus in the spleen cells, many of which circulate in blood and/ or lymph, might allow production of virus that could become inserted into the DNA elsewhere. In the case of MMTV, the trend during postpubertal aging appears to be the opposite of that during early development, which includes a heavy methylation of the enhancers of endogenous viruses. This type of demethylation might be like the tissue-specific demethylation of enhancers seen in later development, or the demethylation might result from the type of stochastic loss of methylated sites that was seen in cultured cells (Shmookler-Reis and Goldstein, 1982a,b; Goldstein and Shmookler-Reis, 1985). One might suggest that ineffective remethylation following repair could explain this phenomenon. A study of the methylation of the IAP repetitive element during aging was performed by Mays-Hoopes et al. (1983). A probe, from the nonLTR portion of the conserved 3' region of the endogenous defective virus genome, detected an age-related progressive loss in methylation in the 1000 copies of this sequence, between 6 and 26 months of age (see Fig. 2B). An increase in cutting by both HpaII and MspI was observed, but no significant change in the IAP sequence copy number was detected. The simplest explanation of these findings is that a progressive loss in methylation in both the 5' and the central C's in the CCGG sequences occurs during aging in mouse liver. It is possible that the demethylation might be related in some way to a progressive rearrangement of the IAP genes that was detected during the same time span. The trend seen in aging-liver DNA for the IAP genes is opposite to the developmental trend

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seen, which involves heavy methylation that is essentially complete by birth. The demethylation affected sites that were shown in this same study and in Morgan and Huang (1984) to be completely demethylated in myeloma cells that are expressing the IAP genes to form the defective R N A virus particles in the endoplasmic reticulum cisternae. No examination of the expression of these genes in aging mouse liver was performed, however. Since this change is not a continuing a developmental process, it might also be explained by incomplete methylation of repaired DNA. A later study of the LIMd sequence, the major long interspersed repeated sequence of mouse with -10’ copies per genome, showed that this sequence also loses methylation in male mouse liver DNA during aging (Mays-Hoopes et al., 1986). By 27 months of age, 8% of these sequences can be cut by HpaII, while in young animals, essentially none of the copies is sensitive to HpaII. In this case, no change in the ability of MspI to cut the sequence occurred during aging. This sequence is the interspersed repeated sequence which is demethylated at these same sites in oogenesis but not in spermatogenesis (Sanford et al., 1984; Fig. 2C); however, the loss of methylation in this sequence in a somatic tissue appears to bear no relationship to the gametic demethylation. Instead, it appears to resemble the losses of methylation observed for the MMTV and IAP sequences in aging. This case appears to be stochastic rather than a continuation of enhancer demethylation seen late in development, since the particular sites examined do not demethylate late in development. Another observation that might be related to these findings is that agerelated graying in some mouse strains is correlated with expression, at the age when graying occurs, of infectious MLV (Morse er al., 1985). Evidently, the virus infects the melanocytes at a critical stage of development. and is expressed much later due to uncharacterized age-related changes in the regulatory system. The expression of virus genes is known to occur in the spleen and the thymus. If this mouse strain is an appropriate model system for endogenous proviruses in general, then more virions should be generally produced in cells from aging animals with integrated proviruses. This is certainly a testable hypothesis. It is rather unlikely that there is an up-regulation of transcription of a large set of endogenous genes in aging, since at both the mRNA level (Arlan Richardson, personal communication) and the protein level (Finch, 1972, 1976; Arlan Richardson, personal communication) there appear to be few quantitative agerelated changes detected in gene expression. Most products that are present in large enough amounts to detect on two-dimensional gels, or by enzymatic activity, undergo very small or negligible changes in concentration with aging.

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V. Methylation of X-Linked Genes A. DEVELOPMENT An event that occurs early in development, at approximately the time of implantation, is inactivation of one X chromosome of the extraembryonic tissues in females, with the paternal X being preferentially chosen for inactivation (reviewed by Riggs ef af., 1985; Gartler ef af.,1985; Monk, 1986). At a somewhat later stage of development, one of the X chromosomes in each cell of the future female fetus is inactivated, but this inactivation is random with regard to the maternal or paternal origin of the X chromosome. One of the primary hypotheses to explain maintenance of X inactivation through multiple cell divisions is marking of the inactive X by means of methylation. This hypothesis is strongly supported by the fact that the only technique presently available to reactivate the X chromosome of cells from the embryonic lineage (as opposed to extraembryonic lineage) cells is to treat cross-species-fused cultured cells with the DNA methyltransferase inhibitor 5azaC (reviewed by Monk, 1986; Riggs er al., 1985). However, as described in Section LA, this drug is not limited in its action to inhibition of DNA methylation, so that these results are not unequivocal proof that X inactivation occurs through DNA methylation. Cloned, unique X-specific sequences used as probes do not reveal a higher overall level of methylation on inactive X than on the active X (Wolf and Migeon, 1982). But it does appear that particular sites in some X-linked genes are methylated when the X is inactivated (Keith er al., 1986). It has been proposed that enhancer sequences near genes could serve as way stations to spread information concerning inactivation over the inactivated area (Riggs ef al., 1985) starting from a center of inactivation, Xce, which has been localized in the human X to a region somewhat below the centromere on the long arm where a fold usually occurs in karyotype preparations (Van Dyke ef af., 1986). However, in a study of X chromosome-regulatory sequences during early development by Lock ef al. (1987), several sites in the first intron of hypoxanthine phosphoribosyltransferase (HPRT) gene (methylation at which was previously shown by the same authors to correlate negatively with gene expression) appeared to become methylated at least 2 days following the time of cessation of gene expression due to X inactivation. There are a few methodological problems in the study described which might have prevented the authors from correctly determining the methylation status of the gene. The two major problems were the use of pools of 20 embryos with an unknown proportion of male and female fetuses [a proportion which could have

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been determined, for example, using Y chromosome-specific probes as done by Krumlauf et al. (1986)],and the use of conditions which barely separated some of the relevant restriction fragments, making the blots difficult to interpret. If the authors have come to the correct conclusions, however, the role of DNA methylation in X inactivation must be secondary to a marking and earlier-inactivation system in the embryonic lineages. Some studies have tested the idea that X-chromosome methylation is different in embryonic and extraembryonic lineages. It is possible to reactivate the inactive human X chromosome in the cells of chorionic villi merely by fusing the cells with mouse A9 cells (Migeon et al., 1986). These authors studied X-chromosome inactivation in cells from human chorionic villi heterozygous for glucose-6-phosphate dehydrogenase (G6PD) A/B isozymes, fused to A9 mouse fibroblasts lacking HPRT. This system is a little unusual in that the isozyme encoded on the late-replicating X chromosome is partially expressed in chorion. Recall that there is evidence from in vivo studies that the extraembryonic membranes utilize non-methylation-dependent regulatory systems and tend to be undermethylated. In the system that Migeon et al. studied, merely fusion of the two cell lines together (without the necessity for 5azaC treatment) and selection for HPRT expression, resulted in the reexpression of G6PD and phosphoglycerate kinase (PGK). Furthermore, the cells replicated both X chromosomes derived from the human cells nearly synchronously. The reactivation evidently did not involve demethylation, however, at least in the case of the G6PD. This result is not too surprising, since the 5‘ cluster of CpG’s is already demethylated in the inactive X in this tissue, so only the 3’-methylated HpaII sites can be assessed. This study showed that the switch from inactivity to activity of the genes was correlated with the normal replication timing for the X chromosome. In addition, it showed that the inactivation of the X chromosome in chorionic villi may not involve a DNA methylation component. In addition, Krumlauf et al. (1986) studied transgenic mice in which the normally autosomal a-fetoprotein gene, altered to distinguish it from the normal copies, had become integrated onto the X chromosome. They found that expression of the gene was repressed in the visceral endoderm of the yolk sac of female embryos, but not repressed in fetal liver, just as the normal gene is regulated, Although the data were not shown, the authors stated that there was no difference in the methylation patterns observed for this transgene in the two different tissues. These two investigations suggested that methylation of the X chromosome had little relevance for expression of X-linked genes in the extraembryonic lineages. This finding is consonant with the previously described low overall methylation of the DNA in these cells (Sections I1,A.Z and 3.)

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We now turn to a consideration of the issue of X-chromosome reactivation in cultured cells. The inactive X in female cells is not easy to reactivate, but fusing human cells with rodent cells has in some cases permitted reactivation. Mohandas et al. (1981) showed first that in a mouse-human cell fusion line, 37-24R-D, a structurally normal inactive X chromosome could be reactivated by treatment with SazaC, with HAT selection for expression of the HPRT gene from the inactive X. They fused the established mouse line deficient in HPRT to fibroblasts from a human female with a balanced X/11 translocation, having the active HPRT gene on the translocated X. Having chosen for study a clone with both the active and the inactive X from the human set, they treated with 8-azaguanine to select against expression of HPRT. The continued presence of the human inactive X could be detected by assaying for the noninactivated gene STS. Such tines were then treated with 5azaC to test for the reactivation of the human X. All such lines that had human STS activity could be induced to express the HPRT gene which was selected for. In 16 clones, only two expressed another X-inactivated gene: one expressed G6PD and one expressed PGK. So, although the treatment could reactivate the selected marker gene, it did not reactivate the entire X chromosome. The authors estimated that 65% of these clones had reactivated one of the other genes from the inactive X that they tested. They interpreted their results to suggest that DNA methylation is involved in maintaining the inactivation of the X chromosome, and that the inactivation is segmental in nature. The results were extended by Jones et al. (1982), who found that treatment of these hybrid lines in the latter half of the S phase, when the inactive X replicates, was most effective in inducing reactivation, and that two cell divisions had to ensue for maximal induction to occur. These findings supported the interpretation that demethylation had to occur to allow reexpression of the inactive HPRT. Additional support for this idea comes from use of genes from inactive versus active X chromosomes for transfection into mouse LTk--derived HPRT- fibroblasts (613 fibroblasts) or 380-6 Chinese hamster fibroblasts that are HPRT-. Venolia et al. (1982) used the mouse-human lines as well as hamster-human lines to examine this issue. In all cases, the transfection was ineffective when the donor DNA was isolated from the inactive X-containing donor cell line, but effective if the HPRT gene had been reactivated by 5azaC and selection. These results again led to the idea that the X-inactivation process includes DNA methylation as at least a part of the mechanism. Paterno el al. (1985), utilized a mutant of the embryonic C86S1 line, which lacks HPRT activity (HPRT-). This line was treated with SazaC, and a transient expression of HPRT and three other X-chromosome-en-

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coded genes was evident. From 5 to 20% of the surviving cells from this experiment expressed HPRT stably. Most cells in the latter category also had two X chromosomes that replicated early in the S phase, instead of the inactive X replicating very late as in the parental line and in other cells with inactivated X chromosomes. No effect on HPRT expression or X-chromosome replication was observed if retinoic acid-induced differentiation occurred before the 5azaC treatment. The authors concluded that X inactivation might include an early methylation step followed by later stabilizing effects that could not be reversed by demethylation. Thus several studies on cell lines have implied that DNA methylation plays a role in gene regulation via X-chromosome inactivation, in cells not derived from extraembryonic lineages.

B. AGINGA N D X INACTIVATION During in vitro-replicating life span, Torrelio and Paz (1979) found that human female lung fibroblasts (1MR90) slowly lost activity for the X-linked enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) compared to the autosomally coded APRT. The authors interpreted their data as mutational changes in the active genes. In contrast, Wareham et al. (1987) found that in mice in vivo, an age-related reactivation of the Xlinked ornithine carbamoyltransferase (OCT) could be detected. The authors used female mice heterozygous for the Spf mutation, which results in abnormal OCT that lacks activity at pH 7.0. Because the heterozygotes had the abnormal allele on an X-autosome translocation and the normal OCT gene on the normal X chromosome, the normal gene was forced to undergo X inactivation in all cells. Liver cells from such female mice were examined for OCT activity at pH 7.0, and a distinctive frequency of positive cells was seen at each of three ages examined. The 8- to 10-weekold mice had 0.0558% positive area, the 6- to 9-month-old adults had 0.2861% positive, and the 14- to 17-month-oldanimals had 2.76% positive, albeit each of these percentages had a standard deviation larger than the number itself. The general impression of an increase in expression was supported by an increase in the number of patches per field, increased from 0.14 ? 0.71 to 7.63 ? 5.61 over the same period. The fact that this effect did not occur in male Spfry mice was an important control ruling out reverse mutation. Holliday (1987a) placed these findings in the context of possible age-related demethylation, and suggested that the evidently random production of variant cells with fully active OCT, rather than intermediate levels of gene expression, could be explained by loss of methylation of critical sites. Data to assess whether the entire inactive X was reactivated were not available from this study.

AGE-RELATED CHANGES IN DNA METHYLATION

20 1

Another study had suggested earlier that X-linked genes could undergo age-related reactivation, although again, the mechanism remained undetermined. Cattanach (1974) studied tyrosinase genes translocated to the X chromosome. In heterozygotes in which, because of X-autosome translocations, the tyrosinase gene was on the inactive X in all cells, Cattanach observed a progressive darkening of the animals with age. He attributed this to a weakening of the X-inactivation mechanism with age, in a manner that depended in part on where in the animal the hair was formed. Holliday (1985) suggested that a progressive loss of DNA methylation with aging could account for this observation. This small but interesting data set implies that X-chromosome reactivation, possibly due to DNA demethylation, might occur in vivo more frequently than in vitro. This change would be the reverse of, rather than a continuation of, development. This idea would be very fruitful to pursue, from both the standpoint of X inactivation and that of aging.

VI. Methylation of Globin Genes

A. DEVELOPMENT 1. Gametogenesis

In spermatogenesis, high levels of methylation are either maintained, if extensive methylation of globin genes marks the germ line permanently, or originated. This is clear from a number of studies on globin genes in sperm of a variety of organisms. Both Weintraub et al. (1981) and McGhee and Ginder (1979) found heavy methylation of ci- and p-globin genes in chicken sperm. Rahe et al. (1983) examined endonuclease sites in genes for adult globin chains and found them to be completely methylated in mouse sperm. Razin et al. (1984) confiied this finding for mouse P-globin gene. An extensive and very careful analysis of sites for methylation-sensitive endonucleases in the human p-globin gene cluster showed that these genes are also highly methylated in sperm (van der Ploeg and Flavell, I 980).

2 . Early Development In the extraembryonic lineages, it seems that the globin genes, similarly to other sequences, tend to be undermethylated. Razin et al. (1984) found low methylation of (3-majorglobin gene in yolk sac and placenta of mouse. Human placenta also has undermethylated p-globin cluster genes (van der Ploeg and Flavell, 1980).

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3. Later Development and Differentiation

In chickens, early evidence indicated that the genes for adult hemoglobin were methylated in tissue-specific ways, and that undermethylation was characteristic of tissues where expression was possible (McGhee and Ginder, 1979). In humans, embryonic (&)-globin is produced first; then fetal (01ly)globin is produced, and finally adult (dpwith some d6)-globin is produced. The earliest erythroid cells come from the megaloblasts of the primitive yolk sac, and circulate from 4 to 1C-12 weeks after conception. Macrocytic erythroblasts arise in the fetal liver at 6-7 weeks; these produce erythrocytes that circulate from 8 weeks on. Finally, bone marrow cells (normoblasts) produce adult globin (Peschle et al., 1985). The pattern of gene expression switches is compatible with a model where a single stem-cell pool changes from megaloblasts to macrocytes upon migration from yolk sac to liver, and from macrocytes to normocytes upon migration from liver to bone marrow. Each of these migrations is nearly, but not entirely, synchronous with switches in chain expression. The first migration is near the 2; -+01 and E -+ y switches, and the second is near the time of y -+ p switching (Peschle et al., 1985). Similar patterns of fetal to adult globin gene switching are found for mice (Leder et al., 1980). In an extensive study of the human p-globin cluster (including G-y, Ay, 6, and p genes, in that order) showed that the y-globin genes are unmethylated in about three-quarters of the DNA in fetal liver, where about half the cells are erythroid. The p- and &chain genes, expressed in adult bone marrow, are lower in methylation there than in other adult tissues (van der Ploeg and Flavell, 1980). In fetal brain and lymphocyte DNAs, these genes were found to be close to totally methylated in the 17 endonuclease sites examined. On the other hand, in adult liver DNA, the p gene was -25% unmethylated in two upstream sites, although most other sites throughout the cluster were fully methylated. These findings suggested that a low level of DNA methylation could be necessary but not sufficient for expression of the globin genes at appropriate times in development. The study was extended to earlier stages and to >90% purified human erythroblasts by Mavilio et al. (1983). A strong correlation was found between DNA hypomethylation in the flanking sequences of the gene cluster and the expression of the genes. The E gene, upstream of the y genes and expressed earlier, was included in the study and also showed this type of correlation. In chicken a-globin gene cluster, endonuclease sites flanking the genes were found to be unmethylated in embryonic, anemic, and adult red blood cells but fully methylated in brain and spleen (Haigh et al., 1982).

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An interesting experiment testing the effect of methylation on human y-globin gene expression was performed by Busslinger et al. (1983b). They methylated this gene in vitro, as part of a clone in the phage MI3 mp8. They then exchanged parts of methylated and unmethylated clones and examined the effect of methylating just the vector sequences, the structural gene but not its flanking sequences, or just the flanking sequences. They transfected the completely or segmentally methylated genes into mouse L cells. The results showed that constructs methylated from (-760 to + 100) in the 5’ region of the y-globin gene were not expressed, while methylation elsewhere did not shut off transcription of the transfected gene. The results were interpreted to indicate a possible direct role for DNA methylation in shutting off globin gene transcription. In humans, Oppenheim et al. (1985)have studied the methylation status of the fetal globin genes in the immature red blood cells of adults. They found that there was a correlation between undermethylation of fetal globin gene and its expression in erythroid precursor cells. A great deal of interest has been generated by the somewhat successful treatment of patients with p thalassemia and sickle cell anemia with SazaC, resulting in reexpression of their fetal globin genes, as reviewed by Ley and Nienhuis (1985). In a follow-up study, Humphries er al. (1985) found that a 4- to 6-fold increase in fetal (y) globin mRNA production in bone marrow cells was followed by an increase in fetal globin synthesis. The fetal globin-producing cells were increased before there was a measurable mortality of the more mature types of cells, resulting from the drug treatment, which supported a direct effect of 5azaC on mRNA transcription rather than a cell selection mechanism favoring the more immature types of erythroid procursor cells. The next group of papers concern the erythroleukemia cell lines that can differentiate in vifro with production of the hemoglobin chains, upon suitable induction. In an early study by Christman er al. (1977), Friend murine erythroleukemia strain 745A was treated with L-ethionine. At concentrations that induced hemoglobin production and differentiation, both DNA and tRNA methylation in vivo were inhibited. Later, Creusot et al. (1982) continued the characterization in showing that 5azaC and its deoxy derivative were potent inducers, and that substitution of 0.3% of the cytosines in DNA with 5azaC resulted in a 95% inhibition of the DNA methyltransferase activity. The analog was washed out and the DNA allowed to replicate, leading to -15% of the cells forming globin. The DNA methyltransferase activity was back to normal in 48 hours, which may explain the low percentage of differentiation observed. Another possible explanation is suggested by the work of Zucker er al. (1983) using T3C 12 line of Friend erythroleukemia cells. They found that adding 5azaC to cells, being induced with butyric acid or DMSO, increased the proportion of fetal to adult globins expressed, and that added hemin increased the syn-

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thesis of both types of globin chains. It is thus possible that hemin synthesis may be the limiting factor in differentiation of some 5azaCinduced cells of these lines. Sheffery et af. (1981) studied Ds19 strain of mouse erythroleukemia cells, induced to differentiate using hexamethylene bisacetamide. Although changes in DNase 1 sensitivity were observed during this differentiation, the sites near the a,-and p-major globin genes that they examined did not appear to change in methylation status. Razin et ul. (1986) have found a transient genome-wide demethylation that occurs early in the induction of the Friend cells line 745 to form hemoglobin, by a number of inducers, and explored its mechanism. They induced the cells with hexamethylene bisacetamide and labeled the DNA using the density label 5-bromodeoxyuridine and the radioactive label [deox.~-S-~H Jcytidine. Newly synthesized DNA in this system should be labeled with tritium and heavy-light DNA. During the induction process, but not in the undifferentiated cell cultures, they consistently observed incorporation of the tritium into light-light DNA, which is most easily interpreted to mean that 5mC has been replaced with labeled C, without much new overall DNA synthesis. In summary, a number of correlations between hypomethylation of 5’flanking sequences and expression of globin genes have been found. 5azaC can evidently induce gene expression, while in vitro methylation can prevent expression of transfected genes. Thus, there is a substantial body of evidence favoring a role of DNA hypomethylation in the expression of globin genes in development and differentiation. Both de novo methylation of sites adjacent to embryonic and fetal globin genes and production of unmethylated sites adjacent to adult globin genes are evidently programmed to occur. However, some sites are not fully methylated in all nonexpressing tissues.

B. CHANGES IN METHYLATIONIN AGING In a study of human fibroblast lines undergoing their replicative life span, Shmookler-Reisand Goldstein (1982a)found that fetal globin genes, which should not be expressed in the cell lines used, lost methylation as detected by methylation-sensitiveendonuclease cutting. While a decrease in methylation occurred at all sites examined in this study, the loss was not equal at the different sites. Whether or not this effect was merely stochastic or reflected some feature of the chromatin organization or repair processes was not clear. The authors suggested that inappropriate (ectopic) gene expression could result from demethylation, and predicted leakiness in the tissue specificity of gene expression in v i m , with possible relevance

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to the situation in vivo. A follow-up experiment showed that methylation of a- and P-globin genes, among others, drifted and often decreased upon long-term culturing of several human lines (Shmookler-Reisand Goldstein, 1982b). The phenomenon of methylation drift during culturing, in fetal globin gene, had also been noticed by Wolf and Migeon (1982). Little evidence has been collected to date concerning globin genes in vivo during aging. Ono and Cutler (1978) found that nuclear, but not cytoplasmic transcripts of globin genes increased with aging in mouse liver. The mouse a,-globin gene (Leder et al., 1978), which is not expressed in liver, becomes demethylated at HpaII sites in and near the gene between 3 and 6 months of age, and remains demethylated thereafter (L. L. MaysHoopes, W. Chao, and L. Spuck, unpublished data). However, analysis of poly(A)' mRNA from these cells showed that no increase in mRNA hybridizing with a,-globin probe could be detected (L. Spuck, S. Lee, and L. L. Mays-Hoopes, unpublished data). Thus, it appears, from rather limited data, that the expression of protein product from at least one globin gene is prevented in postpubertal adults, by mechanisms other than DNA methylation. The results of Ono and Cutler suggested that mRNA processing could be one type of regulation worth exploring. Another could be activation-resistant chromatin packaging, because in the study of Ono and Cutler, the level of nuclear transcription was far lower than expected for a fully demethylated gene. In any case, the status of the methylation process in aging suggests that normal developmental controls are not continuing per se into senescence, but dysdifferentiation is not the obligatory result.

VII. Methylation of the Chicken Vitellogenin I1 Gene A. DEVELOPMENT The chicken vitellogenin I1 gene has been studied using various techniques. Wilks et al. (1982) examined the correlation between demethylation and sensitivity to restriction endonucleases and found that demethylation appeared to follow the onset of transcription of vitellogenin. However, a later study by the same group employed the genomic sequencing technique and came to the opposite conclusions (Saluz et al., 1986). A CpG sequence at -612 (612 bp upstream from the vitellogenin I1 transcription start) had been previously identified as becoming demethylated after the induction of transcription, since it became sensitive to cutting by HpaII at that time (Wilks et al., 1982, 1984; Meijlink ef al., 1983). This site is a part of the estradiol regulatory binding site. There is an additional CpG in this region,

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as well as two more downstream about 50 nucleotides, in the glucocorticoid-responsive sequence and an adjacent short stretch of alternating purines and pyrimidines. The work of Saluz er a / . (1986) showed that when immature roosters were treated with estradiol, in their liver DNA, all four of the CpG sequences on one of the DNA strands became demethylated during the time slightly preceding transcription, continuing until maximal transcription. The other strand was demethylated only 24 hours later, about 22 hours after the start of transcription. The authors pointed out that the demethylated sequence correlated with the presence of steroid-receptor complex in the tissue rather than with transcription per se, since the oviduct also demethylated these sequences but does not transcribe the gene. There are two pairs of DNase I sites bracketing this regulatory region that appear with the same kinetics as the hemimethylation of the regulatory region. The mechanism of the demethylation in this system is currently under investigation, but the limited amount of DNA replication during the short time course of induction, and the lack of amplification of the vitellogenin gene during induction, seem to rule out demethylation mechanisms that prevent postreplicative methylation. The authors propose that an excision repair type of mechanism or a glycosylasemediated exchange of C for 5mC could explain their results. The binding of a steroid-receptor complex to its regulatory sites was predicted to facilitate complete removal of methylation from both strands, since all tissues studied have glucocorticoids and their receptors, and all showed at least partial demethylation of the CpG in the glucocorticoid-responsive element.

B. In Vivo AGING Meijlink ef a / . (1983) examined the ability of HpalI to cut site I , 5' to coding sequences, and site 2, just within the 5' end of the chicken vitellogenin gene in young and 8-month-old chickens. Only one older bird of each sex was examined in this study. In young cockerels, both sites were 90% methylated in liver, while in the older cockerel, site 1 was 70% methylated and site 2 was still 90% methylated. In the older hen liver, site 1 was 20% methylated while site 2 was 30% methylated; this was a laying hen which was actively expressing this gene in the liver. In the oviduct of this hen, which does not express the gene, site 1 was 40% methylated while site 2 was 90% methylated. In cockerels treated with estradiol to induce expression of vitellogenin gene in the liver, demethylation of site 1 progressed from 80% methylated 16 hours after hormone to 20% methylated 32 days after hormone, while site 2 remained 90% methylated. The gene map in Fig. 2A compares the site found to undergo age-related demethylation by Meijlink et al. (1983) with the sites shown to undergo de-

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A

RNA H

H

I

-1 f

,.

2.8Kb

3

2.6Kb

B

w LTR

LTR

C

?

s t e of Aging Demethylation

?

Site of Development or Differentiation Demethylatlon Transcription

H

H

I

I

SEND

H H I

E

E

I ,

3' END

E

Estrogen Receptor Binding Site

R

EcoRl 1.35 Kb Cloned Probe

E

E

0 Lon~Terminal Repeat

LTR

FIG. 2. Comparisons of methylation-sensitive endonuclease sites that demethylate in development and in aging in vivo. (A) Chicken vitellogenin I1 gene HpaIl sites (5' end) that demethylate in development in hen liver and in aging in rooster living. Data used to construct the figure were from Wilks et al. (1982, 1984). Saluz et al. (1986). and Meijlink ef al. (1983). (B) Mouse intracisternal A-type particle genes, showing 5' LTR sites unmethylated when the genes are expressed and 3' non-LTR sites demethylated in aging liver. Data were from Morgan and Huang (1984) and Mays-Hoopes et al. (1983). (C) Mouse L l M d repeated sequence, showing Hpall sites demethylated both in extraembryonic tissues and in aging liver. Data were from V. Chapman et a/. (1984) and Mays-Hoopes et a / . (1983).

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velopmental demethylation correlated with expression in liver of hen by Saluz et 01. (1986). The site is one of those programmed to demethylate in hen liver. In this case, then, the demethylation in older cockerels and laying hens' oviducts might be explained as an inappropriate progression of an estrogen-controlled DNA demethylation in tissues that contain estrogen receptors. VIII. a-Fetoprotein and Albumin A. DEVELOPMENT A pair of genes which have been studied by several groups are a-fetoprotein and albumin, both serum proteins with similar physicochemical properties. The gene for a-fetoprotein is expressed strongly in the yolk sac and the embryonic liver, but in normal adults the serum concentration is 100.000-fold lower than in fetuses. Albumin synthesis is low in fetal life, but increases to high levels in adult life, and is synthesized in the liver. Vedel et al. (1983) used MspI and HpaII restriction to look for methylation changes in fetal and adult liver. Although the authors of this study did not have the correct probes to enable them to study thoroughly the 5' regions of either gene, the results that they did obtain enabled them to conclude that albumin gene became extensively demethylated in adult liver DNA. For example, they studied an MspI site in the first intron of this gene, identifying it as a subfragment of a Hind111 fragment known to occur in this region. This site was 65% methylated in 17- to 19-day embryonic liver, but only 10% methylated in adult liver. The a-fetoprotein gene, in contrast to the albumin gene, was less methylated in the fetal liver than in the adult liver, but was partially methylated in both ages. For this gene, the probes available did not cover the 5' 200 nucleotides, so that the upstream regulatory regions could not be studied. Adult kidney was used as a control tissue which does not express either of these genes, and the genes were fully methylated in the sites under study. Because the 17- to 19-day fetal hepatocytes were all thought to be transcribing the afetoprotein gene, the authors concluded that a partially methylated gene for this protein could be actively transcribed. The major findings of this study have been substantially confiied by subsequent studies of Kunnath and Locker (1983) and Sell et al. (1985). On the other hand, in the a-fetoprotein gene of mice, there is methylation of an MspI site in the first intron just after expression ceases (Papaconstantinou and Church, 1984). Young and Tilghman (1984) studied the induction of differentiation in

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F9 embryonal carcinoma from 129/SV mouse line, either growing as monolayers or as aggregates. Upon retinoic acid induction, monolayers differentiate into parietal endoderm lacking expression of a-fetoprotein and albumin, while aggregates differentiate into visceral endoderm and express these two genes. Retinoic acid induction is accompanied by demethylation of these genes, among others. The a-fetoprotein gene was more methylated in the parietal, than in the visceral, endodermlike differentiated cells. Kuo et al. (1984) studied the methylation of the a-fetoprotein gene in transplantable hepatocellular carcinomas 7777 and 252 of rat. The gene is repressed in normal adult liver and in 252, but is highly active in 7777. The HpaII sites were demethylated in the expressing cell line, but the HhaI sites were found to be demethylated in both carcinoma lines when compared to liver DNA. Ott et al. (1982) studied methylation and expression of albumin gene in clones derived from the Reuber H35 hepatoma. They compared well-differentiated albumin-secreting clones, variants that lack differentiation and do not secrete albumin, and revertants of the variants that do secrete albumin. They identified an HpaII site in the first intron of the albumin gene as being demethylated whenever the gene was expressed, but found that demethylation of this site was not sufficient to permit expression of this gene. It is striking that the undermethylation of the albumin gene in liver that was observed in this study covered the entire length of the gene, while in the expressing hepatoma lines, the 5’ site is the only one which is well correlated with the transcriptional status of the gene. However, the location of the sensitive site is plausible, in that Ott et al. (1984) showed that the 400 bp immediately upstream to the gene are sufficient to permit expression of the CAT reporter gene only in those lines that express the albumin gene itself. These results were called into question by a study of Sell et al. (1985) in which demethylation of a-fetoprotein and albumin genes in rat hepatocellular hepatomas was not correlated with the transcription of the genes. Tratner et al. (1987) later studied both genes in a group of Reuber hepatoma lines using genomic clones covering the 5’ regions of the genes and did find HpaII sites that were demethylated when the gene was turned on. The authors explained their differences from other studies as resulting from use of genomic rather than cDNA probes so as to examine the 5’-regulatory regions more effectively. In summary, not all the evidence concerning a-fetoprotein and albumin genes supports the importance of DNA methylation in their regulation. However, in some experiments, hypomethylation of particular sites appears to occur in differentiation when the genes are expressed.

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B. AGING The albumin gene's methylation status has not been studied during aging, but Richardson (1981)summarized data from six experiments on rat liver, covering analysis in vivo, in isolated hepatocytes, and in a cell-free translation system, all agreeing that between 30 and 300% increase in albumin synthesis occurs in rat liver during aging. In rats, Kroes el al. (1972)found that 9- or 15-month-old rats displayed delayed positive a-fetoprotein responses only beginning at 4 weeks after carcinogen treatment, while 6- to 12-week-old rats responded within 2-3 weeks. The mouse a-fetoprotein gene is the only sequence studied so far that undergoes age-related increases in methylation (Papaconstantinou and Church, 1984; J . Papaconstantinou, personal communication). As mentioned earlier, the gene undergoes methylation in an Mspl site in the first intron just after birth, when expression is extinguished, and there is a progressive methylation thereafter in the second intron, continuing into senescence (ages of animals were not specified, however.) This process appears to be a clear-cut case of senescence continuing processes that have begun in development.

IX. Methylation of Several Other Genes in Development and Aging A. a, U-GLOBULIN

This urinary globulin is synthesized in liver. Information about its regulation in development and aging suggests that methylation does not play a role in its age-related decrease in expression. Roy ef uf. (1984) have examined the azU-globulingenes using the endonucleases HpaII and Mspl in prepubertal rats (before the conformational transition in chromatin that occurs as the gene begins to be transcribed), during the active expression in mature rats, and after the shutoff of the gene. The data are described, rather than presented, but indicate that no detectable change in methylation accompanied either of these transitions.

B. MHC, CLASSI GENES The class I genes of the major histocompatibility complex code for cell surface proteins that function in presentation of antigens to cytotoxic T lymphocytes. They are not expressed in early embryonic tissues, although their expression can be induced by interferons (Ozato ef d.,1985). In adults, the levels of these molecules are controlled in a tissue-specific manner, although they occur on most somatic cells (Hams and Gill, 1986).

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The studies of induction of MHC class I molecules in cultured embryonic cells have given conflicting results concerning methylation. The F9 embryonal carcinoma line was studied by Tanaka et al. (1983). They compared F9 with two subclones isolated after different lengths of induction with retinoic acid. Although several genes (albumin, insulin, and MMTV) demethylated progressively, the class I gene for Kbdid not. Using a specific 3' probe, they found that the gene for the H-2 Kb became progressively more methylated in comparisons of these three cell lines, as well as in samples taken during a time course of retinoic acid treatment of F9 cells. The authors then treated the most differentiated subline with 5azaC for 3 days. Electrophoresis of the proteins present in these cells showed that several were greatly increased by this treatment; however, immunoprecipitation of the Kb antigen molecules and electrophoretic analysis revealed that they decreased to almost undetectable levels by 3 days after 5azaC. This result shows that methylation appears to turn on the transcription of this gene, while demethylation turns it off. Tanaka et al. (1986) showed that methylation of the 3' end of the QlO gene of the mouse MHC was positively correlated with expression. Specific methylation of this region appeared developmentally in a time sequence paralleling that of transcription of this gene, which increased greatly at -1 day after birth. In contrast, Miyada and Wallace (1986) found that the MspI/HpaII sites surrounding the third exon of the gene-which are upstream of those studied by Tanaka et al. (1986)-are less methylated in liver where the gene is expressed than in spleen or thymus where no expression occurs. In the same study, a similar area of the gene for the H-2 Kbtransplantation antigen of the MHC was found to be demethylated in all three tissues, which corresponds to its pattern of expression. This result conflicts with conclusions of Tanaka et al. (1983), who found that the 3' end of this gene is methylated when it is expressed in cultured cells. Young and Tilghman (1984) studied F9 cell differentiation and found that 5azaC induced H-2 mRNA. There is no reason a priori to expect all parts of a gene to behave similarly with regard to methylation in transcriptionally active states, but the conflict in 5azaC effects is difficult to understand. Uitterlinden et al. (1984, 1985) and Vijg et al. (1985) analyzed rat MHC class I genes using MspI and HpaII and found methylation differences between liver and spleen, the spleen showing more cutting by HpaII. Age-related changes in methylation of these sequences in liver were not found, but in pituitary, demethylation occurred in old rats. However, normal pituitaries did not show this demethylation; it was found to be characteristic of pituitary tumors that occur very frequently in aged rats of the strain tested. Interestingly, liver precancerous nodules did not show such a demethylation (Vijg ef al., 1985).

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C. CHORIONIC GONADOTROPIN Goldstein and Shmookler-Reis (1985) studied the methylation status of the gene for the a chain of human chorionic gonadotropin (hCG) in human fibroblasts undergoing their replicative life span. They found that some clones and subclones lost methylation in this gene, detected by increased HpuII endonuclease cutting, while others did not. None of the clones had detectable protein product, and Northern blots of mRNA were negative for these transcripts; yet there evidently were some initial transcripts present.

D. THErus ONCOGENE The c-rus genes are cellular homologs of the viral Harvey (v-Ha-rus) and Kirsten (v-Ki-rus) oncogenes of murine sarcoma viruses. They are transcribed in normal cells, and it has been found that their transcription increases during liver regeneration (Goyette et ul., 1983). Thus, they may play a role in normal and abnormal growth control. Vijg ef ul. (1985) studied the methylation and arrangement of c-Ha-rus genes in female WAG/Rij rats at 24, 30, and 45 months of age. They found no differences in methylation; the sequences were evidently completely methylated at all ages as detected by HpuII resistance. EcoRI digestion revealed no rearrangements. In each case, normal liver was compared to hyperplastic nodules and neither showed DNA methylation o r arrangement changes. The same gene was studied by Uitterlinden et al. (1984, 1985). in pituitaries of this rat strain. In that case, the c - H a m s gene was demethylated in tumors, but not in adjacent normal tissue, regardless of age.

E. THEnzyc ONCOGENE As noted in Section I,A, Swain ef af. (1987) found that when c-myc was used as a transgene, it was methylated and shut off when inherited from a female parent, but unmethylated and turned on when inherited from a male parent. Ono ef ul. (1986) have examined the methylation of sites on the first three exons of c-myc in liver and brain DNA, using actin and dihydrofoiate reductase genes controls. They found hypomethylation in aging spleen DNA but hypermethylation in liver DNA and no change in brain DNA. The control genes did not change in methylation. They used female mice at 2, 14, and 26 months of age. It would be interesting to know whether more 5' sites showed the same trends.

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X. Final Synthesis and Conclusions If at least some animals experience a fairly widespread demethylation of their DNA during normal aging, what might be the results? It is possible that little or no change in gene regulation might follow demethylation. It is clear that demethylation alone cannot turn on any gene, although Keshet et al. (1986) have offered a demonstration that methylation status of transfected exogenous DNA can direct assembly into different types of chromatin structures, as detected by DNase I sensitivity. If the appropriate trans-acting factors are present to repress transcription, or if the tissue is missing specific trans-acting factors that positively regulate transcription, demethylation may have little or no effect. On the other hand, ectopic transcription could result, especially if stress on the cells disturbed the normal regulatory status. It is still possible to consider the suggestion that such transcription could play a role in age-related increases in cancer incidence (Shmookler-Reis and Goldstein, 1982a; Goldstein and ShmooklerReis, 1985; Mays-Hoopes, 1985b). But the lack of transdifferentiation or dedifferentiation in normal aging tissue would argue that whatever processes that DNA methylation may control, these processes must have a degree of redundancy in their regulation that keeps them in abeyance despite demethylation. Presently there are hints that such is the case. For example, the lack of expression of vitellogenin in oviducts of 8-monthold hens or in livers of cockerels 32 days after estrogen, when demethylation of the gene is extensive, suggests that another regulatory process must be preventing expression. It is possible that RNA processing and transport to the cytoplasm may include a regulatory site, since ectopic transcripts were not detected in the cytoplasmic RNAs of aging liver and brain, while they did occur in the nuclear RNAs (Ono and Cutler, 1978). It is also interesting to note that transdifferentiationlike processes have been observed in the mutagen-induced or spontaneous focal lesions (Hartman and Morgan, 1985), and that these focal lesions do increase with age. These results are presently interpreted to mean that somatic mutation increases with aging, but some of the metaplasia might actually result from clonally inherited epigenetic changes-for example, methylation changes. Another type of possible consequence of age-related demethylation might be an increased risk of chromosome breakage during aging. The evidence reviewed that might bear on this question is the increase in rearrangements of IAP genes that accompanies their slow age-related demethylation, and possibly the slow release of splenic MMTV proviruses. Since Hare and Taylor (1985) and Steven S. Smith (personal communi-

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cation) have found evidence that methylation could mark DNA near potential repair sites, it is also possible that repair efficiency might decrease as methylation decreases. This review has identified two trends in DNA methylation changes during aging. The first is a continuation of developmental changes in methylation, as exemplified by vitellogenin of chickens and a-fetoprotein of mice. It is possible that the demethylation of MMTV sequences should be viewed as a continuation of the tissue-specific developmental demethylation of enhancer sequences of proviruses, as seen in development in Mov strains of mice (see Section IV,A,2). In addition, there is a second trend toward possibly stochastic demethylation. This trend is examplified by IAP and LIMd sequences of mice, mouse satellite, and a variety of genic sequences in human fibroblasts undergoing their replicative life spans. Thus. aging may in part continue development, but it appears to include nonprogrammed components possibly related to damage and repair followed by incomplete remethylation. There is some fragmentary evidence to support the idea that tissue specifcity has broken down with aging, predicted by the dysdifferentiation hypothesis (see Section 1,B). In the vitellogenin I1 gene, demethylation has occurred in both liver and oviduct, while only liver should express the gene. Demethylation of male liver DNA L l M d sequences, which should demethylate in oocytes, might be another such example. In these two cases, differentiation control may have been lost. So, the analysis of data concerning the process of DNA methylation in development and aging has revealed evidence that can support both extrinsic and intrinsic hypotheses of aging. It is plausible based on the data reviewed here that many of the changes occurring in aging in DNA methylation may be continuations of the processes of development.

ACKNOWLEDGMENTS The helpful suggestions of Raul Saavedra have been much appreciated by the author. Permission to cite unpublished work of John Papaconstantinou and Steven S. Smith is gratefully acknowledged. The author's research is supported by a William and Flora Hewlett grant through the Research Corporation, a grant from the American Federation for Aging Research. and a NIH-NRSA Senior Fellowship.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 114

Epithelium-Capillary Interactions in the Eye: The Retinal Pigment Epithelium and the Choriocapillaris GARYE. KORTE,*MARGARET S. BURNS,?AND ROY W. BELLHORNS *Department of Ophthalmology, Montefiore Medical Center and Albert Einstein College of Medicine, Bronx, New York 10467, fDepartment of Ophthalmology, University of California at Davis, School of Medicine, Davis, California 95616, and $Department of Surgery, University of California at Davis, School of Veterinary Medicine, Davis, California 95616

I. Introduction Closely apposed sheets of epithelia and plexus of capillaries are found in many organs. In this review we survey the evidence for epitheliumcapillary interactions where the two are apposed, emphasizing structural and functional manifestations such as capillary permeability and cell polarity. We then focus on observations derived from human ocular histopathology and experimental animal models in which interactions are evident between (1) the retinal pigment epithelium (RPE) and its apposed capillary plexus, the choriocapillaris, and (2) between RPE and retinal capillaries experimentally brought into apposition with RPE, from which they are normally isolated. These observations are relevant to the pathogenesis of chorioretinal diseases like age-related macular degeneration and retinitis pigmentosa. They are but two of the causes of reduced vision and blindness that probably arise primarily at the RPE and lead to complicating secondary changes in the adjacent choriocapillaris and neural retina (Hogan, 1972; Green and Key, 1977; Gartner and Henkind, 1982; Young, 1987). Finally, possible mechanisms of RPE-choriocapillaris interactions are discussed in light of current work.

11. Histologic Evidence of Epithelium-Capillary Interactions Histologic evidence for an interaction between epithelia and their capillaries is seen in the correlation between (1) the occurrence of fenestrated endothelia near apposed epithelia, and (2) the polarization of epithelial cells toward their proximate capillary beds. 22 1 Copyright 0 1989 by Academic Press. Inc. All righls of reproduction in any form reserved.

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A. CORRELATION BETWEEN CAPILLARY FENESTRATIONS AND EPITHELIUM Qualitatively, there is a conspicuous correlation between the presence of endothelial fenestrae and a proximate epithelium (although it must be noted that fenestrated capillaries do occur without a proximate epithelium, for example, the capillaries of the atrioventricular node of the mammalian heart: Weihe and Kalmbach, 1978). The correlation between fenestrated capillaries and a proximate epithelium is especiaUy striking where fenestrae are present only where the capillary encroaches on the epithelium. For example, peritubular capillaries in the mouse epididymis contain continuous. unfenestrated endothelium except where the capillaries closely appose the epithelium: where this occurs, fenestrae are formed (Abe et al., 1984). Another example comes from pathology, where brain capillaries that normally have a continuous endothelium become fenestrated near metastasized renal carcinoma cells (Hirano and Zimmerman. 1972). Experimental evidence for the capillary-epithelium interactions suggested by these observations is seen in an ultrastructural study of the guinea pig vas deferens and ureter (Campbell and Uehara, 1972). The authors observed fenestrated capillaries only near the epithelium in both organs, and noted that the fenestrae tended to occupy the side of the capillary facing the epithelium. When the mucosa of either organ was transplanted to the anterior chamber of the eye, fenestrated capillaries were seen only in transplants containing epithelium. Capillaries in transplants stripped of their epithelium lost their fenestrae. The epithelium-capillary interactions suggested by these observations are buttressed by the skewing of endothelial fenestrae at these and other sites toward the adjacent epithelium (Cauna and Hinderer, 1969; CasleySmith, 1971: Heriot ef al., 1986; Mancini ef al., 1986). In a morphometric study of rat choriocapillaris, the latter investigators documented the skewing of endothelial fenestrae toward the RPE, offering this as evidence that the RPE influences the polarity of the choriocapillary endothelium.

B. POLARIZATION OF EPITHELIUM TOWARD CAPILLARIES A characteristic of epithelia is their structural and functional polarization. This is striking where the cells form sheets closely apposed to a capillary plexus. At these sites (the W E is a good example: Figs. lA,B) the epithelial cells develop apical and basal specializations, such as the formation of folds and attachment sites on the basal plasma membrane, opposite the choriocapillaris. The epithelium-capillary interaction suggested by this and similar arrangements at other epithelial sheets (kidney tubules and

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FIG.1. (A) Light and (B) transmission electron micrographs of RPE and choriocapillaris of normal rabbit. (A) Outer retina containing somata of photoreceptors in outer nuclear layer (ONL), their inner segments (IS), and outer segments (0s)abutting RPE. Choriocapillaris (C) is also visible. x 800. (B) RPE basal surface (that facing Bruch's membrane, BM) bears numerous folds (F). Endothelium of choriocapillaris ( C ) has extensive plaques of thin cytoplasm (arrows) bearing many fenestrae. x 13,400.

intestines are examples: Casley-Smith, 1971; Rhodin, 1974) gains credence when an epithelium rearranges its structural and functional polarity in relation to changes in the adjacent capillary bed. An example, detailed in Section III,D, is the incorporation of retinal capillaries into the normally avascular RPE. When this occurs the segments of retinal capillaries become inserted between the lateral plasma membranes of RPE cells. This

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normally flat and morphologically undifferentiated membrane develops structural and functional specializations usually restricted to the basal plasma membrane facing the choriocapillaris.

C. EXPLANATIONS FOR EPITHELIUM-CAPILLARY INTERACTIONS Although these observations suggest an interaction between capillaries and epithelium mediated by soluble molecules or the extracellular matrix (ECM; see Section IV), other explanations for observations like those just described have been proposed. Federman (1982) has suggested that altered blood flow dynamics, such as perfusion pressure, could elicit the change in distribution of choriocapillary fenestrae observed at choroidal melanomas. Abe et al. (1984) believe that the fluid contents of the lumen of the epididymis may be responsible for changes in peritubular capillary fenestrations seen after ligation of the efferent duct of the mouse. In neither case is a signaling mechanism proposed, whereas there is abundant evidence that the ECM or soluble molecules from epithelium or endothelium can cause such changes.

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It has been suggested that the structure of ocular blood vessels “is dictated by the demands of the tissues, and that a change in tissue demand can lead to a change in vessel morphology” (Bellhorn, 1980, p. 328; see also Davson, 1979). This idea has served as a fruitful theme for animal experimentation and the interpretation of human ocular histopathology (e.g., Federman. 1982). It has become an especially attractive idea as evidence documenting dynamic changes in capillary structure and function in response to specific stimuli has accumulated outside the eye and filtered into the field of eye research. Examples are ( I ) the propensity of continuous capillaries to form fenestrae in thrombocytopenia and psoriasis and to lose them upon steroid treatment (Kitchens and Weiss, 1975; Kitchens, 1977; Braverman and Yen, 1977); (2) the increased permeability, due to increased fenestrations, of periovum capillaries prior to ovulation (Okuda et al., 1983); (3) the induction of fenestrae in nonfenestrated capillaries when exposed to a tumor promoter in v i m (Lombard et al., 1986); and (4) the formation of fenestrae by vaginal capillaries in rats treated with estrogen (Wolff and Merker, 1966). These observations bear out the suggestion, made by Bennett et al. (19591, that “capillary endothelial cells may be labile and may change structural characteristics under the influence of circumstances such as anoxia, or under the influence of various reg-

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dating or pharmacological mechanisms” (p. 389). Observations linking numbers of capillary fenestrae with the status of a proximate epithelium, as at guinea pig ureter and vas deferens (Campbell and Uehara, 1972; see Section II,A) or at the RPE and choriocapillaris (see Sections III,C and D), suggest that epithelia may be one of the regulatory mechanisms alluded to by Bennett et al. (1959). A. ORGANIZATION OF THE RPE-CHORIOCAPILLARIS INTERFACE The RPE-choriocapillaris interface consists of a sheet of cuboidal epithelial cells (the RPE) separated from a planar sheet of capillaries (the choriocapillaris)by a connective-tissue lamina called Bruch’s membrane (Fig. lA,B). This composite structure of epithelium, capillary bed, and interposed connective-tissue lamina is coextensive with the neural retina. The RPE, choriocapillaris, and Bruch’s membrane can be considered a structural unit, whose function is to ensure photoreceptor nutrition (Leeson, 1968; Henkind and Gartner, 1983). This concept has been slighted, however, as emphasis in research and pathology has focused on the interface between the RPE and photoreceptors. The impetus for this arose in part from evidence that the RPE serves as an “organizer” of the neural retina during development, aberrations in the RPE resulting in atrophy of the adjacent neural retina, the photoreceptors in particular (Silverstein el al., 1971; Hollyfield and Witkovsky, 1974; Randall et al., 1983). The RPE influence on photoreceptors may work in mature retina as well; the loss of a proximate RPE has been offered as an explanation for the photoreceptor atrophy seen in retinal detachments, where the neural retina splits away from the RPE (Kroll and Machemer, 1968). It has been suggested (among other possibilities) that the influence of RPE on photoreceptors is manifested by an RPE-derived factor, such as a component of the interphotoreceptor matrix (a mucopolysaccharide-richsecretion of both RPE and photoreceptors that occupies the tissue space between them) or an upset in the vitamin A cycle (Kroll and Machemer, 1968; LaVail, 1979; Porrello and LaVail, 1986). Observations from human histopathology and animal experimentation have identified the RPE-choriocapillaris interface as a locus of pathology in some important chorioretinal diseases, such as age-related macular degeneration and retinitis pigmentosa (Gartner and Henkind, 1982; Young. 1987). This has rekindled interest in a suspected trophic influence by RPE on choriocapillaris mentioned as early as 1937 by Mann but never adequately documented. This author later stated (Mann, 1950) that the “choroidal net,” or choriocapillaris, “seems to develop wherever mesoderm is in contact with pigmented epithelium. It appears pari passu with the

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pigment and seems to be in some way related to this, in that if for any reason pigment is absent over an area of the surface of the optic cup, the choroid is absent also” (p. 38). Numerous later histologic observations on the developing eye have documented this correlation in the chick, monkey, human, and rat eye (O’Rahilly, 1%2; Berson, 1%5; Leeson, 1968; Braekevelt and Hollenberg, 1970; Heimann, 1972; Mund et al., 1972; Endo and Hu, 1973; Takei and Ozanics, 1975; Ozanics et al., 1978). In several of these studies the parallel maturation of RPE and choriocapillaris-another manifestation of their proposed interaction (see Section III.C)-is evident in the illustrations, although the authors do not focus on it (Leeson, 1968; Braekevelt and Hollenberg, 1970; Takei and Ozanics, 1975; Ozanics et al., 1978). Other investigators have, however. In a study of rat intestinal capillary development (Milici and Bankston, 1981), a similar tandem maturation of capillary and adjacent intestinal epithelium is described, such that endothelial fenestrae form and become localized toward the epithelium as it matures. The authors note: “Our results may indicate that the maturation of the overlying epithelium is important in the formation of fenestrations” (p. 441). As seen later (Section lll,C), this is true at the RPE-choriocapillaris interface as well.

B. OBSERVATIONS FROM HUMAN HISTOPATHOLOGY Circumstantial evidence that there is an interaction between the RPE and choriocapillaris comes from histopathologic examination of human eyes obtained at autopsy or enucleation. Mann (1937) pointed out that in colobomata (a congenital eye defect in which the choroid fissure fails to close, leaving a gap in the wall of the eye) the atrophy of neural retina and choroid are coextensive with areas where RPE is absent. More definitive observations are those of Sarks (1979, 1980), made on >500 eyes. She observed a correlation between the geographic, or focal, loss of RPE and atrophy of the adjacent choriocapillaris in eyes with age-related macular degeneration. Her conclusions are supported by similar observations on eyes from patients with other conditions, such as retinitis pigmentosa (Gartner and Henkind, 1982; Henkind and Gartner, 1983; for discussions, see also Korte et al., 1984a; Young, 1987) and fundus flavimaculatus (Eagle et 01.. 1980), diseases characterized by progressive RPE degeneration. Similar correlations between RPE and choriocapillaris status have been made in animal models of some ocular diseases, such as a model of gyrate atrophy (a metabolic disease of patients with hyperornithinemia: Takki, 1974) in which rats or monkeys receive intravitreal injections of ornithine hydrochloride (Kuwabara et al., 1981). These studies support a correlation between RPE and choriocapillaris damage-specifically, that choriocap-

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illaris depends on RPE for its survival. Experimental animal models for elucidating this possibility and RPE-choriocapillaris interactions overall were found in rabbits with a sodium iodate-induced retinopathy and in rats that were exposed to excess fluorescent light or dosed with urethane.

c. SODIUM IODATE RETINOPATHYIN RABBITS The retinotoxic effects of iodate were an unfortunate discovery of the 1920s, when intravenous iodine solutions that were used for treating septicemia also caused blindness (Sorsby, 1941). Subsequent studies determined that the cause of the blindness was iodate metabolically derived from the iodine, although the mechanism of iodate’s toxic effect on RPE in particular, and photoreceptors as well, is still not clear (Sorsby, 1941; Potts, 1980).The use of sodium iodate as a research tool began in earnest in the 1950s, when it was noted that intravenous injections of sodium iodate elicit the degeneration of RPE and photoreceptors in cats, rats, rabbits, and other mammals (Noell, I95 1, 1953). Subsequent ultrastructural studies detailed this necrosis, but the choriocapillaris response was not examined other than to note a loss of its fenestrae (Ringvold rt al., 1981). Given the geographic nature of the RPE response, in which areas of RPE can be spared (Flage, 1983; Korte et al., 1984b), it may be expected that only choriocapillaris adjacent to sites where RPE was destroyed would show ultrastructural evidence of atrophy (e.g., loss of fenestrae, thickening of endothelium, necrosis of endothelium)if the relation suggested by ocular histopathology (Section III,B), development (Section III,A), or observations on extraocular tissues (Sections II,A and B) is true. Upon injecting pigmented rabbits and rats intravenously with sodium iodate (sodium iodate is ineffective in albino animals: Sorsby, 1941), a striking geographic correlation was observed between RPE destruction and choriocapillaris atrophy (Korte et al., 1984b, 1986b), illustrated in Fig. 2 and 3. Thin sections taken where RPE was necrotic or destroyed and replaced by scar tissue (Figs. 2B and 3B) showed that choriocapillaris endothelium had thickened and lost its fenestrae. Necrotic endothelial cells were also observed (Korte et al., 1984b).Their removal accounts for the conspicuous atrophy of the choriocapillaris observed in vascular casts examined by scanning electron microscopy (SEM; cf. Fig. 3C and D). Where RPE was spared the adjacent choriocapillaris remained normal in ultrastructure. The sparing of choriocapillaris adjacent to unaffected RPE indicated that the capillary response was not due to a direct effect of iodate on its endothelium, a possible artifactual complication. This was corroborated by observations showing no effect by sodium iodate (at the dosage used in the experiments) on aortic endothelium:

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FIG.2 . ( A ) Light and (B) transmission electron micrographs from area of damaged RPE, 2 days after intravenous injection of sodium iodate into a pigmented rabbit. ( A ) RPE is flattened and depigmented. Choriocapillaris (C) appears normal, but shows ultrastructural changes, as seen in (B). ~ 6 6 0(.B ) Choriocapillaris endothelium adjacent to damaged RPE (cf. normal RPE and choriocapillaris in Fig. 1B) shows early signs of atrophy. such as loss of fenestrae to produce extensive zones of thickened cytoplasm (arrow). Other than some are within normal range separation of disk membrane, photoreceptor outer segments (0.5) of ultrastructural preservation. x 13,400.

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1. Intravenously injected Evans blue (a dye that binds to albumin and acts as a probe of vascular permeability) did not leak into the aortic intima, a test site for endothelial integrity. 2. Interendothelialjunctional complexes were intact and necrotic cells were not observed upon transmission electron microscopic (TEM) examination of the aortic endothelium. 3. SEM of the aortic surface revealed no evidence of endothelial cell loss.

Nor was choriocapillaris atrophy a response to photoreceptor damage (sodium iodate also damages photoreceptors: Noell, 1951, 1953). As gauged by both electrophysiologicand ultrastructural criteria, the RPE response begins within hours of iodate administration (Potts, 1980; Anstadt et af., 1982kpnor to a photoreceptor response (e.g., Fig. 2A and B). Also, when photoreceptors but not RPE are damaged (which does occur in some iodate-dosed rabbits and can be produced purposely in rats by exposing them to fluorescent light or urethane: see Section IlI,D), the choriocapillaris remains normal in appearance. These observations led to a hypothesis that RPE influences the structure and function of choriocapillaris(Henkind and Gartner, 1983; Korte et al., 1984b), and supported the observations of Bellhorn and co-workers on rats in which retinal capillaries were experimentally brought into apposition with RPE (Bellhorn et af., 1980). Their observations (see Section III,D) led them to conclude that “a factor(s) within the retinal pigment epithelial layer determines the morphology of vessels within their environment” (p. 584). The loss of fenestrae by atrophic choriocapillaris suggested that these structures and at least one function-permeability-are influenced by WE. Evidence for this was obtained when rabbits that had received sodium iodate were injected intravenously with the vascular tracer horseradish peroxidase (HRP) prior to euthanasia (Korte et al., 1987). Choriocapillaris profiles adjacent to spared RPE retained their fenestrae, and their normal permeability to HRP. Atrophic choriocapillaris that had lost its fenestrae retained the tracer in its lumen (Fig. 4). Ohkuma and Ryan (1983) also made a correlation between HRP permeability and the degree of endothelial fenestrations at experimentally induced subretinal neovascularizations in the monkey. The influence of RPE on choriocapillaris structure and function suggested by these observations was also seen during their subsequent regeneration (Korte ef al., 1987). Starting - 1 week after administration of iodate in rabbits, the RPE begins to regenerate from the edge of spared RPE. Light-microscopic examination of series of sections of paraffin- and plastic-embedded tissue, augmented by adjacent thin sections, revealed

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that choriocapillaris regeneration paralleled RPE regeneration (Fig. 5A and B). The advancing edges of the regenerating RPE and choriocapillaris were approximately in register in sections, as may be predicted from observations on the developing eye (Mann, 1937, 1950; Heimann, 1972; see Section 111,A). Some regenerating capillary profiles that had advanced beyond the edge of the regenerating RPE showed evidence of secondary atrophy, such as endothelial necrosis. One interpretation of this observation is that the regrown capillaries were dying back where they outranged a trophic influence by the RPE,resulting in the striking geographic match between RPE and choriocapillaris observed in the late stages of the retinopathy (Korte et al., 1984b), and illustrated in Fig. 3A. Since the response of the choriocapillaris endothelium was examined over the course of its atrophy and regeneration, the attendant ultrastructural changes seemed related to loss and subsequent re-formation of cellular polarity in the choriocapillaris, apparently in response to the presence of the adjacent epithelium. Normal choriocapillaris has its fenestrations preferentially located on the side of the endothelial tube facing the RPE, as seen in Fig. 1B. When RPE is destroyed, this polarity disappears as the endothelium thickens and fenestrae are lost (Figs. 2B and 3B). During regeneration of the choriocapillaris, however, this polarity returns once more where the capillaries lie adjacent to newly formed RPE (cf. Fig. 5B and C). Ultrastructural examination of the endothelium of regenerating choriocapillaris showed a discrete series of changes leading to this. First, isolated fenestrae were formed, with no particular localization about the formative endothelial tube. With maturation of the capillary, clusters of

FIG.3. Atrophy of choriocapillaris as seen in sections (A,B) and in vascular casts examined by SEM (C,D). (A) Light micrograph obtained 1 1 weeks after iodate administration, showing border between zone of spared retina (right of arrow) and atrophic retina (left of arrow). RPE stops near arrow and is replaced by retinal scar tissue (S).Choriocapillaris (C) adjacent to scar is atrophic (detailed in B) as compared to that adjacent to spared RPE at right. x390. (B) Transmission electron micrograph of choriocapillaris from area to left of arrow in A, showing advanced capillary atrophy. Endothelium is thickened and bears no fenestrae. RPE has been replaced by retinal scar tissue (S). BM denotes Bruch's membrane. x 13,400. (C) Scanning electron micrograph of vascular cast (viewed from retinal side) of choriocapillaris at far periphery of rabbit that received sodium iodate 6 days prior to euthanasia. Choriocapillaris here is an extensive network of spared capillaries, and corresponds to ones like that to right of arrow in (A). Arrows denote peripheral edge of choriocapillaris. which stops abruptly near base of ciliary body, above. ~ 4 0(D) . Vascular cast more centrally in same specimen as in (C), showing border between spared choriocapillaris (at upper left) and zone of capillary atrophy at lower right; atrophy exposes underlying venules, V. This picture corresponds to zones where spared RPE and choriocapillaris border zones of atrophy or scar formation (e.g., at arrow in A). ~ 6 6 .

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FIG.4. Transmission electron micrograph of atrophic choriocapillaris adjacent to retinal scar (S) of rabbit euthanized 5 weeks after administration of sodium iodate. The animal received HRP prior to euthanasia. Due to thick. unfenestrated endothelium (one manifestation of atrophy). the capillary retains tracer reaction product (black) in its lumen: normally the endothelium is thin and fenestrated, making it permeable to peroxidase (cf. Figs. IB and 6A). Bruch's membrane (BM) is thickened due to deposition of connective tissue, which occurs coincident with capillary atrophy. x 17.000.

FIG.5. Tandem regeneration of RPE and choriocapillaris in rabbits that received sodium iodate at varying times prior to euthanasia. (A) By light microscopy. regenerating RPE consists of flattened cells. with some mitoses evident (arrow). Adjacent choriocapillaris (*) is also . Immature RPE and enregenerating. as seen in (B). Seven days after iodate. ~ 8 5 0 (B) dothelium of choriocapillaris (C) as seen by TEM from area like that seen in ( A ) . KPE as yet lacks basal specializations such as folds (cf. Fig. IB). Choriocapillaris has small plaques of thin. fenestrated cytoplasm (arrows) scattered about its perimeter. BM denotes Bruch's membrane. which contains a portion of a monocyte, M. x 10.000. (C) Regenerated choriocapillaris adjacent to regenerated RPE, in tissue obtained I I weeks after iodate administration. New capillaries are ensheathed in remnant basement membrane (encircled) and bear extensive plaques of thin. fenestrated cytoplasm (arrows) polarized to the side facing the RPE. unlike less mature capillary profiles (cf. part B). x 7000.

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fenestrae became associated with small plaques of thinned cytoplasm, still scattered, however, about the endothelial tube (Fig. 5B). Eventually, as the capillary reached maturity, these plaques of thin, fenestrated cytoplasm enlarged and became concentrated on the side of the endothelial tube facing the regenerated RPE (Fig. 5C). Where regenerating choriocapillaris exceeded the edge of regenerating RPE, the new endothelial tube remained unpolarized in its fenestrations and eventually atrophied (Korte et al., 1987). This contributed to the “end-stage” retinopathy, in which areas where RPE had not regenerated were occupied by a dial scar, the adjacent remnant choriocapillaris consisting of atrophic capillary profiles embedded in a dense, collagenous connective tissue (Korte et al., 1984b, 1986b). A similar correlation between retinal scar formation and choriocapillaris atrophy is seen in rats with photothermal or phototoxic retinopathies (Kuwabara, 1979; Burns et al., 1986). These observations suggest that choriocapillaris responds to the presence of RPE. They obscure, however, an equally important response on the part of the RPE to choriocapillaris. This response was documented in rats that were exposed to fluorescent light or urethane, treatments that selectively destroy the photoreceptors and cause retinal capillaries to become embedded in the RPE-a common response when photoreceptors are lost in the rodent retina (LaVail, 1979). Observations on these intraepithelial capillaries clarify those obtained in iodate rabbits and rats (Korte el d., 1984b. 1986b) by showing that the situation at the RPEchoriocapillaris interface is one of interactions between these components, and not merely the trophic influence of RPE on the capillary.

D. INTRA-RPECAPILLARIES IN RATS When young rats are exposed to fluorescent light or receive subcutaneous injections of urethane, the photoreceptors atrophy and are lost several months later (O’Steen et al., 1972; Bellhorn ef al., 1973, 1980; Shiraki et al., 1982). When and where this occurs, retinal capillaries, normally separated from the RPE by the photoreceptors, become inserted among the RPE cells. These foci of intraepithelial capillaries are excellent sites at which to examine the response of capillaries to an epithelium, and vice versa. The structural and functional characteristics of the intraepithelial capillary segments can be compared to their parent capillaries remaining in the neurosensory retina. These latter are of the continuous type, having a thick, nonfenestrated endothelium that is impermeable to intravenously injected HRP. Bellhorn and co-workers have published a series of articles documenting the structural and functional transformations in these capillaries when they become embedded in the RPE, as well as the response

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of RPE cells to them (Bellhorn ef al., 1973, 1980; Kritzinger and Bellhorn, 1982; Shiraki el al., 1982; Bellhorn and Korte, 1983; Korte et al., 1983, 1984a, 1986a). The endothelium of the intraepithelial capillary segments changes its structure and function (Figs. 6, 7). It thins, develops fenestrae, and becomes permeable to intravenously injected HRP (Korte ef d., 1983, 1984a). These observations buttress the notion that RPE influences choriocapillaris structure and function, for the capillaries are normally fenestrated and permeable to HRP. Retinal capillaries are normally unfenestrated and impermeable to HRP (Fig. 7D). In the course of these investigations it became obvious that the RPE also responded to the presence of the retinal capillary. The RPE cells abutting the segments of capillaries embedded in the epithelium rearranged the structural and functional polarity of the basal surface, which is normally oriented toward the choriocapillaris (Korte et af., 1986a). This was most evident where capillary segments were interposed between the normally flat, undifferentiated lateral plasma membranes of RPE cells (Fig. 7A-C). This lateral membrane developed the attachment sites, infoldings, and tubules normally restricted to the basal plasma membrane facing the choriocapillaris (Miki et al., 1975; Korte, 1984; Korte and Goldberg, 1986). It also assumed two functions it normally does not have: secretion of basement membrane and endocytosis. The latter function was of particular interest, because coated pits are rare on the lateral plasma membrane of rat, rabbit, and human RPE cells, being restricted to the basal and apical plasma membranes (Orzalesi ef al., 1982; Perlman et al., 1984). However, coated pits (where endocytosis occurs) are frequent on the lateral plasma membrane facing an intraepithelial capillary, and numerous HRP-labeled coated vesicles are observed in the adjacent cytoplasm when this tracer is administered (Korte ef af., 1986a). The reorganization of the RPE cell’s polarity toward these intraepithelial capillaries suggests that, in the normal eye, the choriocapillaris exerts a similar influence on the RPE, inducing in it several structural and functional specializations that give the RPE its polarity. This could have important functional implications, since the RPE is the “gate” separating two important tissue spaces: that between the RPE apical plasma membrane and the photoreceptors, and that between the choriocapillaris and the RPE basal plasma membrane. The exchange of ions and molecules such as vitamin A between these compartments is controlled by the RPE. This transport is directional-perhaps due to the polarizing influence of choriocapillaris on RPE plasma membrane constituents or the cytoskeleton. Conversely, transport across choriocapillaris must be polarized due to the influence RPE has on the numbers and distribution of endothelial fenestrae. That proximate cells can affect

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endothelial permeability is seen when glial cells influence the directionality (i.e., polarity) of transport across brain capillaries (Beck et al., 1984), although the effect is on the molecular constitution of the plasma membrane, and not due to changes in endothelial fenestrae.

E. RELATEDOBSERVATIONS DERIVEDFROM ANIMAL EXPERIMENTATION Corroborative evidence for the notion that choriocapillaris and RPE interact comes from several quarters. A morphometric study of normal rat RPE cells shows that infoldings of their basal plasma membrane are most extensive opposite a capillary profile; the basal plasma membrane spanning the tissue space between choriocapillaris profiles has less infolding (Heriot et al., 1986). Other morphometric observations in rats with a spontaneous hypertensive retinopathy documented the polarization of choriocapillaris endothelial fenestrae toward the RPE and showed a decrease in fenestrations and increase in endothelial thickness with increasing distance from the RPE (Mancini et al., 1986). Moreover, when RPE cells begin to migrate across the retina (a common phenomenon in retinal disease), they maintain their normal structural polarity where apposing a retinal capillary or the internal limiting membrane (the basement membrane that lines the vitreal surface of the retina and is probably secreted by the Miiller glial cell), as in rats with a hypertensive retinopathy (Frank and Mancini, 1986). This suggests that basement membranes and, more broadly, the ECM, contribute to the control of RPE-choriocapillaris interactions.

FIG.6 . RPE and intraepithelial capillaries from rats exposed to excessive fluorescent light or urethane, which selectively destroy the photoreceptors. The electron micrographs are taken from rats that received HRP I5 minutes prior to euthanasia. (A) When photoreceptors are lost, inner retina (INL denotes inner nuclear layer) encroaches on RPE. RPE appears normal; numerous folds (F) face Bruch’s membrane (BM) and choriocapillaris (C), and apical villi (V) adorn opposite side of the cells. Black reaction product of peroxidase stains Bruch’s membrane and outlines folds due to escape from choriocapillaris (its lumen appears clear due to perfusion fixation). x 5000. (B) Where intraepithelial retinal capillaries (L, lumen) occur. folds (F) form on RPE lateral plasma membrane where it faces the capillary. The capillary endothelium develops fenestrae (encircled) that permit intravenously injected HRP to penetrate into pericapillary space. Parent segments of retinal capillaries in underlying neural retina still retain peroxidase (cf. Fig. 7D). V, apical villi of RPE. BM, Bruch’s membrane. ~ 8 3 0 0 .

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IV. Mechanisms of RPE-Choriocapillaris Interactions The idea that cells of different types influence each other is not new. Consider, for example, the massive literature on epithelium-mesenchyme interactions (see Bissell et al., 1982). The putative inductive influence by W E on ocular mesenchyme (Mann, 1937, 1950; Reinbold, 1%8; Newsome, 1976) probably falls into this category of interaction. There is much more debate, however, on the intermediary of such cell-cell interactions. Abundant evidence indicates that basement membranes, and ECM generally, influence cell structure and function, in part via the cytoskeleton (Bissell et al., 1982; Hay, 1983). The importance of basement membranes in controlling tissue organization at levels beyond just the structural and functional polarity of individual cells has been documented as well (Vracko, 1974; Montesano et al., 1983a,b). In so far as it has been shown that RPE and endothelium (though not choriocapillaris endothelium) are responsive to ECM components (Mandelcorn et al., 1975; Crawford, 1983; Madri et al., 1983; Montesano el al., 1983a,b; Vidaurri-Leal et al., 1984; Milici et al., 1985; Herman, 1987), we may propose the ECM as an intermediary in RPE-choriocapillaris interactions described in Section 111. However, evidence is accumulating that soluble factors released from RPE and choriocapillaris endothelium also contribute to the “status quo” at Bruch’s membrane (Campochiaroand Glaser, 1985b; Glaser er al., 1985); that is, these cells exert paracrine effects on each other in a way similar to the vascularization of corpus luteum in response to the basic fibroblast growth factor of the luteal granulosa cells (Gospodarowicz et al., 1987) or the control of pancreatic islet secretion via local hormonal effects (Bauer, 1983). It is also possible that RPE-choriocapillaris interactions are controlled by the ECM and soluble factors acting in concert-for example, the ability of hematopoietic growth factors (Gordon et al., 1987)

FIG. 7. Details of (A-C) intraepithelial capillary endothelium and RPE lateral plasma membrane facing it; and (D) retinal capillary. (A) RPE lateral plasma membrane facing intraepithelial capillary (E, its endothelium) forms rudimentary folds (arrows) that are outlined by peroxidase reaction product. x 35,000. (B) Fenestrae (arrows) in endothelium of intraepithelial capillary (L, lumen). Lateral RPE plasma membrane (to right) has not formed folds but has formed attachment sites (encircled), which are normally restricted to the basal plasma membrane. x 31,OOO.(C) Fenestrae (encircled) in endothelium of intraepithelial capillary (L, its lumen) permit passage of HRP, contributing the black reaction product in pericapillary space. RPE lateral plasma membrane above bears two coated pits (arrows), which are normally restricted to the basal or apical plasma membranes. ~ 3 1 , 0 0 0 .(D)Capillaries in neural retina are unfenestrated and retain HRP, as seen by restriction of black reaction product to lumen (L). x 17,000.

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or the fibroblast growth factors (Gospodarowicz et al., 1987) to bind to ECM components and maintain their bioactivity. An increase or decrease in the binding of a molecule derived from the RPE, choriocapillaris endothelium, or other cells such as macrophages, by the ECM separating the WE and the choriocapillaris, could create the geographically localized milieu linking secondary changes in RPE or choriocapillaris endothelium to primary changes in one of them.

A. EXTRACELLULAR MATRIX in Section III,D it was noted that W E cells orient themselves in relation to basement membranes (Mandelcom et a / . , 1975; Frank and Mancini, 1986). The ECM components responsible are not known with certainty, although several investigators have attempted to identify them. In one study it was shown that RPE cells in culture could reorient their polarity when a serum-soaked filter was placed on top of them-that is, over their apical surface (Crawford, 1983). The apical surface lost its structural specializations, such as villar projections, and transformed into a basal surface that secreted new basement membrane material. It has been shown that type 11 collagen (the collagen of vitreous) causes cultured RPE cells to lose their polarity and become migratory. They transform into fibrocytelike cells similar to those in the intravitreal “membranes” of human proliferative vitreoretinopathy (Vidaurri-Leal et a / . , 1984). (These membranes are derived in part from RPE cells that migrate across the retina and onto its vitreal surface, or are exposed to vitreous at a retinal tear.) As rudimentary as these observations are, they support the idea that RPE can respond to ECM and basement membrane components. Also, RPE can probably be influenced by a proximate capillary basement membrane, as evidenced by the maintenance of structural polarity when RPE cells arrange themselves along retinal capillaries (Frank and Mancini, 1986; Korte et al., 1986a). There is no information on the involvement of ECM components in the choriocapillaris responses to RPE damage (e.g., the loss of endothelial fenestrae with W E loss), or their re-formation and repolarization adjacent to regenerating RPE (see Sections III,B,C, and D). RPE contains or releases several of the candidate molecules to which endothelium responds, such as type 1V collagen, fibronectin, glycosaminoglycans, and the basic fibroblast growth factor (Turksen et al., 1985; Pino, 1986; D’Amore et ul., 1987; Herman, 1987; Schweigerer et al., 1987; Stramm, 1987). The response of the choriocapil/ary endothelium to these molecules is not known. Other endothelia, however, do interact with these ECM com-

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ponents, as with the formation of tubular structures when rat epididymal fat pad endothelial cells are cultured on basement membrane (as opposed to interstitial) collagen (Madri et al., 1983; see Herman, 1987, for review), and the induction of fenestrae in cultured endothelial cells by the basement membranes of epithelial cells (Milici et a / . , 1985).

B. SOLUBLE FACTORS For years, ill-defined “factors” have been implicated in the biologic economy of the eye and the genesis of ocular disease, especially those involving abnormal blood vessel growth (Henkind, 1978; Glaser et al., 1980; Gamer, 1986). Some sense is being made of these with the realization that disparate factors such as the retina-derived and eye-derived growth factors are both really the acidic fibroblast growth factor (Baird et a / . , 1986; Gospodarowicz et a / . , 1987). The acidic and basic fibroblast growth factors have been characterized (D’Amore et al., 1981 ; Gospodarowicz et a / . , 1986, 1987) and, although their distribution in the retina remains undefined, their influence on blood vessel growth in situ, and thus neovascularization, is suspected. For example, the basic fibroblast growth factor occurs in cultured bovine RPE and stimulates cell division in cultured bovine adrenocortical endothelial cells (Schweigerer et al., 1987). The retina-derived growth factor (i.e., acidic fibroblast growth factor: Gospodarowicz et al., 1987) elicits neovascularization in the cornea (Gospodarowicz et al., 1979) and loss of stress fibers and migration in cultured adrenocortical endothelium (Herman and D’Amore, 1984). Such changes would probably be manifestations of this factor in the retina in situ, which, however, remain undocumented (see Gamer, 1986, for review). Basic fibroblast growth factor, which stimulates the proliferation of endothelial cells, is expressed by bovine RPE cells in culture (Schweigerer et al., 1987). This raises the possibility that RPE cells in situ can produce an autocrine and paracrine factor capable of regulating growth of nearby endothelium and RPE cells. Studies in vitro have suggested other factors working at the RPE-choriocapillaris interface: an inhibitor of endothelial growth released by RPE, and a chemoattractant for RPE that is released by endothelial cells (Glaser et a / . , 1985; Campochiaro and Glaser, 1985b). In the study by Glaser et a / . (1983, culture medium “conditioned” by the growth of human RPE cells caused the regression of new blood vessel growth when applied to chick embryonic yolk sac; it also inhibited mitosis of cultured fetal bovine aortic endothelium exposed to a mitogenic extract of adult bovine retina. The factor(s) involved, as yet uncharacterized or isolated biochemically, could suppress choriocapillaris-derived neovas-

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cularization ( I ) in the normal eye and (2) at sites where neovascularization occurs subsequent to RPE damage but is then suppressed with RPE regeneration. The study by Campochiaro and Glaser (1985b) extends the notion that RPE inhibits capillary growth. It rests on the observation that RPE tends to surround choriocapiUaris-derived neovascularizations that erode through Bruch’s membrane and begin to invade the retina, as if attracted to the new capillaries by their endothelium. They observed that cultured fetal bovine aortic endothelium produces a protein that, in the Boyden chamber assay, acts as a chemoattractant for human RPE cells. Thus, choriocapillaris-derived neovascularizations that cross Bruch’s membrane may attract RPE to them; and the RPE may then inhibit the growth of their endothelial ceUs and suppress the neovascularization. It has been suggested that the RPE that surrounds neovascularizations in situ influences endothelial characteristics other than growth-for example, numbers of fenestrae, and thus permeability (Ohkuma and Ryan, 1983).This possibility is supported by the abundant histopathologic evidence cited earlier (Sections I1,A and II1,C and D). Several considerations complicate interpretation of these in vitro studies. The experiments do not give the results predicted by histopathologic and experimental observations in situ (e.g., see Section 111,C). The studies from animal experimentation and human histopathology show that loss or damage of RPE is followed by choriocapillaris atrophy (see Sections IU,B-D). They are part of a body of observations that supports the idea that RPE is an “organizer” of the chorioretinal interface not only during development (see Section III,B) but in the mature eye as well. Primary damage to RPE by many means, both experimental and during disease, is followed by atrophy of the adjacent choriocapillaris and the adjacent photoreceptors (e.g., Green and Key, 1977; LaVail, 1979; Eagle e l al., 1980; Sarks, 1980; Kuwabara et al., 1981; Ishibashi et a / . , 1986; John et al., 1987). Yet, the in vitro observations cited earlier would lead to the opposite prediction, that is, that loss of RPE would be followed by choriocapillaris growth. [However, one observation has been published that does predict this; in rats whose RPE was selectively damaged by controlled light exposure the adjacent choriocapillaris began sprouting (Heriot et a / ., 1984).] Clearly, additional observations are needed to explain the gamut of RPE-choriocapiIlaris interactions seen in situ and suggested by in vitro observations. A major problem in interpreting the in vitro observations is the technical inability to use choriocapillaris endothelium in culture experiments, although advances are being made in this direction (e.g., Morse et a / . , 1987). This is an important requirement in light of observations that the endo-

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thelium of capillaries and large vessels, or the endothelium of the same class of vessel in different organs or sites, differs in its biochemical and growth characteristics (e.g., the expression of organ-specific antigens: Auerbach et al., 1985). An example of the conflicting observations that can arise in this respect is the observation by Boulton et al. (1987) that human WE-conditioned culture medium has a mitogenic effect on cultured bovine retinal capillary endothelium and on isolated capillaries maintained in vitro. Glaser et al. (1985), however, observed an inhibitory effect by human RPE-conditioned medium on a known mitogen (an extract of adult bovine retina) for cultured bovine fetal aortic endothelium. Other investigators have identified the basic fibroblast growth factor in cultured bovine RPE-a factor with documented mitogenic effects on endothelia (Schweigerer et al., 1987). Repetition of these experiments using RPE and choriocapillaris endothelium from the same species will be most instructive. Further disarray arises from the growing appreciation that either side of the RPE-choriocapillaris equation can be influenced by, or can interact with, the cells of the monocyte-macrophage line (e.g., Penfold et al., 1986; Pollack et al., 1986; Burke and Twining, 1987; Rosenbaum et al., 1987). For example, RPE cells release a chemoattractant for monocytes, which transform into the macrophages seen in the outer retina when RPE or photoreceptors are damaged (Rosenbaum et al., 1987; Penfold et al., 1986; Lai and Rana, 1986). These cells, in turn, may produce a host of factors (e.g., prostaglandins, leukotrienes, platelet-derived growth factor, macrophage-derived growth factor) that could influence RPE and choriocapillaris endothelium (e.g., Campochiaro and Glaser, 1985a; BenEzra, 1978; Folkman and Klagsbrun, 1987). Macrophages, for example, can stimulate capillary growth (Polverini et al., 1977; Werb, 1983). The factor responsible, macrophage growth factor, may really be fibroblast growth factor (Baird et al., 1986). The presence of basic fibroblast growth factor in RPE (Schweigerer et al., 1987) and its ability to cause endothelium in vitro to produce plasminogen activator and organize into capillarylike structures (Montesano et al., 1986)--two counterparts to capillary formation in simsuggests that healing and neovascularization at the RPE-choriocapillaris interface may proceed without the presence of inflammatory cells (i.e., macrophages). The vexing complexity that may characterize the relationship between W E and endothelium is enhanced by observations that RPE can transform into cells with the cytologic characteristics of macrophages and fibrocytes (Machemer and Laqua, 1975; Mandelcorn et al., 1975; Mueller-Jensen et al., 1975; Johnson and Foulds, 1977; Vidaurri-Leal et al., 1984; Lai and Rana, 1986). It has been suggested that capillary segments near these types

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of transformed RPE cells behave differently. Pollack rt al. (1987) observed “macrophagic” RPE near neovascular choriocapillaris, and a cytologically different type of RPE covering capillaries undergoing secondary atrophy, in their investigation of laser-induced subretinal neovascularization in rats. Campochiaro and Glaser ( 1985b) have proposed that transformed RPE cells contribute to the chorioretinal scars that form after laser photocoagulation for treatment of neovascularizations, and are correlated with the regression of neovascularization. Clearly, a biochemical balancing act may control events at the RPEchoriocapillaris interface in a way similar to that proposed between the angiogenic retina-derived (or fibroblast) growth factor and an antiangiogenic vitreal factor. Their imbalance has been offered as one explanation for the neovascularization seen in ischemic retina, and which leads to retrolental fibroplasia and proliferative diabetic retinopathy (Michaelson, 1948; Ashton et al., 1954; Henkind, 1978; Glaser et a/., 1980; Lutty et al., 1983). V. Conclusion Abundant evidence from histology, pathology. and animal experimentation indicates that the RPE and choriocapillaris interact. They probably work as a unit that provides for photoreceptor nutrition. A major challenge to our understanding of the biology of the RPE-choriocapillaris interactions will be to determine the relative contributions of ECM components, soluble factors, and phenotypically different types of RPE cells to observations made in situ. Such information will elucidate the role of the RPE-choriocapillaris interface in retinal, especially photoreceptor, physiology and pathology, as well as in new treatments for diseases resulting from RPE damage or destruction, such as efforts to transplant RPE (Gouras ef al., 1985; Lopez et a/., 1987). The latter are ultimately attempts at restoring the normal interactions between RPE, photoreceptors, and choriocapillaris. ACKNOWLEDGMENT Supported by grants from the National Eye Institute. Research to Prevent Blindness, Inc.. and the National Society for the Prevention of Blindness.

REFERENCES Abe. K . . Takano. H . . and Ito. T. (1984). Anar. Rec. 209, 209-218. Anstadt. B.. Blair, N.. Rusin. M . . Cunha-Vaz, J . , and Tso. M . (1982). Exp. Eye Res. 35. 635-662.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 114

Dinoflagellate Sexuality LOISA. PFIESTER Department of Botany-Microbiology, University of Oklahoma, Norman, Oklahoma 73019

I. Introduction Dinoflagellates are protists, the majority of which are photosynthetic. Some live as endosymbionts and others as parasites. They are ubiquitous but are most abundant in marine waters where they are second only to diatoms as the dominant phytoplankters. Dinoflagellates are claimed by protozoologists and phycologists, with each developing their own terminology and system of classification. Dinoflagellates are well represented in the fossil record and are used extensively in dating cores. Thus palynologists have also developed a system of classification and terminology for the fossil forms. Dinophycean literature, therefore, is complicated by the use of three distinct sets of terminology and systems of classification. I will here consider dinoflagellates as algae and where possible give the comparable protozoan or palynological term in parentheses for the trait or phenomenon described. Dinoflagellates were long considered to be either unarmored (i.e., to have a cell covering consisting only of membranes) or armored (i.e., to have structural cellulose or other polysaccharides which form plates within vesicles: Fig. 1). It is now known that there is an unbroken continuum of increasing complexity in the cell covering from the “naked” to the “armored” dinoflagellates. Various terms have been used to refer to this cell covering. In 1970 Loeblich reintroduced the term “amphiesma,” first used by Schutt in 1895, for the dinoflagellate cell covering to replace the term theca which is still used by many phycologists. Netzel and Durr (1984) have introduced the term cell cortex for the cell covering exclusive of the pellicle, a chemically resistant layer, which may be present underneath the cell cortex. The dinoflagellate nucleus is unique in that the chromosomes are condensed during interphase and attached to the nuclear envelope. Histonelike proteins are present but differ in quality and quantity from those of other eukaryotes (Rizzo and Nooden, 1972). Because of their unique nuclear structure they have been called mesokaryotes (Dodge, 1966). 249 Copyright Q 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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LOIS A . PFIESTER

FIG.1 .

Plated theca of frridiniirm wi//ei. C , Cingulum: S, sulcus.

The dinoflagellate cell has two grooves, the cingulum and sulcus, present at some time during their life history. The cingulum encircles the organism while the sulcus is found only on the ventral side from the cingulum to or near the antapex of the cell (Fig. 1). Dinoflagellates have two flagella; one, the longitudinal flagellum, lies along the sulcus extending posteriorly, while the other, the transverse flagellum, is oriented around the cell. A. HISTORYOF STUDIES ON S E X U A L REPRODUCTION

Early dinoflagellate descriptions were based on living or preserved field samples. Only a small percentage of the -1500 named dinoflagellate taxa are even now available in culture. Thus, descriptions of their life cycles found in the older literature are complete to the degree that the author was able to collect frequently andor be fortuitous enough to have collected a population in all stages of its life history. The sexual life histories (Table I) of the 31 dinoflagellates studied thus far require hours to months to complete their cycle (ffiester. 1984; ffiester and Anderson, 1987). Sexual phenomena such as cellular fusion was often mistakenly interpreted as cell division. Hypnozygotes were observed but thought to be a vegetative,

TABLE I REPORTSON DINOFLAGELLATE SEXUAL REPRODUCTION Mode of reproduction"

Reference

Habitatb

Amphidinium carteri Amphidinium klebsii Ceratium cornutum

I I A

Cao Vien (1967, 1968) Barlow and Tnemer (1988) von Stosch (1965)

M M

Ceratium horridurn Crypthecodinium cohnii

A I

von Stosch (1964)

Cystodinium bataviense Glenodinium Iubiniensiforme Gloeodinium montanum

-

ffiester and Lynch (1980) Diwald (1937) Kelley (1988) Kelley and ffiester (1988) Walker and Steidinger

Species

Beam and Himes (1974); Tuttle and Loeblich

Thally

Mode of induction

F

Homo ? Hetero

M M

Homo Homo

Aging cultures Deluted sample N + P limitation; reduced temperature; short day Aging cultures P + N limitation

F F F

Hetero Homo

( 1974)

Gonyaulax nionilata

1

I A I

Hypnozygote

-

M

-

M

-

Increased temperature

-

Decreased food

( 1979)

Gonyaulax lamarensis

A

Turpin et al. (1978); Anderson and Wall

Gymnodinium fungiforme Gymnodinium paradoxum Gymnodinium pseudopalustre

I I I

Spero and Moree (1981) von Stosch (1972) von Stosch (1973)

M F F

A. I

Coats et al. (1984); Anderson et al. (1985) von Stosch (1972)

M

-

M

Homo

( 1978)

Gyrodinium uncatenum Helgolandinium subglobosum

A

Nutrient depletion Aging culture

Hetero

-

Low temperatures; short day; N + P limitation P limitation Aging cultures

TABLE I (continued) Species

Mode of reproduction"

Reference

Habitath

Hypnozygote

Thally

Zingmark (1970); Hofker (1930) von Stosch (1972) Chesnick (lY86); Chesnick and Cox (1987) Pfiester (1975, 1984); Spector ef ul. (1981) Sako et ul. (1984)

M

-

M M

-

Homo, Hetero Homo

F

+

Homo

N deficiency

F

+

Homo

ffiester (1977) ffiester et 01. (1984) ffiester and Skvarla (1980) Sako et a / . (1987)

F F F F

+ + + +

Homo Homo Homo Homo

Watanabe et a / . (1982)

M

7

F F

+ +

Protogonyuulux cutenello Ptychodiscus brevis

ffiester and Skvarla (1979) Pfiester (1976) Morey-Gaines and Ruse (1980) Yoshimatsu (1981) Walker (1982)

+ P-deficient medium N deficiency N deficiency N deficiency N + P-deficient medium N. P limitation; NaHCO? enrichment N deficiency N deficiency

Woloszynskiu upiculutu

von Stosch (1973)

Noctilucu miliuris (syn. scintilluns) Oxyrrhis murinu Peridinium balticium Peridinium cinctim (UTEX 1336) Peridinium cunningtonii Peridinium Peridinium Peridinium Peridinium

garunensc~ inconspicuum limhutum oenurdi

Peridinium trochoideum (Scrippsiella trochoideu) Peridinium volzii Pcridinium willei Polykrikos kofoidi

"A. Anisogarny: I, isogarny. 'F. Freshwater: M . marine.

M

7

Hetero Homo -

M M

+ ?

Hetero Hetero

F

+

Hetero

Mode of induction

Change in food source N deficiency

N

N limitation; cold temperatures; blue light Lower light; N + P limitation

DINOFLAGELLATE SEXUALITY

253

overwintering stage, and were referred to as thick-walled cysts (Biecheler, 1952). In some instances zygotes were described as distinct taxa. There are numerous references to cysts which appear to be descriptions of zygotic stages in the dinoflagellate life cycle (Endo and Nagata, 1984; Owen and Norris, 1985). Many of these zygotes are capable of being fossilized. Palynologists, who use them to date cores, refer to these fossilized zygotes as cysts (Evitt, 1985). In fact, dinoflagellate sexual reproduction was long disputed in the literature until recent years (Grell, 1973). Dinoflagellate sexuality is now well documented and established (Pfiester, 1984; Pfiester and Anderson, 1987). A generalized life cycle appears to be emerging with minor differences between taxa. However, one must bear in mind that as of now the sexual life histories of only 31 of the -1500 taxa have been observed and reported on in the literature. B. GENERALIZED PATTERN OF SEXUALITY To date, sexuality can be induced in most dinoflagellates in culture by lowering or eliminating the nitrogen and/or phosphorus in the culture medium. This may occur in nature when a bloom occurs. That is, nutrients may be lowered in natural waters as they are incorporated into the biomass of a large population. The author has observed fusing cells in many natural collections of dinoflagellate blooms. Most dinoflagellates studied thus far are homothallic, although some heterothallic species have been noted (see Table I). Under conditions of N and/or P limitation vegetative cells divide producing gametes. Such divisions may occur within a single culture flask over a 3-week period. These divisions begin within 24 hours following inoculation into N- and/or P-deficient medium and occur during the dark phase of the light-dark cycle. The latter probably accounts for the fact that sexuality was rarely observed and then rarely accepted until culturing became more common. In most species, gametes fuse laterally and are either naked when fusing or have a very thin theca or cell covering not visible at the light-microscopic (LM) level (Spector et al., 1981). A fertilization tube has been reported in the sulcal region of Peridinium cinctunz (Spector et al., 1981), and in Crypthecodinium cohnii (D. L. Spector, personal communication). This is the same area where von Stosch (1973) observed the “copulation globule” in Gymnodinium pseudopalustre. Fusion takes -45 minutes with cells retaining their mobility. Nuclear fusion occurs before cytoplasmic fusion is complete. Once fusion is completed, the sphere so formed develops the morphology typical of a vegetative cell of that species and a theca becomes visible at the LM level within 24 hours. The zygote is motile at this stage, having two trailing

254

LOIS A. PFIESTER

and two transverse flagella, and is referred to as a planozygote. Depending on the species, the planozygote remains motile for 3-14 days, during which time it enlarges to twice the size of a vegetative cell and becomes warty in appearance. The original theca formed by the young planozygote accommodates cell enlargement by growth in the intercalary bands between plates. The fact that growth is not equal in all intercalary bands accounts for the “warty” appearance (Pfiester and Skvarla, 1979, 1980) observed in Peridinium species. During this motile zygotic phase the theca thickens greatly and the protoplast becomes extremely dark brown in color. Once the planozygote reaches maximum cell size it loses its motility; the protoplast contracts and lightens in color, and one to three large red accumulation bodies form which contain lipids (L. A. Pfiester and F. Schmitz, unpublished research). The cell in this phase is referred to as the hypnozygote (cyst or hypnocyst) (Fig. 2). Its cell wall continues to thicken during dormancy (L. A. Pfiester, unpublished observations on Peridinium spp.). Nuclear stains readily penetrate the planozygote, but once it develops into the hypnozygote (cyst) it becomes virtually impossible to penetrate the dormant cell with fixatives or stains (von Stosch, 1973; Pfiester, 1975).Thus nuclear phenomena such as meiosis have eluded researchers until recently (H.A. von Stosch, personal communication; Coats et ul., 1984; Ptiester et al., 1984). Under culture conditions the hypnozygote (cyst) remains dormant for -3 months. This dormancy can, however, be shortened by subjecting hypnozygotes (cysts) to cold temperatures (4°C) for several weeks, following which they readily germinate (H. A. von Stosch, personal communication). The two meiotic divisions (which will be described in greater detail elsewhere in this paper) are thought to be long separated in time (Hiester, 1984; Ptiester et al., 1984), with the first division occurring in the late planozygotic or early hypnozygotic (cyst) phase and the second meiotic division taking place immediately prior to or following germination. There is commonly one germination product, but some few species such as Crypthecodinium produce more than one. The postzygotic cell retains the large red accumulation body through the first division. 11. Selected Life Cycles The life cycles of the following dinoflagellates are herein described in detail: Peridinium cinctum, Crypthecodinium cohnii, Peridinium inconspictrum, Noctiluca scintillans, Ceratium cornutum, and Gloeodinium montunum. These taxa are either representative of the various life history patterns reported thus far or represent unique descriptions of cellular phenomena associated with the sexual cycle.

DINOFLAGELLATE SEXUALITY

255

FIG.2. Peridinium willei hypnozygote (cyst). Arrow points to endospore wall; asterisk on exospore wall. Oil droplet (od)or accumulation body. N , Nucleus. Reprinted from Timpano and Pfiester (1986).

A. Peridinium cinctum (UTEX 1336)

The sexual life history of P . cinctum, henceforth referred to as UTEX 1336 (Table I), since its identification is in question (Pfiester and Carty, 1985), has been documented by light (Pfiester, 1975), scanning, and transmission (Spector et al., 1981; Pfiester and Skvarla, 1980) microscopy. Thus its sexual life history is the most fully documented of the 31 reported on thus far.

1 . Gamete Formation Gamete formation is induced in UTEX 1336 when exponentially growing vegetative cells are inoculated into N-deficient medium. Under these conditions smaller vegetative cells divide. In N-deficient medium the two cells produced by this division may act as gametes. However, if they are placed in a N-rich environment within 15 minutes of formation they produce a vegetative population that can later be induced to function as gametes. Thus vegetative populations are haploid and homothallic. TEM studies show the parent ceU’s protoplast to be surrounded by three membranes internal to the thecal plates (Spector and Triemer, 1979). Mucilage is deposited between the outer two of these membranes during gamete formation (Spector et al., 1981). Gametes are released by rupture of the parent theca, which appears to occur as the deposited mucilage

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swells with water imbibition. Viewed through the light microscope, these gametes appear naked; however, SEM and TEM observations showed some fusing gametes with definite thecal plates forming a walled cell covering and others covered solely by membranes (Spector e t al., 1981). One probable explanation is that gametes begin developing thecal plates upon formation. Some fuse soon after release from the parent cell, while others may swim about for several hours before fusing. The latter would have partially to wholly formed plates in the thecal vesicles. However, the author has seen obviously walled gametes begin fusing at the LM level, although these gametes have never completed fusion. 2 . Gamete Fusion

Gametes have few chloroplasts as compared to vegetative cells, numerous membrane-bound storage bodies, and many starch grains (Spector et af., 1981). Gamete fusion in UTEX 1336 appears to be lateral at the LM level, but TEM observations (Spector et al., 1981) have shown that it occurs in the sulcal region in an area devoid of reticulate thecal plates, with a fertilization tube initially forming beneath the basal bodies. Both nuclei migrate into the fertilization tube where fusion occurs. Fusing "naked" gametes are surrounded by three membranes. Fusion is completed in -45 minutes, resulting in a spherical zygote. Within another 45 minutes this zygote develops a cingulum and a sulcus.

3 . Pfunoiygotic Stage The planozygote develops a plated theca and is motile for 2 weeks. During this time it swims actively and enlarges to twice its vegetative size. Cell enlargement is accommodated by an increase in the width of intercalary bands between plates. Bands do not enlarge equally, resulting in an increasingly warty appearance as the planozygote increases in size and age (Pfiester and Skvarla, 1980). The planozygote also darkens considerably in color during this motile stage (L. A. Pfiester, unpublished observations). At the end of the 2-week motile phase the protoplast shrinks slightly, rounds up, lightens in color, and develops one or more large red accumulation bodies. These bodies contain long-chain lipids (L. A. Pfiester and F. Schmitz, unpublished research). The cell wall appears extremely thick. At this phase the zygote is referred to as the hypnozygote (cyst). A secondary wall is observed in TEM preparations of planozygotes (Spector e f al., 1981). 4. Hypnozygotic Phase

The UTEX 1336 hypnozygote (cyst) i s much lighter in color than the planozygote. Presumably, storage materials present in membrane-bound bodies throughout the planozygotic cell may account for the dark appear-

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ance. These bodies may fuse forming the red accumulation bodies and result in a tighter cell color in the hypnozygote. The hypnozygote (cyst) remains dormant for 3 months under ordinary culture conditions (Mester, 1975). If it is subjected to cold temperatures for at least 3 weeks, excystment can occur within a week when the cells are returned to 20°C. Researchers have been unable to penetrate the dormant hypnozygote (cyst) with nuclear stains or fixatives for TEM despite repeated attempts and the use of various fixatives and stains (Wiester, 1984). Three cell layers surround the protoplast (Wester, 1975; Timpano and Wiester, 1986): a thick-walled exospore, a thin-walled mesospore, and a thick-walled endospore. The third wall is laid down during hypnozygote (cyst) formation (Spector et al., 1981). Chitin has been reported in the exospore (Wiester, 1975). Spector et al. (1981) suggested that sporopollenin may be contained within the endospore wall. This would account for both its preservation in the fossil record (cysts) and its impervious nature.

5. Meiosis Meiosis per se has not been observed in UTEX 1336. However, the first meiotic division is thought to occur after syngamy as nuclear fusion precedes plasmogamy and because all planozygotes observed are binucleate (Wiester, 1984). The second meiotic division is long separated from the first and occurs immediately prior to or after excystment, a fact supported by the segregation of mating types in Peridiniirm volzii (Pfiester and Skvarla, 1979). Four nuclei have been observed in the single postzygotic cell that germinates from the hypnozygote. This cell retains the large red oil body present in the hypnozygote through its first division. Thus Pfiester (1975) assumed that meiosis occurred either in the hypnozygote (cyst) or in the postzygotic cell, with two or three of the four nuclei aborting.

B. Crypthecodinium cohnii Cryprhecodinium cohnii, a marine dinoflagellate, is hologamous and homothallic. Cells of the same size and morphology usually fuse, but the cell size range is large and unequal. Tuttle and Loeblich (1974) induced it sexually by N and P limitation, but Beam and Himes (1974) reported spontaneous sexuality even in young cultures. Crypthecodinium cohnii has been studied extensively. It has lent itself to genetic studies, since mutants can be obtained and grown on agar. In C . cohnii a fertilization bridge connects the two gametes soon after plasmogamy (D. L. Spector, personal communication). A nucleus from one gamete then migrates to the other nucleus and karyogamy occurs (D. L. Spector, personal communication). The chromosomes in each nucleus appear slightly unwound

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before fusing as they do in Peridinium cincfitm (UTEX 1336) (Spector et a f . , 1981). Once nuclear fusion is complete, the chromosomes rewind and appear as banded structures (Beam and Himes, 1980). The planozygote of C. cohnii is first quadriflagellate, then biflagellate, as are vegetative cells. Tetrad analysis of motility mutants showed that only parental or nonparental combinations of genes were produced. Segregations never resulted in cells containing both (i.e., tetratypes). This means that segregation is performed in the first meiotic division only. The second and sometimes third divisions within the zygote are not segregational and therefore not meiotic (Beam and Himes, 1980). The planozygote encysts immediately when isolated on agar and undergoes meiosis. After 24-36 hours the zygotic wall softens and the division products can be separated and transferred individually to liquid medium. Four progeny are usually produced, but two or eight are sometimes observed from zygotic cysts.

C . Peridinium inconspicuum Sexual reproduction in P . inconspicuum (Fig. 3) is triggered in culture by N limitation (Pfiester et a / . , 1984).Thecate gametes are then produced by mitotic division of small vegetative cells. Gametes shed their theca shortly after fusion begins. Nuclear fusion occurs before plasmogamy begins. Once plasmogamy is completed the resulting spherical zygote develops a plated theca within 24 hours. The armored zygote can be motile, but eventually the cell settles on the substrate and sheds its theca. The immobile zygote becomes spherical, enlarging to a maximum of 20 krn in diameter. An unarmored theca (amphiesma, cell covering) develops which is continuously shed and re-formed every 48-72 hours. During this process the zygote continues to enlarge and elongate. It eventually constricts in the middle, resulting in a peanut-shaped cell that measures 35-40 prn in length. Elongated hypnozygotes (cysts) have also been reported for the marine dinoflagellates Gonyaulax famarensis (Anderson, 1980) and Protogonyaulas catenella (Yoshimatsu, 198I ). Elongated, peanut-shaped zygotes of Peridinium inconspicuum have been observed in Lake Kinneret (Pollingher, personal communication). Previous researchers have been unable to penetrate the dormant hypnozygotic dinoflagellate cell wall with nuclear stains. The same is true concerning the armored thecate zygote of P. inconspicuum. However, once the armored theca is ecdysed, nuclear stains readily penetrate the zygote. Thus, detailed L M details of meiosis can be observed. Meiosis begins in the spherical zygote. The diploid nucleus becomes enlarged and diffuse as the chromosomes further condense. This stage appears to correspond to eukaryotic prophase I. A distinct metaphase is lacking. Without

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FIG. 3. Diagram of meiosis in Peridinium inconspicuum: (a) plated zygote: (b) zygote after ecdysis: (c) first binucleate stage; (d) peanut-shaped cell with two C-shaped nuclei; (e) trinucleate stage; (0 second binucleate stage resulting from cytokinesis of a trinucleate cell; (g) cell with an arrested meiotic product; (h) four motile haploid cells. Reprinted from ffiester ei al. (1984).

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apparent orientation of chromosomes the nucleus begins to invaginate and cytoplasmic channels appear coursing through it. An anaphase I-like stage then occurs. The two daughter nuclei, each with its own membrane, can be seen lying in close proximity. As the zygote begins to elongate the two daughter nuclei appear C shaped, resembling a vegetative nucleus. Each nucleus migrates to opposite poles as cytoplasmic infurrowing begins. The elongated binucleate cell then becomes peanut shaped. Cytoplasmic infurrowing resulting in the formation of two daughter cells is completed -2 weeks following fertilization. One daughter nucleus may divide again before the infurrowing is completed. The second meiotic division thus may be asynchronous, resulting in a trinucleate cell. Once cytoplasmic infurrowing is complete, one cell contains a nucleus which has not yet undergone the second meiotic division while the other cell may contain two haploid nuclei. N o distinct phases similar to eukaryotic meiosis I1 have been observed. Both daughter cells divide again within 24 hours. In one cell only cytoplasmic division takes place, while the other undergoes both cytoplasmic and meiosis I1 divisions. Yoshimatsu (1984) studied excysted hypnozygotes (meiocytes) of Protogonyaulax cutenella and followed the distribution of mating types in chains they developed by meiotic and subsequent mitotic divisions. He found that following the first division one cell would be the plus strain and the other the minus. Plus cells would subsequently divide, always producing other plus cells, as the minus cells would other minus cells. Consequently, in an eight-cell chain one-half of the chain would carry the plus factor, the other half the minus; hence the first meiotic division occurs with segregation of the plus and minus factors. Some variations have been observed: (1) The thecate zygote may be nonmotile: (2) the two daughter cells may form and separate before either nucleus completes the second meiotic division, in which case the trinucleate stage is absent; (3) the arrested meiotic cell may not become motile: (4) the second meiotic divisions may occur in each of the arrested meiotic cells, followed immediately by cytokinesis, resulting in a total of four haploid cells.

D. Noctiluca scintillans Noctiluca scintillans is diploid in the vegetative state. At present it is the only dinoflagellate known in which meiosis is gametic rather than zygotic. Both homothallic and heterothallic (Zingmark, 1970; Hofker, 1930) strains have been reported. Cells first undergo a meiotic division in gamete production, followed by several to many synchronous mitotic divisions, resulting in as many as 1024 mature uniflagellated dinokaryotic gametes. These gametes are attached upon the surface of the parent gametophyte

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cell (Zingmark, 1970). The isogametes appear to be formed and released by a budding process. Gametes fuse with their flattened sides at 90"angles and may remain motile throughout the fusion process. The flagella shorten as they are absorbed into the zygote. The single zygote which survived to maturity during Zingmark's study enlarged from 25 to 200 pm in diameter and developed a coiled tentacle. Zingmark further showed the vegetative nucleus to be eukaryotic rather than mesokaryotic, as in most dinoflagellates. Because of its diploid, eukaryotic vegetative cell nucleus, R. Zingmark (personal communication) does not believe Noctiluca is a true dinoflagellate. It was originally assigned to the Dinophyceae on the basis of the morphological resemblances of the gametes to some freeliving gymnodinoid dinoflagellates. Noctiluca's systematic position remains unresolved. E. Ceratium cornutum H. A. von Stosch, Thiel, and C. Happach-Kasan (personal communication) have found C. cornutum to be a heterothallic freshwater dinoflagellate that reproduces anisogamously. It forms a planozygote which increases in size over several weeks, eventually developing into a hypnozygote. Under their laboratory conditions one uninucleate swarmer is released. It differs from vegetative cells in size, shape, and flagellation. The two subsequent nuclear divisions of this cell are thought to be meiotic. According to their observations, aberrant thecal halves of this meiocyte are transmitted to the offspring. Using these aberrant halves as markers, von Stosch et al. were able to isolate ordered tetrads and to raise clones from them. They then tested their sexual determination by subjecting them to conditions favorable to sexual reproduction in clonal cultures or in combination with each of the standard female and male clones and recording the number of hypnozygotes formed. From the analysis of 125 tetrads they recorded that 69 had the sex factors segregated in the first meiotic division and 59 in the second meiotic division, while 7 tetrads were 1-3 segregants or nonclassifiable.

F. Gloeodinium montanum KLEBS Gloeodinium montanum is a nonmotile dinoflagellate.t! has large, subspherical cells united in small packetlike colonies by a common, stratified, gelatinous envelope. Kelley's study represents the first report of sexuality in the dinoflagellate order Dinocapsales (Kelley, 1988; Kelley and Pfiester, 1988). In clonal cultures G . montanum produces motile cells similar in appearance to Hemidinium ochraceum. These motile cells may fuse or

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develop into vegetative cells. Fusion takes 6-8 days to complete. The resulting zygote is large, nonmotile, spherical, and thick walled. Meiosis occurs in the zygote but nuclear cyclosis has not been observed. Four haploid, nonmotile vegetative cells are produced as a result of either one division or two successive divisions. The zygote, however, may remain dormant for months before these divisions take place. 111. Nuclear Phenomena

Dinoflagellate hypnozygote walls are highly resistant to fixatives and stains, hence little is known concerning the meiotic process. Von Stosch (1972). however, observed an enlargement and rapid swirling of hypnozygotic nuclei in Gymnodinium purudoxum. He referred to this phenomenon as “cyclose nucleaire” and associated it with meiosis, as did Pouchet (1883, 1885) and Biecheler (1952). Nuclear cyclosis has also been observed in Paridinirrrn bafricurn(Fig. 4 ) (J. Chesnick and E. R. Cox, personal com-

FIG.4. SEM of Peridiniurn hnlfirctrn vegetative cell. Reprinted from Chesnick and Cox (1985).

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munication). E. R. Cox (personal communication)noted that the nucleus would swirl in one direction, stop, and then swirl in the opposite direction. It may be associated with the unwinding of the chromosomes. H. A. von Stosch, Theil, and C. Happach-Kasan (personal communication), working with ordered tetrads in Ceratium cornutum, have shown that meiosis is a two-step phenomenon, with nuclear cyclosis associated with meiotic prophase I. In Cyrodinium uncatenum meiosis occurs in divisions of the postzygotic cell termed a planomeiocyte. At 24-48 hours following excystment the planomeiocyte’s nucleus increased dramatically in size and eventually occupied much of the anterior portion of the cell (Coats et al., 1984). Enlarged nuclei had only one nucleolus as opposed to four present in newly excysted cells, and large, paired chromosomes. Coats et al. (1984) associated this stage with Borgert’s (1910) “Knauelstadium,” nuclear cyclosis, and the postzygotene phase of meiosis (Skoczylas 1958; von Stosch, 1964, 1972). Cells with the “Knaue1”-stage nuclei first appeared -24 hours following the start of excystment. Coats et al. (1984) believe that in G. uncatenum the first and second meiotic divisions were not closely associated in time, since they only encountered cells in first-division stages in stained preparations. Hiester (1975, 1976, 1977, 1984; Hiester and Skvarla, 1979) has also reported on a meiosis in which the first and second meiotic divisions are long separated in time. See Section II,C on Peridinium inconspicuurn for details. Barlow and Triemer (1988) have reported in detail on nuclear cyclosis and the physical nature of the dinoflagellate chromosome in planozygotes of Amphidinium klebsii. They described the vegetative nucleus of A . klebsii as crescent shaped and located in the posterior portion of the cell. The condensed chromosomes resemble thickened sausages. However, in the planozygote the nucleus is large and spherical, and it displaces the chloroplast toward the anterior of the cells. The chromosomes appear elongate and threadlike. They observed some cells in which these threadlike chromosomes appeared to be paired. During nuclear cyclosis in A . klebsii the planozygote’s nucleus and its contents rotate within the cytoplasm, but the location of the nucleus itself within the posterior of the cell remains unchanged. Barlow and Triemer observed that a complete nuclear revolution required from 30 to 60 seconds and that they were able to differentiate two regions within the nucleus. In A. klebsii the outer third of the nuclear matrix rotated most rapidly, whereas the central portion of the nucleus exhibited comparatively little rotation. The arms of the chromosomes were oriented toward the center of the nucleus and trailed behind their attachment sites at the nuclear envelopes. According to Barlow and Triemer (1988), this suggests the mo-

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tive force lies at or near the nuclear envelope. They stained a nucleus in the rotation process with acetocarmine and noted that the chromosomes appeared as elongate paired threads with several points of apparent contact between them. TEM observations of such chromosomes showed that they were aligned side by side and in some cases appeared to consist of two chromatids. The chromosomes along the periphery of the cell appeared to curve around the nuclear perimeter. Occasional points of contact were observed between chromosomes which were attached at one end to the nuclear envelope. Neither microtubules nor a layer of fibrous or electrondense material was observed associated with either the cytoplasmic or nucleoplasmic side of the nuclear envelope. Barlow and Triemer (1988) were unable to follow the fate of these planozygotes further than described previously, but they believe the sexual process occurs entirely within the motile condition and does not involve the formation of nonmotile zygotes (cysts). Peridinium balricum (Fig. 4) is a binucleate dinoflagellate that contains both a dinokaryotic and a eukaryotic nucleus (Tomas and Cox, 1973). The eukaryotic nucleus has been ascribed to an endosymbiotic member of the Chrysophyceae, Bacillariophocae, or Rhaphidophyceae, since it contains chlorophylls A and C (Tomas and Cox, 1W3; Withers er al., 1977; Chesnick and Cox, 1987). According to Chesnick and Cox (1987), this symbiotic relationship most likely evolved in P. balticum following engulfment of the photosynthetic alga by a phagotrophic dinoflagellate (Tomas and Cox, 1973). Results from TEM studies on the sexual reproduction of P. balticum by Chesnick and Cox (1987) showed that plasmogamy of the two gametes occurs by dissolution of plate material and rupture of a membrane between them (Fig. 5a). The dinokaryotic nuclei fuse first, and the resulting diploid nucleus at first extends through the middle of the zygote. Plasmogamy of the symbionts ensues, but their eukaryotic nuclei remain at opposite ends of the zygote separated by the dinokaryotic nucleus (Fig. 5b and c). The two eukaryotic nuclei move toward a central position in the nucleus as zygote development progresses. The eukaryotic nuclei fuse and the resulting diploid nucleus remains centrally positioned on the cell (Fig. 6ac). Darkly pigmented plastids and cytoplasm cluster around this nucleus. At this stage of development dinokaryotic nuclei can be seen at both ends of the zygote (Fig. 6b). Presumably these nuclei are the products of meiosis 1. Unfortunately, Chesnick and Cox were unable to observe further development of the diploid eukaryotic nucleus or the two meiosis I dinokaryotic nuclei. Thus, the ultimate fate of the P . halficum zygote is unknown. Isolated zygotes either died or ceased development under culture conditions.

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FIG.5. Serial sections through two fusing gametes of Peridiniurn balticum. Dissolution of thecal plates (T) occurs, allowing plasmogamy and karyogamy of the dinoflagellate host nuclei (D). C, Chloroplast; E, eukaryotic nuclei. Courtesy of J . Chesnick.

FIG.6 . Serial sections through later developmental stage zygote of Peridinium hdfiutrrr. Following karyogamy of the dinoflagellate host nuclei. division (presumably meiosis I ) occurs resulting in two sibling nuclei ( 0 ) .The symbiont moves centrally and fuses, with karyogamy

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The evolved synchrony of the sexual cycles of the host and endosymbiont are of particular interest to cell biologists and those interested in symbioses. Perhaps further studies on the sexual life histories of this host. and endosymbiont may lead to a greater understanding of cellular communication cues as well as symbiotic relationships.

IV. Cyst Formers versus Non-Cyst Formers The majority of dinoflagellates studied thus far produce hypnozygotes that function as resting cysts. This thick-walled resting stage may enable both marine and freshwater forms to survive unfavorable conditions in their environment (see P . cinctum, C. cornutum life histories, Sections II,A and E). Red tides are thought to originate when cysts (hypnozygotes) are brought up to the surface waters by hurricanes (Steidinger, 1975a,b; Anderson and Wall, 1978; Anderson and Morell, 1979). Wall et a f . (1970) demonstrated that the photosynthetic dinoflagellate Peridinium trochoideum (Scrippsiefla trochoidea) formed cysts in culture spontaneously without any apparent stress. Watanabe et al. (1982) also demonstrated cyst (hypnozygote) formation in controlled nutrient-rich cultures of P . trochoideum but was able to increase the percentage of encysting cells by nutrient depletion. Hiester et al. (1984) also demonstrated hypnozygote formation in nutrient-rich cultures of P . inconspicuum. She too was able to increase the percentage of hypnozygotes produced by limiting the nitrogen available in the media. Dormant stages referred to as cysts or hypnocysts have been reported for other dinoflagellates (Endo and Nagata, 1984; Owen and Norris, 1985). Endo and Nagata (1984) reported on Peridinium sp. recovered from the sea-bottom muds following a red tide in coastal waters near Fukuyama, Japan, located in the central part of the Set0 Inland Sea. From their description these cysts were probably hypnozygotes, since they required a 3- or 4-month resting period prior to germination. Vegetative cysts are not known to require a dormant period prior to germination. Owen and Norris (1985) isolated two types of morphologically distinct cysts of the thecate dinoflagellate genus Fragifidium from sediments of the Indian River Lagoon in Florida. They suggested that the “hypnocyst” might be a hypnozygote. Thus, even though relatively few sexual life cycles have been reported in the literature, there is a growing recognition that many thick-

of its nuclei (E). Crystalline rods (r) lead up to condensed chromatin areas in the eukaryotic nucleus. C, Chloroplast; F. flagellar insertion. Reprinted from Chesnick and Cox (1987).

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walled cyst stages observed in natural populations are probably hypnozygotes. The following dinoflagellates are also known to produce parthenospores identical in appearance to hypnozygotes: Ceratium cornutum, Peridinium limbarum (H. A. von Stosch, Theil, and C. Happachan-Kasan, personal communication; Pfiester and Anderson, 1987), and Ceratium hirundinella (Happach-Kasan, 1980). There are dinoflagellates which do not produce resting (dormant) zygotes in their life cycles such as Noctiluca scintillans (see life history, Section I1,D) (Zingmark, 1970), Ceratium tripos (von Stosch, 1%9), and Peridinium gatunense (Pfiester, 1977). The first two are marine dinoflagellates, while P. gatunense is a freshwater form. The significance of this is at present unknown.

V. Environmental Control of Sexuality The most common way to induce dinoflagellate sexual reproduction in the laboratory is by nitrogen limitation (Pfiester and Anderson, 1987). Exponentially growing cells are removed from their growth medium and placed into medium which is either low in or lacking nitrogen. Several studies have induced sexuality by simultaneously deleting both nitrogen and phosphorus. Sexuality has also been observed in old cultures and in collections of natural populations in bloom. In the latter two, nutrient limitation due to population growth is proposed (Cao Vien, 1967; von Stosch. 1973; Pfiester, 1976, and unpublished observations). Von Stosch (1964, 1965) induced sexuality in Ceratium cornutum by decreasing temperature, day length, and light intensity. Temperature is not known to induce sexuality directly. However, it is known to affect the process once it is initiated by nutrient depletion. Anderson and Lindquist ( 1985) showed that cyst formation of Gonyairlax tamarensis was more sensitive to temperature than was growth rate, with optimal cyst production occurring over a relatively narrow temperature range and no encystment at temperatures that permitted growth. Dissolved COz is also linked to sexuality. Sexuality and cyst formation of Peridinium trochoideum was observed under nutrient stress (separate N and P depletion). However, the addition of < I mM bicarbonate enhanced the phenomenon significantly (Watanabe et a / ., 1982). Sexuality does not appear to require a direct environmental change in some species. It has been observed in exponentially growing phagotrophic species not subjected to low light, temperature, or nutrient conditions. Zingmark (1970) observed sexuality in Noctiluca scintillans cultures fed

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on Dunaliella. Morey-Gaines and Ruse (1980) reported spontaneous cyst formation in phagotrophic Polykrikos cultures. Spero and Moree (1981) observed sexuality in Gymnodiniumfungiforme once the algal food source was depleted. In the latter this’may be due to the effects of starvation. Spontaneous sexuality has also been observed in photosynthetic species. Peridinium trochoideum formed zygotes (cysts) in cultures without any apparent stress (Wall et al., 1970). Nonstressed sexual stages have also been observed in cultures of Peridinium inconspicuum and Prorocentrum sp. (L. A. Hiester, unpublished observations). Both Wall et al. (1970) and Sandgren (1983) have suggested that encystment in some algae may not occur in response to an adverse environment but occurs naturally in the life history, usually at a low rate within a population. Watanable et al. ( 1982) reported encystment in nutrient-rich cultures of P. trochoideum. He was, however, able to increase the percentage of cyst formation within the population by nutrient limitation. The time to gametogenesis varies. Induction periods, however, as short as 30 minutes or as long as 7 days have been reported (Hiester, 1975; Walker and Steidinger, 1979). Some gametes have functioned as vegetative cells when placed in nutrient-replete media (von Stosch, 1973; Hiester, 1975). VI. Sexuality: Its Function and Significance Genetic exchange is usually given by most biologists as the function of sexuality. Such an exchange is thought to lead to increased variation important for species survival. Such variation undoubtedly occurs in dinoflagellate populations as a result of sexual reproduction. However, sexual reproduction is known to play other important roles in dinoflagellate populations. Researchers now know that red tides (blooms of toxic marine dinoflagellates) are carried over from year to year by hypnozygotes (cysts), which can remain viable in ocean sediments for years (Steidinger, 1975a,b; Anderson and Wall, 1978; Anderson and Morell, 1979). These hypnozygotes act as “seeds” which when brought to surface waters during meteorological disturbances germinate and produce the red tide. Once hypnozygotes settle in the sediments of an area, that area may then be subject to periodic red tides. In culture, dinoflagellates may undergo polyploidy (Holt and Pfiester, 1982). More recently, as yet unpublished research (J. R. Holt, personal communication) has shown that the chromosome number is reduced to the base number in postzygotic cells. Thus, one function of dinoflagellate

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sexual reproduction appears to be to return the organism to its base chromosome number. To date, sexual induction has not been studied per se. Rather, studies have used induction merely as a means of studying sexuality.

VII. Future Research

The ability to culture dinoflagellate taxa not only allows us to study their vegetative and sexual life histories but enables us to examine all aspects of the biology of these protists claimed by botanists and zoologists alike. Aspects of sexual reproduction which have yet to be examined include the hormonal attraction of gametes to each other and membrane recognition sites. Such hormones have been identified and studied in the Volvocales and Oedogoniales (Darden, 1966; Starr and Jaenicke, 1974; Hoffman, 1960). Membrane recognition sites have been little studied in the algae. Yet dinoflagellate gametes, with few exceptions, attach to each other at a specific site. Advances in immunology have paved the way to look at these receptor sites in the algae. Within the next few years applications of molecular biology, genetic engineering, and immunology to the dinoflagellates should tremendously increase our knowledge of these protists and increase our desire to explore them further.

REFERENCES Anderson, D. M. (1980). J. Phycol. 16, 166-172. Anderson, D. M., and Lindquist, N. L. (1985). J. Exp. Mar. B i d . Ecol. 86, 1-13. Anderson, D. M., and Morell, F. M. M. (1979). Esiuarine Coastal Mar. Sci. 8, 279-293. Anderson, D. M., and Wall, D. (1978). J . Phycol. 14, 224-234. Anderson, D. M., Coats, D. W., and Tyler, M. A. (1985). J . Phycol. 21, 200-206. Barlow, S., and Triemer, R. E. (1988). Phycologia (in press). Beam, C. A., and Himes, M. (1974). Naiure (London) 250, 435436. Beam, C. A., and Himes, M. (1980). I n “Biochemistry and Physiology of Protozoa” ( M . Levandowsky, S. H. Hutner, and L. Provasoli, eds.), 2nd ed., Vol. 3, pp. 171-206. Academic Press, New York. Biecheler, B. (1952). Bull. Biol. Fr. Belg., Suppl. 36, 1-149. Borgert, A. (1910). Arch. Proiisienkd. 20, 1-46. Cao Vien. M. (1%7). C . R. Hebd. Seances Acad. Sci., Ser. D 264, 1006-1008. Cao Vien, M. (1968). C. R. Hebd. Seances Acad. Sci., Ser. D 267, 701-703. Chesnick, J. (1986). Dissertation, Texas A&M University, College Station. Chesnick, J., and Cox, E. R. (1985). Trans. Am. Microsc. SOC. 104, 387-394. Chesnick, J., and Cox, E. R. (1987). BioSysiems 21, 69-78. Coats, D. W., Tyler, M. A., and Anderson, D. (1984). J . Phycol. 20, 351-361. Darden, W. H. (1966). J . Proiozool. 13, 239-255.

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Diwald, K. (1937). Flora (Jena) 132, 174-192. Dodge, J. D. (1966). In “The Chromosomes of the Algae” (M. B. E. Godward, ed.), pp. 96-115. St. Martins Press, New York. Endo, T., and Nagata, H. (1984). Bull. Plankton SOC.Jpn. 31, 23-33. Evitt. W. R. (1985). “Sporopollenin Dinoflagellate Cysts.” Am. Assoc. Stratigraphic Palynologists Found., Austin, Texas. Grell, K. G. (1973). “Protozoology.” Springer-Verlag, Berlin and New York. Happach-Kasan, C. (1980). Dissertation, Philipps University, Marburg. Hoffman, L. R. (1960). Southwest Nat. 5, 11 1-1 16. Hofker, 1. (1930). Arch. Protistenkd. 71, 57-78. Holt, J. R., and ffiester, L. A. (1982). A m . J . Bot. 69, 1165-1 168. Kelley, 1. (1988). Dissertation, University of Oklahoma, Norman. Kelley, I., and ffiester, L. (1988). J. Phycol. (submitted for publication). Loeblich, A. R., Ill (1970). Proc. North Am. Paleontol. Conv., Part G pp. 867-929. Morey-Gaines, G., and Ruse, R. H. (1980). Phycologia 19, 230-236. Netzel, H., and Durr. G. (1984). In “Dinoflagellates” (D. L. Spector, ed.), pp. 43-105. Academic Press, New York. Owen, K., and Norris, D. R. (1985). Coastal Res. 3, 263-266. Hiester, L. A. (1975). J. Phycol. 11, 259-265. Hiester, L. A. (1976). J. Phycol. 12, 234-238. Hiester, L. A. (1977). J . Phycol. 13, 92-95. Hiester, L. A. (1984). In “Dinoflagellates” (D. L. Spector, ed.), pp. 189-199. Academic Press, Orlando, Florida. Hiester, L. A., and Anderson, D. (1987). In “Biology of Dinoflagellates” (F. J. R. Taylor, ed.), pp. 61 1-648. Blackwell, Oxford. ffiester, L. A., and Carty, S. (1985). J. Phycol. 21, 509-51 1. Ptiester, L. A., and Lynch, R. A. (1980). Phycologia 19, 178-183. Hiester, L. A., and Skvarla, J. J. (1979). Phycologia 18, 13-18. ffiester, L. A., and Skvarla, J. J. (1980). Am. J . Rot. 67, 955-958. Hiester, L. A., Timpano, P., Skvarla, J. J., and Holt, J. R. (1984). Am. J . Bot. 71, 11211127. Pouchet, G. (1883). J. Anat. Physiol. 19, 399-455. Pouchet, G. (1885). J . Anat. Physiol. Norm. Pathol. Homme Anim. 21, 28. Rizzo, P. J., and Nooden, L. D. (1972). Science 176, 796-797. Sako, Y., Ishida, Y., Kadota, H., and Hala, Y. (1984). Bull. Jpn. SOC. Sci. Fish. 50, 743750. Sako, Y., Ishida, Y., Nishijima, T., and Hata. Y. (1987). Nippon Suisan Cakkaishi53,473478. Sandgren, C . D. (1983). In “Survival Strategies of the Algae” (G. Fryxell, ed.). pp. 23-49. Cambridge Univ. Press, London and New York. Schutt, F. (1895). Ergeb. Plankton-Exped. Humboldt-Stif. 4, M.a. A, 1-170. Skoczylas. 0. (1958). Arch. Protistenkd. 103, 193-228. Spector, D. L., and Triemer, R. E. (1979). A m . J . Bot. 66,845-850. Spector, D. L., Pfiester, L. A., and Treimer, R. E. (1981). Am. J. Bot. 68, 34-43. Spero, H. J., and Moree. M. D. (1981). J. Phycol. 17, 43-51. Starr, R. C., and Jaenicke, L. (1974). Proc. Natl. Aclad. Sci. U.S.A. 71, 1050-1054. Steidinger, K. A. (1975a). Proc. Int. Con$ Toxic Dinoflagellates, Blooms, 1st 1974 pp. 153162. Steidinger, K. A. (1975b). Environ. Lett. 9, 129-139. Timpano, P., and ffiester, L. A. (1986). Trans. Am. Microsc. Soc. 105, 381-386.

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Tomas. R. W., and Cox, E. R. (1973). 1. fhycol. 9, 304-323. Turpin, D. H . , Dobell, P. E. R.. and Taylor, F. J. R. (1978). J. Phvcol. 14, 235-238. Tuttle, R. C.. and Loeblich. A. R.. 111 (1974). Science 185, 1061-1062. von Stosch. H. A . (1964). Helgol. Wiss. Meeresunters. 10, 140-152. von Stosch. H . A. (l9fS). Natumissenschafien 52, 12-1 13. von Stosch. H . A . (1%9). HeIgol. Wiss. Merresunters. 19, 569-577. von Stosch. H . A. (1972). Mem. Soc. Bot. Fr. pp. 201-212. von Stosch. H . A. (1973). Br. fhycol. J . 8, 105-134. Walker. L. M . (1982). Trans. Am. Mirrosc. Soc. 101, 287-293. Walker. L. M . , and Steidinger, K. A. (1979). J . fhycol. 15, 312-315. Wall. D.. Guillard. R. R. L., Dale, B., Swift. E.. and Watabe, N . (1970). Phycologiu 9, 15 1-156. Watanabe, M. M.. Watanabe, M.,and Fukuyo. Y. (1982). Res. Rep. Nail. Insr. Environ. Stud. (Jpn.) 30, 2741. Withers. N . W.. Cox. E. R., Tomas, R. W.. and Haxo, F. T. (1977). J. Phyc.ol. 13, 354358. Yoshimatsu, S. (1981). Bull. Plankton Soc. Jpn. 28, 131-139. Yoshimatsu. S. (1984). Bull. Plankton Soc. Jpn. 31, 107-1 11. Zingmark, R. (1970). J . fhvcol. 6, 122-126.

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 114

Water Exchange through the Erythrocyte Membrane GHEORGHE BENGA Department of Cell Biology, Medical and Pharmaceutical Institute ClujNapoca, Faculty of Medicine, 3400 Cluj-Napoca, Romania

I. Introduction

One of the main functions of the plasma membranes of living cells consists of the control of movement of various molecules into or out of the cell. These transport processes permit the entry of oxygen and cell food as well as the exit of cell wastes; they also regulate the cell volume and the composition of intracellular fluid. Indeed, continued existence of the cell is critically dependent on a functional cell membrane having a selective permeability, that is, the ability to discriminate among various molecules so that some are kept inside or outside the cell, while others are allowed to permeate via specialized and controlled pathways. Consequently, the intracellular fluid contains solutes at concentrations which are quite different from their corresponding values in the extracellular medium. The movement of water and solutes through the membrane is a dynamic process, and the living cell is never in equilibrium with the environment in terms of materials across its membrane. The cell achieves a state of equilibrium only when it is dead (Sha’afi, 1977). It is therefore clear that the transport of water across biological membranes is essential for cell functions. Aside from being of considerable theoretical importance, the water transport is of practical importance in a broad range of processes, from the protection of cells undergoing cryogenic preservation to the effects of certain hormones in some tissues. Because of its availability and simple structure, lacking internal membranes, the red blood cell (RBC) is ideally suited for investigating water permeability. In fact it has been for many years one of the most favored cells for studies in this field. The rather detailed knowledge of molecular structure of RBC membrane is important in this cell’s continued preferred use in investigations. 213 Copyright Ci 1989 by Academic Pres. Inc. All rights of reproduction in any form reserved.

274

GHEORGHE BENGA

11. Osmotic and Diffusional Permeability of Red Blood Cells

There are two basic strategies for measuring water exchange through the RBC membrane: nonstationary and stationary methods. The nonstationary methods involve subjecting the cells to an osmotic gradient that creates a net flux of water in one direction or the other depending on whether the cells swell or shrink. By these methods one can estimate the osmotic permeubility (Pas) of RBC, and the results are generally expressed in terms of an osmotic permeability constant L,. I n case of stationary methods, the diffusion movement of water is measured and there is therefore no net flux of water through the membrane. The cells remain in their normal state, which is often considered an advantage over nonstationary methods. By stationary methods one can estimate the diffusional permeability (P,) of RBC. A. NONSTATIONARY METHODS FOR W A T E R MEASUREMENTS

PERMEABILITY

1. Hemolysis Method

This method is based on measurement of the time required for a system of RBC to hemolyze either completely, or to any specific extent, when placed in a solution in which NaCl has been replaced by the permeating molecule under study. Hemolysis occurs because entry into the cell of a permeating solute moving down its concentration gradient causes an imbalance of water activity, which is quickly compensated for by movement of water into the cell. The process continues until the cell swells to a critical volume and hemolysis occurs. End points for hemolysis are easily detectable, because as hemoglobin escapes the cell, the refractive index of the medium increases and the refractive index of the cell decreases until both medium and cell have similar indices. At this point what was once a turbid solution becomes clear because the cells no longer scatter light. Permeability coefficients for either water or solutes can be calculated from the time of hemolysis as described by Jacobs (1952). This type of measurement is no longer in use, because such calculations are not completely accurate.

2. S t o p Flon~and Rapid Injection More modern techniques have relied on the use of light scattering or transmission to measure actual changes in cell volume as a function of time. As cells shrink (or swell), hemoglobin becomes more (or less) con-

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

275

centrated. As a result, light scattering increases and transmission decreases as cell volume decreases. In application of this principle, various approaches have been developed for rapid mixing, and also for recording (for reviews, see Forster, 1971; Sha'afi and Gary-Bobo, 1973; Macey, 1979). Most often the rapid mixing is accomplished with some form of stop-flow apparatus. Two solutions. one containing a dilute suspension of RBC and the other containing the permeating molecule, are rapidly mixed. When steady flow is achieved. the flow is stopped abruptly and the fluid is isolated in an observation tube through which light passes. The time course of cell volume changes can be measured indirectly from the changes in the intensity of either 90"scattered light or 180"-transmitted light.

B. METHODSFOR MEASURINGWATER PERMEABILITY UNDER STEADYSTATECONDITIONS In all methods of this type, exchange measurements are performed in a system at equilibrium, where no net movement of water takes place in the system. I . Radioactive-Isotope Techniques a. Continuous Flow. This method was used by Paganelli and Solomon (1957) to measure the rate of influx in human RBC. A suspension of RBC at a relatively high hematocrit was mixed in a rapid-flow mixing chamber with isotonic buffered solution containing tritiated water, and the mixture was forced down a tube with ports that permit axial sampling of the suspension medium. These ports are covered with filter paper to permit passage of the suspending medium while retaining the cells. Thus samples of suspending medium can be obtained at each port, which is located at a preset distance from the mixing chamber. Knowing the velocity of flow down the tube, the time between mixing and sampling can be calculated for each port. Conversion of distance to time by means of velocity is the underlying principle of this method. In an updated version of the continuous-flow method (Brahm, 1982), the rate of efflux of tritiated water from a very dilute (cytocrit 0.6%) suspension of labeled cells is measured. Distance is related to time of sampling because the velocity of flow is constant, and it is thus possible to follow the increase of radioactivity in the filtrates collected simultaneously at six precisely determined distances from the mixing chamber along the tube.

276

GHEORGHE BENGA

b. Linetir Diflilsion (or Bulk Diffitsion). This method was reported first by Redwood et t i l . (1974) for use with RBC and subsequently developed for use with other cell types. The RBC are packed by centrifugation inside polyethylene tubing, and the cell column is pulsed at one end with tritiated water. After diffusion proceeds for several hours, the distribution of label along the column is measured by sequential slicing and counting. The data are analyzed assuming a diffusion model with intracellular and extracellular pathways. From the measured diffusion coefficients the red cell membrane permeability coefficient P (in centimeters per second) is derived. The theoretical and practical aspects of this method are described in detail by Garrick ( 1987) and by Klosgen et al. ( 1987). The linear diffusion method has been applied in current research not only to RBC (Garrick et al., 1982, 1986; Osberghaus et al., 1982) but also to isolated lung cells (Garrick and Chinard, 1982), endothelial cells (Ganick e f al., 1986). rat hepatocytes (Alpini el al., 1986), and Novikoff hepatoma cells (Polefka et al., 1981). 2 . Merrsitrements of Water Permeability by Solvent Isotope Eflects A new technique for measuring water permeation across membranes based on optical differences (different indices of refraction) of H,O and D?O was described by Lawaczek (1984). It was applied to RBC by Pitterich and Lawaczek (1985); however, the value of Pd obtained by this method is about half of that obtained by other techniques. Details on the new technique are described by Lawaczek (1987).

3 . Nictleur Magnetic Resonance ( N M R ) Techniques The N M R techniques for measuring water diffusion can be classified into doping methods and other variants. The doping methods employ the relatively impermeable paramagnetic Mn” , added in high (12-40 mM) or low (1-2 mM) concentrations to the medium in which the RBC are suspended, to determine the relaxation times of water protons inside and outside the RBC. An NMR method, using high Mn” concentrations for measuring water exchange times, has been developed for RBC by Conlon and Outhred (1972).This method is based on the following considerations. When water protons are placed in a static magnetic field, their spins become oriented. Application of a brief intense radiofrequency pulse reorients the spin, and this reorientation serves as a label which can be detected by NMR techniques. Following application of the pulse, the label decays: this decay can be followed either in the x-y plane or along the z axis. In the former case the decay can be described by the spin-spin (or transverse) relaxation time (7’:). the rate constant for the decay being l/Tz. In the latter case the

WATER EXCHANGE THROUGH T H E ERYTHROCYTE MEMBRANE

277

decay can be described by the spin-lattice (or longitudinal) relaxation time (TI), the rate constant for the decay being l/T,. The nuclear relaxation times TI and T2 are thus parameters that characterize the return to equilibrium after a suitable radiofrequency perturbation of the nuclei in an NMR experiment (Farrar and Becker, 1971). Long relaxation times are often associated with nuclei that are part of molecules with fast motion. We will next assume a system consisting of two compartments A and B with the same type of molecules distributed in both compartments. The system can be characterized by two nuclear relaxation times (T, and T,,) of the same type of nuclei residing in each of the compartments (Fig. 1). If, for some reason, the corresponding NMR relaxation times differ, so

B Tb

‘b

FIG. 1. Two nuclear compartments having the same type of nucleus. (1) No exchange between compartments: The nuclei relax in each compartment with the relaxation times T, and Tbrrespectively (T, is assumed to be much higher than Tb). (2) Fast exchange of nuclei from A to B. The observed relaxation of nuclei in compartment A, which also relax in B due to the exchange process, will be shortened compared to T,. Reproduced with permission from Morariu and Benga (1984).

278

GHEORGHE BENGA

that T, >> Tb, two cases can be considered: ( I ) As shown in Fig. I( I ) , there is no exchange of molecules between the two compartments; and (2) as shown in Fig. 1(2), there is a relatively fast exchange process transfemng molecules between compartments (Morariu and Benga, ( 1984). The question is how the exchange process will affect the relaxation times of the two compartments. In case ( I ) , the two relaxation times T, and Th will be detected in an actual NMR experiment (Fig. 1). (In fact, even if there is a very slow exchange so that the nuclei will have time to relax in each compartment, the result will be much the same.) However, in case (2), the nuclei in A will start relaxing with Ta but will end up in compartment B where its relaxation Th will be faster. As a result, the observable relaxation time of phase A will be T i , which is shortened compared to T, (Fig. I , case 2). The faster the exchange, the shorter T2,. The equations describing this phenomenon have been derived by Woessner (l963), enabling the calculation of exchange times. Let us see how this model can be applied to an RBC suspension (Fig. 2). The nucleus of concern in this case is the water proton, which can reside either inside (compartment A) or outside the cell (compartment B). There is an exchange of water molecules between these two compartments. However, if we perform an NMR experiment on such a system we will only detect a single relaxation time. This is because the relaxation times of the protons in both compartments are only slightly different (of the order of hundreds of milliseconds), and the rapid exchange between compartments makes the distinction between the two compartments impossible [Fig. 2(1)]. Obviously some way is needed of making Ta >> Thas described previously. One way of doing this is the method of paramagnetic doping. If we add a paramagnetic ion such a s Mn” to the cell suspension, then the proton relaxation time (T,,)of water molecules in the suspending solution will become much shorter by a mechanism known as electron-proton interaction (see. for example, Dwek, 1973, for an explanation). Then we will have a system with T, >> Tb as described in Fig. 1(2), and the same type of experimental approach can be applied to RBC. Of course, a prerequisite to this experiment is that the manganese ions d o not penetrate the RBC. Fabry and Eisenstadt (1975) showed that the penetration of manganese ions is hampered in the presence of albumin, and indeed, we and others could not detect any penetration of the Mn” into RBC (Morariu and Benga, 1977; Getz et al., 1979). So far the general term ‘Lnuclearrelaxation time” has been used, whereas, (as mentioned before) in reality there are two different measurable relaxation time processes: T , and Tz. Both of these relaxation times can be used for the determination of water exchange times.

WATER EXCHANGE THROUGH T H E ERYTHROCYTE MEMBRANE

-1 B

279

A

Ta- Tb

- 100ms

TA- 10 ms

FIG. 2. Illustration of the NMR paramagnetic doping method for measuring the water exchange through RBC membranes. A is the blood cell compartment and B is the suspending solution compartment. ( I ) In a suspension of RBC the relaxation times of water protons are similar in the two compartments (T, = Tb= 100 msec), and fast exchange occurs between them. As a result, a single relaxation time is detected and therefore this experiment cannot be used for the measurement of water exchange. (2) If manganese ions are added to the suspending solution, then Tbbecomes much shorter (-0. I msec). In the absence of exchange between A and B, T, should remain unchanged (-100 msec). However, fast exchange of water occurs through the membrane, and T, becomes T'. (-IOmsec). The water exchange time can be calculated from T,.Reproduced with permission from Morariu and Benga (1984).

111. Characterization of Diffusional Water Permeability in Human RBC and Ghosts

One major aim of this review is to describe recent work in our laboratory, including the presentation of unpublished results on diffusional water permeability of human RBC. In order to ensure the comprehensibility of the data it will be helpful to describe briefly the methods we have used .

280

GHEORGHE BENGA

A. METHODOLOGY I . Blood Sample Preparations

NMR measurements of diffusional water permeability and labeling experiments with ['u3Hg]p-chloromercuribenzenesulfonate(PCMBS), an inhibitor of water diffusion, have been performed on human RBC or resealed ghosts. Human blood was obtained by venipuncture in heparinized tubes; the RBC were isolated by centrifugation (10 minutes at 1200 g) and washed three times in 166 mM NaCI. Finally the RBC were suspended in 150 mM NaCI-5.5 mM glucose-5 mM HEPES (pH 7.4) and 0.5% bovine serum albumin at a cytocrit of 50%. Two types of ghosts resealed after hemolysis have been prepared having a membrane permeability very close to that of the intact cell: (I) pink ghosts (Schwoch and Passow, 1973), which retain a small amount of the original hemoglobin, and (2) white ghosts (Bjerrum, 1979), which are free from visible contamination with intracellular components including hemoglobin. The concentration of hemoglobin in resealed ghosts was estimated spectrophotometrically a s described by Antonini e f a f .(1963). The hemoglobin content of the pink ghosts in our preparation was reduced to 47% of that of RBC, and in the white ghosts it was even further reduced. Finally, the ghosts prepared by any of the procedures already described have been suspended in the same solution as the RBC at a cytocrit of 50%.

2. NMR Measurements of Water Diffirsion We have used the doping NMR method (Conlon and Outhred, 1972) for the determination of water exchange time (Morariu and Benga, 1977; Benga ef a / . , 1983a). The observed relaxation time T2Aof RBC suspended in an isotonic medium doped with Mn" is dominated by the exchange process through the RBC membrane and is related to the water diffusion exchange time (T,) and the relaxation time of the cell interior (TJ by the equation (Conlon and Outhred, 1978):

The membrane permeability for water diffusion, Pd, is related to 1/Te, the cell water volume V, and the cell surface area A , as follows: V

I

A

Te

P * = - x -

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

28 1

Since different authors have used different values for V and A , in order to compare our results with previous ones we have used two sets of values. On one hand, we have taken a value of 65 pm3 for the intracellular solvent volume of RBC and 86 pm3 for that of resealed ghosts and a value of 1.42 x for the membrane area, after Brahm (1982). These give ratios of 4.58 x lo-' cm and 6.06 x lo-' cm for RBC and ghosts, respectively. On the other hand, we used a slightly higher VIA ratio, after Dix and Solomon (1984); for example, 5.33 x lo-' cm for RBC and the corresponding value for ghosts. In their initial work, Codon and Outhred (1972) diluted 1 ml of blood with 0.5 ml of doping solution containing 20-100 mM manganese chloride. In later work (Conlon and Outhred, 1978) 0.4 ml of whole blood was diluted with 0.9 ml of manganese solution (50 or 100 mM manganese chloride made isotonic with sodium chloride). It was found necessary to keep the packed-cell volume (PCV) <20% in order to eliminate any dependence of relaxation time on the PCV. We have performed the measurements of T2: on washed RBC or resealed ghosts suspended in 150 mM NaCI-5 mM HEPES (pH 7.4) containing 0.5% albumin and 5.5 mM glucose. The presence of albumin is important to prevent the entry of manganese ions into the cells. In our earlier experiments (Morariu and Benga, 1977) we have used 1 ml of blood or suspension of RBC and 0.5 ml of a 40 mM manganese chloride solution made isotonic with sodium chloride; the NMR measurements have been performed with a Bruker-Physik SXP spectrometer, at 90 MHz. Now we are routinely using 0.2 ml of RBC or ghost suspensions and 0.1 ml doping solution. The NMR measurements are now performed with an AREMI-78 spectrometer (manufacturedby the Institute of Physics and Nuclear Engineering, Bucharest-Mfigurele, Romania) at a frequency of 25 MHz. The temperature is controlled to k0.2"C by air flow over an electrical resistance using the variable-temperature unit attached to the spectrometer. The actual temperature in the sample was measured with a thermocouple connected to a microprocessor thermometer (Comark Electronics Ltd, Rustington, Littlehampton, England). T2Lis evaluated by the spin-echo method (Farrar and Becker, 1971) using a computer unit coupled on line with the spectrometer. T2iwas measured by the 9Oo-18O0 method using the Carr-Purcell-Meiboom-Gill sequence (Farrar and Becker, 1971) on packed cells or ghosts from which the supernatant, with no added Mn, had been removed by centrifugation at 50,000 g for 60 minutes. The inhibition of water diffusion across human RBC membranes was calculated assuming that the permeability coefficient is inversely related to T2L,according to the formula (Benga et al., 1983b)

282

GHEORGHE BENGA

1 % Inhibition =

1

-

T,: (control)

T7L (sample)

1 T2: (control)

x 100

B. COMPARISON OF DIFFUSIONAL WATER PERMEABILITY OF RBC GHOSTS

AND

Comparative values of parameters characterizing the diffusional water permeability in RBC and pink resealed ghosts at various temperatures are listed in Table I. The temperature values were chosen so as to facilitate comparison with values reported by other authors for measurements performed at various temperatures. It is obvious that for all temperatures the T, values were higher in ghosts compared to RBC. However, when the permeability values were estimated it appeared that resealed ghosts have a similar permeability to RBC. No difference between pink and white ghosts could be detected; this finding is in agreement with data of Bjerrum (1979). who reported for white ghosts a water permeability similar to that of RBC. Our data (Table I ) show that resealed ghosts have a slower exchange rate compared to RBC. A similar finding was reported by Brahm (1982). who has followed the diffusional water permeability of RBC and ghosts by an isotopic technique. The exchange rate in ghosts is slower because the cellular solvent volume was increased after removal of the cell hemoglobin. It appears that the isotopic and N M R techniques are in agreement in revealing that both RBC and ghosts have a similar water permeability. Comparative values of water permeabilities of RBC and ghosts reported by various authors are listed in Table 11. C. pH EFFECTSON WATERPERMEABILITY Verma and Wallach (1976) have suggested, based on Raman spectroscopic studies, that RBC membranes undergo cooperative, pH-sensitive transitions between pH 5.5 and 7.0 in the physiological temperature range. In order to understand better the molecular mechanisms of water diffusion in RBC membranes, it was important to know if these transitions have any effects on the water transport. We have performed the first studies concerning the effect of pH on the water transport (Morariu el ul., 1981). A marked increase of the T, values in the acid range of pH (7.0 to 5.5) was noticed. A much smaller variation of the same parameter occurred between pH 7.0 and 8.0. It was interesting

TABLE I DIFFUSIONAL WATERPERMEABILITY OF HUMANRBC

RBC

AND

RESEALED GHOSTS".~

pd

Temperature ("C)

T. (msec)

pd

( c d s e c x 10')

I

Statistical significance of the difference

Resealed ghosts

II

T. (msec)

( c d s e c x 10')

I

Pd

I

II

p < 0.001 p
NS NS

NS

p < 0.001 p
NS NS NS

NS

II

T C ~

15 2G 25 30 37

15.4 rf: 1.8 13.321.3 11.4 2 1.3 9.6k0.8 7.4 0.7

2.8 2 0.2 3.4k0.3 4.0 2 0.3 4.820.6 6.2 f 0.7

3.2 2 0.3 4.0k0.5 4.7 f 0.5 5.620.8 7.2 k 0.9

23.4 i-: 4.3 19.323.3 17.0 f 2.9 13.5k2.1 10.4 t 1.4

2.6 & 0.1 3.1ZeO.I 3.6 t 2.9 4.5k0.2 5.8 2 0.3

3.0 2 0.1 3.620.2 4.2 k 0.2 5.220.3 6.8 2-0.4

~~~

NS NS NS

"Reproduced with permission from Benga ef a/.(1986a). ?he measurements were performed on duplicate or triplicate blood samples from 12 donors a s described in Section 111. Results are expressed as mean ? SD. The permeability was calculated from T, using a VIA ratio of 4.58 X lo-' cm for RBC and 6.06 x lo-' cm for ghosts (Brahm, 1982) in column I, and the slightly cm for RBC) given by Dix and Solomon (1984) in column 11. The statistical significance was calculated using higher value for the VIA ratio (e.g., 5.33 x unpaired r-test. NS. Not statistically significant.

DlFFkJ\lONAl

TABLE I I ( p , )O C

PFKMkAHII.ITY

IHI

HLIMAN RBC” ~

reported by parameter determined

D d a correcled

Data a\

Sample R BC

RBC ghosts

Reference Paganelli and Solomon ( 1957, corrected by Barton)’ Barton and Brown (1964) Vieira er u / . (1970) Brahm (1982) Osberghaus er ul. (1982) Klosgen QI u / . (1987) Andrasko ( 1976) Shporer and Civan (1975) Fabry and Eisenstadt (1978) Pirkle er (I/. (1979) Conlon and Outhred (1972) Conlon and Outhred (1978) Morariu and Benga (1977) Chien and Macey (1977) Chien and Macey (1977) Dix and Solomon (1984) Moura er a / . (1984) Benga er a / . (l986a) Brahm (1982) Pitterich and Lawaczek (1985) Benga er a/. (1986a)

Method

Temperature (“C)

7,

f’d

(msec) (crnhec x 10’)

T H O , continuous flow

23

11.5

THO. continuous flow THO. continuous flow THO. continuous flow T H O , linear diffusion THO, linear diffusion NMR, pulsed gradient NMR, ”0, T, NMR, T , . T2*TI,, 2 mM Mn” NMR, T2, 1.7 mM Mn” NMR, Tz, 33.3 mM Mn*’ NMR, T2, 34.6 mM Mn” NMR, T I . 13.3 mM Mn” NMR, T?, 20 m M Mn” NMR, TI, 20 mM Mn” NMR, TI, 22.6 mM Mn” NMR, T2, 20 mM Mn2’ NMR, T I , 13.3 mM Mn” THO, continuous flow DzO, scattering NMR, T2, 13.3 mM Mn”

23 22

10.9

2s

19.0

20 20 24 25 25 25 25 20 37 25 25 21 25 20 25 20 20

70°C H,O. \tmdard cell (cmAec x 10’) to

5.0 2.4 3.7 4.5

17.0 16.7 12.2 21.0 11.0

12.2 6.0 11.0 17.2 10.9 12.4 13.3

11.0

3.7 2.9 1.2

19.3

________ f’,

P.

3.6

4.2

3.9 3.3 2. I 2.5 3.0 2.5 2.3 3.3 2.0 3.6 3.4 4.6 3.6 2.2 3.4 3.2 3.4 2.9 0.8 3. I

4.5

3.8 2.4 2.9 3.5 2.8 2.7 3.8 2.3 4.2 4.0 5.4 4.2 2.6 4.0 3.7 4.0 3.4 0.9 3.6

‘Conversion to 20°C was carried out using the activation energy of 5.3 kcaVmol (Fabry and Eisenstadt, 1978). The value of PI was calculated using a V / A ratio of 4.58 x cm for RBC and 6.06 x 10-j cm for ghosts (Brahm, 1982);that of P2 was calculated using the slightly higher value for the VIA ratio (e.g.. 5 . 3 3 x cm) for RBC given by Dix and Solomon (1984). ‘As shown by Dix and Solomon (1984), there was an error in the original Paganelli and Solomon (1957) paper that arose from including a calculated zero-time point. Subsequently, Barton and Brown (1964) repeated the Paganelli and Solomon experiment using the original apparatus and corrected this error; their corrected value is used in this table.

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

285

to find significant changes of the water exchange time values around the pH values which induce state transitions of the RBC membranes. However, Brahm (1982) pointed out that changes in parameters characterizing diffusional water permeability of RBC in the acid range of pH can be due to a decrease of cellular solvent volume caused by the titration of intracellular buffers, mainly hemoglobin. Consequently, if ghosts relatively free of hemoglobin are used one would expect a much smaller variation of T, with pH. As shown in Fig. 3, for all temperatures explored there was indeed no significant increase in T, of resealed ghosts in the acid range of pH. These findings indicate again that the NMR and isotopic methods are in agreement when used for measuring the diffusional water permeability of RBC. Furthermore, the advantage offered by investigations on resealed ghosts is revealed.

D. TEMPERATURE DEPENDENCE OF WATERPERMEABILITY There are several reasons for studying the temperature dependence of water permeability in RBC. Such studies can be useful in comparing the permeability of different cells or tissues measured at different temperatures. Besides, to interpret most transport studies with tissues and organs

I1

5.5

6.0

6.5

7.0

7.5

8.0 pH

FIG.3. The values of the water proton relaxation time (TA) in ghosts as a function of pH at different temperatures (10, 20, 30, and 37°C). Reproduced with permission from Benga et a / . (1986a).

286

GHEORGHE BENGA

it is necessary to make assumptions about the permeability characteristics of the cell membrane based on data available for isolated cells. However, most of the studies of the permeability characteristics of isolated cells have been conducted at 20°C, while in vivo organ studies are conducted at 37°C the average body temperature for mammals (Ganick and Chinard, 1982). Studies on the effects of temperature on water permeability can also contribute to the understanding of molecular mechanisms of water transport in RBC. The apparent activation energy of water permeation across fluid lipid bilayers is 10 kcal/mol (Fettiplace and Haydon, 1980; Siddiqi et al., 1987). The activation energy of the water exchange through RBC membrane is much lower (Vieira ct a / . , 1970; Shporer and Civan, 1975; Morariu and Benga, 1977; Brahm, 1982). This shows that the mechanisms of water permeation which operate in the case of RBC are different from those in lipid bilayers; that is. the major role in RBC water transport is played by hydrophilic channels in proteins. On the other hand, there are conflicting data regarding the occurrence of significant deflections of the activation energy at a certain temperature, that is, of "break" points or discontinuities in the Arrhenius plots of water exchange parameters. While some investigators (Vieira et al., 1970; Shporer and Civan, 1975; Morariu and Benga, 1977; Brahm, 1982) reported a linear temperature dependence of water exchange, other authors revealed breaks in the Arrhenius plot of water exchange in human RBC measured by the doping N M R method (Conlon and Outhred, 1978; Morariu et al., 1981). It was interesting that such breaks occurred in the range 20"-30°C. This is the range where other authors revealed breaks in the temperature dependence of the following RBC membrane parameters: viscosity or microviscosity (Zimmer and Schirmer, 1974; Feinstein ef al., 1975); osmotic fragility (Aloni et al., 1977); enzymic incorporation of 32Pinto polyphosphoinositides (Buckley and Hawthorne, 1972); transport of chloride and bromine (Brahm, 1977); exchange transport of glucose (Lacko et al., 1973). These phenomena have been discussed by some authors in terms of phase transitions in RBC membranes, occurring around 2Oo-25"C, based on the interpretation of discontinuities in the temperature dependence of various membrane processes as indicating conformational changes of the membrane proteins induced by a temperature-dependent phase change in the membrane (for reviews, see Benga, 1979, 1985; Benga and Holmes, 1984; Chapman and Benga, 1985). A systematic analysis of the temperature dependence of water transport in human RBC should reveal the effect of such transitions on this transport process. In our earlier studies, which in fact represented the first application of the doping N M R method to study the temperature dependence of the water

-

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

287

exchange in human RBC (Morariu and Benga, 1977), the measurements were performed in the range 25"-37"C and the activation energy was found to range between 6 and 8 kcal/mol for different samples. These values are intermediate between the activation energy of molecular rotation in ice (13.3 kcal/mol) and that of liquid water (4 kcal/mol) and are similar to the activation energy of water in the adsorbed state in other systems having statistical monolayer or submonolayer coverages: 7.2 kcal/mol for molecular rotation and 6.0 kcal/mol for diffusion in the H,O-SiO, system, or 6.5 kcal/mol for molecular rotation in the H,O-starch system. It was concluded that water in the RBC membrane behaves like water in adsorbed monolayers and that the mobility of the water molecules is changed as a result of their interaction with the RBC membrane. Conlon and Outhred (1978) reported that a single activation energy did not give the best fit to the observed temperature dependence of the water diffusion; two activation energies, one for low and one for high temperature, gave a better fit. They found values of 5.26 kcal/mol for the mean overall activation energy, and 4.21 kcah'mol and 6.72 kcaVmol for the mean activation energies at lower and at higher temperatures, respectively. Morariu et al. (1981) examined, by the same doping NMR method, the temperature dependence of the water exchange time for five samples isolated from different donors. An obvious discontinuity, around 26"C, was noticed in the Arrhenius plot of the water exchange. Two values of the apparent activation energy for water diffusion could be calculated on the basis of this plot. The mean values of the apparent activation energy of the water exchange time at temperatures higher than that of the discontinuity was 5.7 kcal/mol, while at lower temperatures the values of the apparent activation energy were < 1.4 kcal/mol. It was suggested that the temperature effects on water diffusion reflect cooperative processes and possible conformational changes of the membrane proteins induced by a temperature-dependent phase change in the membrane. However, the authors emphasized that it is not clear whether a phase transition of RBC membrane lipids actually occurs between 0" and 37°C. While some investigators have found evidence for a phase change occurring around 20°C (Johnson, 1975; Zimmer and Schirmer, 1974; Bieri and Wallach, 1976), in other studies it has not been possible to detect any phase transition of the acyl chains of the RBC lipids (Davis et al., 1979). On the other hand, Bond and Baumann (1978) emphasized that it is not likely that an entire membrane will exhibit a phase transition analogous to lipid phase transitions, but interaction between the lipids surrounding a protein channel and the protein complex per se could l e d to rather abrupt changes in membrane permeability. It is also worth mentioning the studies of Chow et al. (1981), who have measured the positron lifetimes in human red-cell

288

GHEORCHE BENGA

ghost membranes as a function of temperature. A marked sudden change in the ortho-positronium annihilation rate was found around 20°C. Such a change in the microenvironment in the membrane sensed by ortho-positronium was attributed to a change in water diffusion rate through the membrane, considered to be a consequence of the sudden change in free volume, or fluidities in the lipid bilayer. These are microscopic morphological changes, occurring usually at the less dense area (Chow et af., 1981), instead of the macroscopic morphological change, such as the rigidto-liquid crystalline phase change in the membrane structure. In view of these conflicting reports, it was important to clarify whether any possible temperature-dependent changes in membrane structure occurring between 20” and 30°C have indeed a specific influence on water diffusion. that is, whether the aforementioned “break” in the Arrhenius plot does really reflect a sudden change in water permeability of RBC at a certain temperature. Therefore, we have undertaken in our laboratory an exte_nsivestudy on many subjects. We noticed that an apparent “break” occurred only in some of the samples investigated. Moreover, the discontinuity was much less pronounced compared to that reported previously (Morariu et al.. 1981). I t was clear that some experimental factors were responsible for the “break.” While our study was in progress, Morariu et al. (1985, 1986) showed that apparent thermal transitions are expected to occur when the NMR manganese doping technique is used for the determination of the water diffusion in RBC. These transitions represent a shift from intermediate exchange rates where water diffusion through the membrane is dominant to either fast or slow exchange rates where proton relaxation is the controlling process. An apparent transition will occur around 26°C if a fraction of RBC are permeated by Mn”. We have previously shown (Morariu and Benga, 1977) that no penetration of Mn” into the red cells occurs during brief storing of the blood in the presence of the doped plasma at room temperature. However, the penetration of Mn2+ into the cells at higher temperatures and longer incubation time has not been checked. When the measurements on RBC suspended in the isotonic medium doped with Mn” were performed (Morariu et al., 1981), each sample was run from room temperature to 42°C and then from room temperature to 3°C. This means that the RBC were incubated with MnZ+ for > 1 hour at temperatures above room temperature, sufficient for a penetration of Mn” in the cells to occur. When the sample was then run at temperatures lower than room temperatures, a lower T2Aoccurred because of the increased concentration of Mn” inside the cells, and consequently an artifactual “break” was noticed. We found an easy procedure to avoid this artifact. Three samples were prepared from RBC of the same donor. The first was run at temperatures

TABLE 111 ACTIVATIONENERGY OF

HUMANRBC DIFFUSIONAL PERMEABILITY Number of points per measurement

E, (kcalhnol), mean 2 SE

3

2-4

8.7 2 1.0

3 3

25

3

4.79 2 0.03 5.26 2 0.14

2-37

5

S

16

3-42 7-37 3-37

10

20

7-25

Temperature range ("C1

Number of donors

23-37

2

NMR (T,, 1.7 mM MnCI?) NMR (T2,34.6 mM MnCI,)

3-37 4-37

1

NMR (T,, 13.3 mM MnC12)

Sample and method RBC T,) NMR ("0,

NMR ( T z , 13.3 mM MnCI,) Radiotracer Radiotracer Ghosts Radiotracer NMR (T,, 13.3 mM MnCI,)

3-37 3-42

10

Number of measurements

20

Reference

4

5.84 k 0.21 6.0 2 0.2 5.0 2 0.5

Shporer and Civan (1975) Pirkle el a / . (1979) Conlon and Outhred (1978) Morariu el a/. (1981) This article Vieira et al. (1970) Brahm (1982)

8 7-25

7.2 f 0.5 6.03 2 0.18

Brahm (1982) This article

25

5.7

?

0.4

290

GHEORGHE BENGA

from 5" to 30"C, the second from 30" to 37"C, and the third from 37" to 42°C. lo this way a prolonged incubation of RBC with Mn'+ at high temperatures was avoided. Under these conditions no "break" in the Arrhenius plot of water exchange time occurred. The values of the activation energy of water diffusion in RBC and resealed ghosts are listed in Table 111. For comparison, values obtained by other authors are included.

IV. Conditions for Inhibition of Water Diffusion in RBC and Ghosts A. EFFECTSOF

INHlBlTORS A N D OF

CHEMlCAL MODIF~CATION OF

MEMBRANES An important characteristic of the water permeability of RBC is its inhibition by sulfhydryl-binding mercurial reagents. Macey and Farmer (1970) first showed that the sulfhydryl (SH) reagents p-chloromercuribenzoate (PCMB) and p-chloromercuribenzenesulfonate (PCMBS) can produce a dramatic decrease in osmotic permeability (90% inhibition), and this could be reversed by adding excess cysteine. They suggested that the action of PCMB and PCMBS may be to block water flow through aqueous channels (pores), leaving the lipid bilayer as the only pathway for water permeation. Naccache and Sha'afi (1974) have further characterized the inhibition of osmotic permeability induced by SH reagents. In addition to PCMBS, which produced 80% inhibition, the SH reagent 5,Sf-dithiobis(2-nitrobenzoic acid), or DTNB, was also found to inhibit (by 60%)the water transport in human RBC. Other SH reagents such as N-ethylmaleimide (NEM) and iodoacetamide (IAM) had no signifcant effect on the rate of water transfer in these cells. In subsequent studies Sha'afi and Feinstein (1976) have investigated the effects of a large number of SH reagents on water permeability. It was found that only mercury-containing compounds in which the mercury is close to a hydrophobic, aromatic ring are inhibitors. Among these compounds, PCMBS and fluoresceinmercuric acetate (FMA) were the most potent inhibitors. It was suggested that the SH groups involved in water permeability lie in a hydrophobic protein region, possibly containing an aromatic amino acid. An exception among the mercury-containing SH reagents was reported to be mersalyl, which apparently produced no inhibition of water transport in RBC. In contrast to many studies on osmotic permeability, fewer NMR data on the changes in water permeability following exposure to inhibitors of this transport process have been reported. Consequently we have per-

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

29 1

formed systematic studies on the effects of a variety of reagents and chemical manipulations of membranes (Benga e l al., 1982, 1983b). An important issue was to investigate whether the SH-reducing reagents that have been applied in osmotic experiments showed similar effects on diffusional permeability. As can be seen from Fig. 4, the mercury-containing compounds substantially inhibited the water exchange time through RBC membranes. The degree of inhibition appeared to be dependent on the temperature and time of exposure of RBC to the mercurial as well as on the reagent concentration. Under optimal conditions a similar degree of inhibition was found with all reagents, ranging between 42 and 50%. Maximal inhibition with PCMBS (-45%) was reached in 30-60 minutes with 1 mM and in 20-30 minutes with 2 mM when the incubation was performed

FIG.4. Inhibition of water diffusion in human RBC by SH-reacting reagents. All incubations were performed for 60 minutes at 37°C at a hematocrit of 10% in ISOmM NaCLS mM HEPES, pH 7.4, 5 mM glucose, except for mersalyl and cysteine, where incubations of 15 minutes at 37°C were used. After incubation three washes in the same medium were performed. The results are the means (columns) and the standard deviation (bars on top of columns) for 4-50 determinations. Inhibition was calculated as described in Section 111. based on NMR measurements. Reproduced with permission from Benga et al. (1986b).

292

GHEORGHE BENGA

at 37°C. A similar degree of inhibition was obtained with FMA, but it was much more potent than PCMBS in that maximal inhibition at 37°C was obtained with 125 p M FMA in 15 minutes and half-maximal inhibition occurred at -60 p M (Benga et af., 1982). An important difference in the behavior of FMA compared to other mercurials became apparent when the reversibility of inhibition by cysteine was investigated (Fig. 4). The effect of PCMBS was fully reversible; 10 mM cysteine in 15 minutes returned the water permeability to control values. However, cysteine did not reverse the inhibition induced by FMA, even with 100 mM cysteine o r a longer incubation period. It was evident that even the smaller inhibition (15-17%) induced by 50 p M FMA could not be reversed by cysteine (Benga et al., 1982). A new kind of experiment reported by us (Benga ef af., 1983b) was that in which the effects of two mercurials on the water exchange time were studied. From Fig. 4 it can be seen that maximal inhibition is obtained with either FMA or PCMBS. N o further inhibition could be obtained after incubation with both reagents, regardless of the order in which these agents were used. However, in all cases where FMA was present in the incubation media the inhibition of water diffusion could not be reversed. even by a large excess of cysteine. A comparison of the structures of PCMBS and FMA (Fig. 5) illustrates differences which may account for the greater potency of FMA and the irreversibility of its inhibition by cysteine. The FMA molecule can be divided into two almost symmetrical halves, each containing a reactive atom adjacent to an aromatic ring. The inhibition by FMA suggests that there are a pair of SH groups in close proximity that react with the SH reagents. This would offer an explanation for the greater potency of FMA and the failure of cysteine to reverse its inhibition, since FMA would bind

PCMBS

FMA

FIG. 5 . Structures of PCMBS and FMA.

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

293

more tightly and cysteine molecules would have to gain access to two SH groups to release the inhibitor. Such a hypothesis would suggest that to block water transport, the binding of two molecules of PCMBS may be required. From the time course of the inhibition of diffusion by PCMB and the effect of pH on this inhibition, Ashley and Goldstein (1981a) concluded that there were probably two SH-reactive sites: one in a hydrophobic environment and one in the anion channel. Whether there is any relationship between the two SH groups suggested by FMA inhibition and these two reactive sites is not yet known. An alternative explanation is that the larger aromatic ring structure of FMA produces a higher affinity for an SH group at the active site. Other SH reagents, aside from mercurials, did not appear to inhibit significantly the diffusional permeability (Fig. 4). It should be emphasized that this also applies to DTNB, for which an inhibition of only 10% was noticed, in contrast to a 60% inhibition of osmotic water permeability reported by Naccache and Sha’afi (1974). From Fig. 4 it can be seen that none of the SH reagents which do not contain mercury prevented the inhibitory effect of a mercurial. This suggests that the SH groups involved in water transport do not react with any of these SH reagents. However, it seems that preincubation with NEM potentiated the inhibitory effects of mercurials. We have also studied the effects of inhibitors of other transport processes. None of these markedly increased water exchange time. It should be emphasized that H, DIDS, a specific inhibitor of the anion transport system in RBC membranes, does not inhibit the diffusional water permeability. H, DIDS does not prevent the inhibitory effect of PCMBS. Phloretin, which also inhibits anion transport, as well as other facilitated transport processes in the RBC membrane, also did not change the water permeability. Other compounds that react with amino groups, such as Nsuccinimidyl-3-(2-pyridylthio)propionate (Carlsson et al., 1978) or methoxy-nitro-tropone had no significant inhibitory effect on water exchange. Water diffusion appears not to be energy dependent, since blocking the glycolysis with NaF did not affect the water exchange time. In view of further labeling experiments (with radioactive PCMBS), the kinetics of inhibition induced by PCMBS on water diffusion in RBC and ghosts has been studied in detail (Benga et al., 1986b,c). The time course of development of the water diffusion inhibition by 1 mM PCMBS on RBC at 20°C is shown in Fig. 6a. It appears that inhibition develops in two steps: the first rapid step is reached in 15 minutes, and this amounts to about half of the maximal inhibition. The inhibition rate then slows, reaching maximum inhibition after 60 minutes. With longer incubation times some hemolysis occurs, giving an apparent drop in the degree of inhibition.

294

GHEORGHE BENGA

50 40 0 c

G 30

n .-

-s .c

20

10 10

20 PCMBSIrnMl

FIG.6 . (a) The time course of inhibition of water diffusion in RBC induced by I mM PCMRS at 20°C. RBC were incubated for various durations at a hematocrit of 10%. and then washed as in Fig. 4. The inhibition was calculated on the basis of N M R measurements as described in Section 111. (b) Dependence of the inhibition of water diffusion in RBC, induced by various concentrations of PCMBS at 37°C. on a preincubation with NEM, a noninhibitory SH reagent. For samples marked by empty symbols, RBC were preincubated for 60 minutes at 25°C with 2 mM NEM at a hematocrit of 25% in 150 mM NaCI-5 mM sodium phosphate buffer. pH 7.5; they were then diluted with medium containing 2 mM N E M to a hematocrit of 10% with PCMBS to give the final concentrations indicated, and incubated for IS minutes (0) or 30 minutes (0)at 37°C. Other samples were incubated only with PCMBS (and no NEM) for 15 minutes (.)and 30 minutes ( 0 )at 37°C. After incubation, three washes of RBC in the same medium containing 2 mM NEM were performed, and the inhibition was calculated on the basis of NMR measurements as described in Section 111. Reproduced with permission from Benga er (I/. (1986b).

The two-step manner of the development of water inhibition induced by PCMBS, in agreement with similar features of osmotic permeability described by Naccache and Sha’afi (1974), may correspond to two populations of SH groups which participate in the control of water transport, differing in location. Some would appear to be located close to the outer surface of the membrane. being readily accessible to PCMBS, and others

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

295

located deeper in the membrane interior. Different populations of membrane S H groups have been described by other authors using various techniques (Abbot and Schachter, 1976). The inhibition is dependent not only on the temperature and time of exposure of RBC to the mercurial but also on preincubation of the cells with noninhibitory SH reagents. As shown in Fig. 6b, the maximal inhibition of water exchange could be induced at 37°C in 15-30 minutes by incubating the RBC with 0.2-0.5 mM PCMBS after a preincubation with NEM, or in 30-60 minutes with 1.0-2.0 mM PCMBS if no preincubation with NEM was employed. This indicates that treatment of RBC with NEM prior to exposure to PCMBS results in the inhibition of water exchange occurring faster and at a lower concentration of mercurial. Studying the effect of PCMBS on the inhibition of water diffusion across ghost membranes, we found that maximal inhibition was obtained at 37°C in 30 minutes with 0.1 mM (Fig. 7). This is 10 times less than the concentration required for maximal inhibition of water diffusion in RBC. At concentrations >0.2 mM, shorter values of T2L were noticed, indicating the penetration of manganese inside the ghosts. A similar finding could be noticed in RBC at concentrations of PCMBS >10 mM. When the reversibility by cysteine of the PCMBS-induced inhibition of water diffusion was studied, it appeared that a >lo-fold excess of cysteine is required to remove the inhibition. This is again in contrast with studies on RBC in which 10 mM cysteine fully reversed the inhibition induced by I mM PCMBS. It may be concluded that PCMBS is -10 times more potent in inhibiting water diffusion in ghosts compared to RBC (Gh. Benga et al., 1985a). This can be due to the absence in ghosts of hemoglobin, which probably binds the excess of PCMBS in RBC.

A 3 7 37'C . C

30. 301 c 0

5 20. c

10. 0

10

20

0'

c

30 min

FIG. 7. The time course of inhibition of water diffusion in resealed ghosts induced by 0. I mM PCMBS alone (0,0 ) and after a preincubation with 1 mM NEM (A).

296

GHEORGHE BENGA

The time course of the inhibition induced by PCMBS on resealed ghosts was also studied at 0°C (Fig. 7). At 0°C no significant inhibition occurred with as much as 30 minutes of incubation. At 37°C at least 15 minutes of incubation were necessary for a significant inhibitory effect to occur, and the maximal inhibition was obtained in 30 minutes with 0.1 mM PCMBS, without preincubation with NEM. If a NEM preincubation is used, a nearly maximal inhibitory effect occurs in 5 minutes at 37°C with 0.1 mM PCMBS. B. EFFECTSOF PROTEOLYTIC ENZYMES The effects of SH reagents on the water permeability strongly suggested that membrane proteins are involved in this transport process. Consequently, it was interesting to see whether the incubation of RBC with proteolytic enzymes, known for their effect on membrane proteins, would affect the water diffusion. In the conditions of incubation used in our studies (Benga er al., 1982), trypsin digested glycophorin without significantly changing the pattern of other polypeptides in RBC membrane. In contrast, with chymotrypsin an extensive digestion of the band 3 protein occurred. This is in agreement with Passow ef a/. (1977), who showed that only chymotrypsin and not trypsin digests band 3 protein in intact RBC. However, neither trypsin nor chymotrypsin treatment significantly inhibited water diffusion through RBC membranes (Table 1 in Benga et al., 1983~).At the same time the enzymic treatment of membranes did not prevent the inhibition induced by mercurials. In contrast, the effect of mercurials appeared to be slightly potentiated by the enzymic treatment. As far as papain is concerned, in some experiments exposure of RBC to papain appeared to prevent the inhibitory effect of subsequent incubation with PCMBS (Benga er ai.,1983~).However, we have reevaluated the effects of papain and found that it does not hamper the inhibitory effect of subsequent incubation with PCMBS, if a lower concentration of inhibitor is used. It appears, therefore, that neither kind of proteolytic enzyme that digests glycophorin (such as trypsin) or band 3 protein (papain and chymotrypsin) influences the water diffusion in RBC or prevents the inhibitory effect of subsequent incubation with PCMBS.

V. Uptake and Binding of [Z03Hg]PCMBSby RBC

In order to understand better the development of water inhibition induced by PCMBS and the reason for the potentiating effects of NEM preincubation, we have studied the uptake of [*03Hg]PCMBSby RBC and

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

297

10.

b

30

60

90

120

rnin

FIG. 8. (a) Time course of the uptake of ['''HglPCMBS by RBC (solid lines) and the binding to the membrane proteins (dashed lines) after incubations at 20°C with 1 m M ['O'HglPCMBS without (0, 0 ) or after a preincubation at 20°C for 60 minutes with 1 mM NEM (0,W). (b) The yield of binding of [Z03HglPCMBSto the membrane proteins expressed as percentage of the total uptake of [''Hg]PCMBS by the RBC, following incubations at 20°C with the I mM mercurial preceded (0)or not (0) by a preincubation with I mM NEM after 60 minutes at 20°C. All incubations were performed at a hematocrit of 10% in 150 mM NaCI-5 mM HEPES, pH 7.4.5.5 mM glucose, followed by three washes in the same medium. When an NEM preincubation was used, 1 mM NEM was also present during the washes. Reproduced with permission from Benga et a / . (1986b).

298

GHEORGHE BENGA

its binding to the membrane. Uptake of the PCMBS is slightly slower in cells preincubated with NEM compared to those with no exposure to NEM (Fig. 8a), but exposure to NEM prior to PCMBS actually enhances the binding of the inhibitor. With no NEM preincubation, PCMBS binding ranges from 3 nmol/mg protein (an amount that is bound within 2 minutes at 20°C, when very little inhibition of water diffusion occurs) to a constant 7 nmol PCMBS/mg protein after 60 minutes of incubation. In contrast, following incubation with NEM the binding of PCMBS continuously increases over the 30- to 90-minute period of time explored. NEM increases the yield of binding (expressed as percentage PCMBS bound to the membranes in relation to the total amount of PCMBS captured by the RBC; Fig. 8b). The results indicate that the potentiating effect of NEM on

c 40. ._ W +

e

a W

t

m

k 30 E

W

E

-F z 2o v)

m

n

I 10

20

30

40

50

60

70

80

90 rnin

FIG.9. The binding of [m'Hg]PCMBS to RBC membranes in r.nrious conditions of incubation. Washed RBC or resealed ghosts were suspended in a wash m d u m (150 mM NaC1-5 mM phosphate buffer, pH 7.5) containing 2 mM NEM at a cytocrit of 25%. and incubated for 60 minutes at 25°C. They were then diluted with the same medium containing NEM to a cytocrit of 10% with [%'Hg]PCMBS added to RBC to give final concentrations of 0.5 mM (0,A,0 ) and 1 mM (W. V). and to resealed ghosts of 0.1 mM (0, A ) and incubated at the temperatures indicated. After completion of the incubation. resealed ghosts and RBC were washed three times in 20 volumes of I50 mM NaCI-5 mM sodium phosphate, pH 7.5. 2 mM NEM. by centrifugation at 8000 g and 2000 g , respectively, for 10 minutes at 4°C. Purified membranes were prepared from the intact RBC and resealed ghosts to remove ["'HglPCMBS that may have bound to hemoglobin and other cytoplasmic components. Other details are described in Section 111. Reproduced with permission from Benga er a / . ( I986b t .

WATER EXCHANGE THROUGH T H E ERYTHROCYTE MEMBRANE

299

PCMBS-induced inhibition of water diffusion may be a result of an enhanced binding of PCMBS to the membrane. The actual binding of PCMBS to the membrane appears to be strongly dependent not only on the duration but also on the temperature of incubation of the RBC and resealed ghosts (Fig. 9). For RBC an incubation time of 15 minutes with 0.5 mM PCMBS results in a binding ranging from 3 nmol/mg protein (at 0°C) to 19 nmol/mg protein (at 37"C), while for an incubation time of 30 minutes the amount of PCMBS is higher and increases with temperature from 7 nmol/mg protein at 20°C to 11 nmol at 27°C and 27 nmol/mg protein at 37°C. For longer incubation times at 37°C the binding further increases, reaching 37 nmoUmg protein in 90 minutes. In the temperature range 20"-27"C, changes in the slope of the binding curve occur, perhaps indicating changes in membrane organization (Benga and Holmes, 1984) favoring the access of mercurial to the SH groups involved in water transport. VI. Identification of Membrane Proteins Involved in the Water Permeability of Human RBC

A new approach to the study of transport processes in RBC membrane has been the use of chemical probes (Cabantchik et al., 1978). This has allowed the identification of one major protein of the membrane, the band 3 protein, as being involved in anion transport, based on the selective binding to this protein of a radioactively labeled inhibitor of anion transport (Cabantchik and Rothstein, 1974). The use of various SH reagents for the study of water transport through the RBC membrane provides valuable information on the particular membrane-associated SH groups involved. From the data already mentioned it is clear that SH reagents that d o not contain mercury (DTNB, IAM, and NEM) do not inhibit water transport. Neither do they prevent inhibition by those S H reagents that do contain mercury (PCMBS, PCMB, FMA, and HgCI2). This suggests that the S H groups involved in water transport exhibit some specificity to mercurials, a finding that is important for evaluating the experiments aimed at associating water channels with specific membrane proteins using radioactive-SH labeling methods. To identify particular proteins involved in the specific binding of PCMBS, it is important to carry out the [203Hg]PCMBS-bindingexperiments under conditions in which inhibition of water diffusion is known to occur. Brown et al. (1975) were among the first to report the results of labeling experiments using [I4C]DTNBafter preincubation of the cells with NEM and IAM. A binding of [I4C]DTNB to the band 3 protein was found and they suggested that band 3 is involved in water transport, on

300

GHEORGHE BENGA

the assumption that DTNB is an inhibitor of this process. Although Naccache and Sha'afi (1974) report an inhibition of osmotic permeability by DTNB, work in other laboratories as well as the present findings show no inhibition of water diffusion by DTNB. Later work by Sha'afi and Feinstein (1976) presented evidence for selective labeling of band 3 with ['4C]PCMBS after preincubation of RBC with IAM, NEM, and mersalyl, compounds that were considered not to inhibit transport. However, as already shown. mersalyl is a strong inhibitor of diffusional water permeability. Solomon et al. (1983) have subsequently reported the localization of radiolabeled PCMBS on band 3 following incubation with human RBC ghosts at 0°C for 2 minutes. In our experiments we also found that a small amount of PCMBS binds under these conditions, but longer incubation times or higher temperatures are needed before binding can be quantitatively associated with any inhibition of water diffusion. A problem in using radioactive probes to identify transport components is that of recognizing a relatively small number of specific transport sites against a large background of nonspecific sites (Gh Benga et al., 1985b). Accordingly, we have designed procedures to minimize the number of nonspecific sites. A relatively high concentration of NEM was used for preincubation of RBC and resealed ghosts before and during the treatment with [zo3Hg]PCMBS.NEM was also present in the washing medium after incubation with mercurial. To avoid displacing the mercurial during electrophoresis, disulfide-reducing agents were omitted from the sample while NEM was added. This avoided the possibility that some thiol groups, not capable of reacting with either reagent in the absence of detergent undergo some exchange on adding sodium dodecyl sulfate, or SDS (Ralston and Crisp, 1981). Taking these precautions, it was interesting to find that under conditions of inhibition of water diffusion the [203Hg]PCMBS-labeling pattern of membrane proteins revealed significant binding of the inhibitor only to polypeptides migrating as band 3, band 4.2, and band 4.5. Such a pattern of labeling. obtained with resealed ghosts incubated at 37°C for 5 minutes, is shown in Fig. 10. A similar pattern was obtained with RBC incubated at 37°C for 15 minutes. These conditions are the same as those under which maximal inhibition of water diffusion occurs and which a minimal amount of ['03Hg]PCMBS is bound per milligram of membrane protein. The distribution of radioactivity in the various polypeptide fractions of resealed ghosts and RBC membranes labeled with [203Hg]PCMBSunder conditions to block nonspecific binding with NEM is presented in Table IV. Assuming values of MW 95,000 for band 3 and 55,000 for band 4.5, it was possible to estimate the amount of PCMBS bound per mole of polypeptide. Under conditions of inhibition of water diffusion, when the major

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

301

I500

1.E

1000

1.c

m >

U

t.

A

2 I-

-

U

a

0

n

500

0.:

c

2

+ 10 MIGRATED DISTANCE ( c m )

FIG. 10. The binding of ["'HglPCMBS to proteins in resealed ghosts resolved by polyacrylamide gel electrophoresis. After a preincubation with 2 mM NEM for 60 minutes at 25°C resealed ghosts were incubated in the same medium containing 0. I mM ["'HglPCMBS for 5 minutes at 37°C or 15 minutes at 0°C. Purified membranes were prepared as described in the legend to Fig. 9. Membrane polypeptides were separated by electrophoresis, gels were cut into 2-mm slices. and the radioactivity was measured as described previously (Benga ef a / . , 1986b). Densitometric scans of Coomassie blue-stained gels are shown by the continuous tracing. The radioactivity, illustrated by the bar graphs. is the difference for the incubation of resealed ghosts for 5 minutes at 37" C (i.e., when a maximal inhibition of water diffusion occurs), and for IS minutes at 0°C (when no inhibition is noticed). The nomenclature derived from Fairbanks ef al. (1971)is used to identify membrane proteins, and F represents the migration of the tracking dye. Reproduced with permission from Benga er al. (l986b).

part of the radioactivity is distributed in band 3 and 4.5 we found - 1 mol PCMBS bound per mole of polypeptide. The total amount of SH groups in RBC titrated by mercury has been mol per cell (Rothstein, 1981; Sutherland et al., reported as 4.1 x 1967). Of these, membrane SH groups constitute <5%, with an equal quantity attributable t o reduced glutathione. The remaining groups are

302

GHEORGHE BENGA TABLE IV DISTRIBUTION OF PROTEIN AND SH GROUPS AMONG MEMBRANE POLYPEPTIDES OF RESEALED GHOSTSINCUBATED WITH NEM AND [203Hg]PCMBSUNDER CONDITIONS OF NO INHIBITION (A) OR MAXIMAL 1NHlBlTlON (B) OF WATER DIFFUSION'

Band number

I+2 2.1 3 4.1 4.2 4.5 5 6 7

8

Protein

Radioactivity

(%I

(%)

A

B

23.0 10.7 28.9 2.9 4.5 9.3 8.0 I .2 1.1 0.5

21.0 8.9 27.2 2.5 6.0 10.7 9.0 I .3

I .o

0.3

A

1.5 1.o

24.3 4.7 3.6 19.2 8.8 4.1 8.1 3.6

Number of SH groups per protein molecule

B

A

B

0.1 I .9 46.8 6.2 11.2 19.9 3.7 1.2 2.4 0.5

0.02 0.02 0.08 0.14 0.06 0.12 0.05 0.14 0.23 0.18

0.06 0.35 1.38 1.66 1.14 0.86 0.15 0.26 0.61 0.35

"Resealed ghosts were incubated for 60 minutes at 25°C with 2 mM NEM in 150 mM NaCI-6 mM phosphate buffer (pH 7.5). at a cytocrit of 25%. They were then diluted with the same medium containing NEM to a cytocrit of 10% with ["'Hg]FCMBS added to give a final concentration of 0. I mM. After an incubation of 5 minutes at 0°C (A) or 37°C (B), the ghosts were washed three times in 20 volumes of 150 mM NaCI-5 mM sodium phosphate (pH 7.5)-2 mM NEM, by centrifugation at 8000 g for 10 minutes at 4°C. Purified membranes were prepared and analyzed as described in the legend to Fig. 10. Total binding of ["'HglPCMBS was I. I 1 nmoUmg protein in experiment A and 8.37 nmoVmg protein in experiment B.

predominantly associated with hemoglobin. Using a conversion factor of 1 mg protein being equivalent to I. I x lo9cells, the total amount of membrane SH groups found in the present study turns out to be 165 nmoVmg protein. Alternatively, using the conversion factors of Dodge et al. (1963) or Lepke et al. (1976) of 1 mg protein equivalent to 1.4 x lo9 cells or 1.95 x lo' cells, respectively, then the SH-group concentrations in the membrane would be 129 nmoVmg or 93 nmol/mg protein. Reported values for this concentration vary from 89 to 144 nmoYmg protein. PCMBS reacts with only 25% of these mercurial-titratable groups in hemoglobin-free ghosts (Vansteveninck et al., 1965), which is in close agreement with the value of 37 nmol/mg protein found in the present experiments. Sutheriand et al. (1967) reported that the binding of PCMBS to intact RBC was associated with an inhibition of glucose uptake, a loss of K ', and decreased osmotic fragility. Indeed, we noticed a degree of

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

303

hemolysis occurring in parallel with the decrease in the inhibition of water diffusion at longer than optimal incubation times or higher concentrations of PCMBS. When PCMBS is used on RBC samples preincubated with NEM, inhibition of water diffusion occurs when only 10 nmol of PCMBS are bound per milligram of protein. Subtracting the amount of PCMBS bound at 0°C in 15 minutes, when no inhibition of water diffusion occurs, the minimum number of SH groups apparently involved in water diffusion would be 7 nmoVmg protein. The present work shows that when conditions of maximal inhibition of water diffusion are chosen, together with minimal PCMBS binding to the membrane, the mercurial binds to band 3 and the polypeptides in band 4.5. These findings strongly point to the proteins in these bands being associated with water channels in RBC. Using an average conversion factor of 1 mg protein corresponding to 1.5 x lo9cells, the 7 nmol/mg protein of SH groups associated with water diffusion would correspond to -2.8 x lo6 SH groups per cell. The reactivity of SH groups in band 3 protein has been studied by a number of authors (Ramjeesingh et al., 1983; Rao, 1979; Rao and Reithmeier, 1979), and there seems to be a general agreement that this protein has six groups, five of which are reactive to NEM and the sixth reactive only to mercurials such as PCMBS. Indeed, in the present work, under conditions where all the NEM-reactive SH groups in band 3 are blocked, 1 rnol of PCMBS binds per mole of band 3. The number of these monomers per cell is considered to be 1.2 x lo6 (Wheeler and Hinkle, 1981). Consequently, of the 2.8 x lo6 SH groups per cell involved in inhibition of water diffusion, approximately half are binding to proteins other than band 3. The other SH groups appear to be associated with bands 4.2 and 4.5. The former is an extrinsic protein located in the cytoplasmic region of band 3 (Haest, 1982) and is reported to be highly reactive to SH reagents (Rao, 1979). Under the conditions of inhibition of water diffusion used in the present work, - I mol of PCMBS binds per mole of protein. Since this protein occurs as 0.23 x lo6 copies per cell, an equivalent number of SH groups could be accounted for in PCMBS binding. Band 4.1, another extrinsic protein in contact with the intrinsic membrane domain (Haest, 1982), occurs at the same frequency and also seems to bind 1 mol PCMBS per mole of protein. However, this binding could be a reflection of its close proximity on the gels to both band 3 and band 4.2. The remaining PCMBS-binding protein is band 4.5, which occurs at -0.9 X lo6 copies per cell. Again, under conditions of inhibition of water diffusion, this band binds - I mol PCMBS per mole of protein. It can be seen, therefore, that there is good agreement between the total number of SH groups reactive to ['03Hg]PCMBS when water diffusion is inhibited and the number of copies of the individual polypeptides binding the reagent.

-

304

GHEORGHE BENGA

It could be assumed that only intrinsic proteins crossing the lipid bilayer would provide a transmembrane pathway for facilitated water diffusion. The labeling of bands 4.1 and 4.2 may be explained mainly by their association with band 3, the main route of PCMBS permeation into the cell (Brahm, 1982; Macey el ul., 1972; Rothstein, 1981). Our results clearly associate this protein, together with the polypeptides migrating in the region of band 4.5. as the intrinsic protein components involved in water channels. Band 4.5 has previously been identified with other transport functions. notably the transport of glucose (Jones and Nickson, 1981 ; Wheeler and Hinkle. 1981; Young et d.,1983). Reconstitution studies using purified bands 3 and 4.5 incorporated into liposomes may be useful in order to clarify further their role in water transport.

VII. Electron-MicroscopicStudies Freeze-fracture electron microscopy (EM) could, in principle, aid in identification of those membrane-spanning proteins associated with the water channels. Pinto da Silva (1973) has shown that water sublimes from the membrane in regions beneath the intramembrane particles (IMP). He interpreted this finding as evidence that the water initially below the particles may be able to escape through aqueous channels associated with the particles. The IMP observed on the freeze-fracture faces of RBC membranes are thought to correspond to the main integral proteins set in the lipid bilayer (Pinto da Silva and Branton, 1972). It has been suggested that the IMP are primarily band 3 tetramers or dimers (Glaubensklee rt ul., 1982; Margaritis et ul., 1977; Weinstein er a / . , 1980). There are also reports of changes in particle size and distribution in pathological conditions associated with a decrease in water permeability of RBC. In the abnormal RBC of the McLeod phenotype, a reduction in the average size and an increase in the IMP density in parallel with a decreased water permeability have been reported (Galey et ul., 1977).This was interpreted (Glaubenskleeet a / . , 1982) in terms of an abnormality in band 3 association. In Duchenne muscular dystrophy (DMD) a reduced number of IMP has been observed (Wakayama et ul., 1979) while the water permeability of the RBC in such patients is also decreased (see Section VIII). Some typical freeze-fracture aspects of membranes in RBC treated with NEM or NEM plus PCMBS are shown in Fig. 11. The distribution and sizes of the IMP are illustrated in Table V. It can be seen that neither the treatment with the noninhibitory SH reagent nor the subsequent inhibition of water diffusion by PCMBS produces a significant alteration in these parameters. Similarly, scanning electron microscopy (SEM) showed there

FIG. 11. Freeze-etched human red cell membranes. Washed RBC suspended at a hematocrit of 25% in basal medium containing 150 mM sodium phosphate buffer, pH 7.5, were incubated for 60 minutes at 25°C with (a) no addition, or with (b, c) 10 mM NEM. They were then diluted with the medium at a hematocrit of 10% and incubated for 60 minutes at 37°C with (a) no addition, (b) 10 mM NEM, or (c) 2 mM NEM and I mM PCMBS. After incubation, three washings of the cells in the basal medium were performed. Thereafter an aliquot from each sample was taken, brought to pH 5.5, and incubated for 60 minutes at 37°C. These are the samples: (d) control; (e) treated with NEM and PCMBS. The cells were frozen on the specimen mounts by rapid immersion in “slushed” nitrogen ( -210°C). Samples were fractured and “etched” for 10 minutes at - IOOT, 1 x torr, before replication using a Polaron E7500 freeze-fracture apparatus. The replicas were cleaned for 10 minutes in sodium hypochloride solution (10-14% available chlorine) followed by two washes in distilled water, mounted on uncoated m m e s h copper grids, and examined and photographed at 100 kV using a Philips EM 301G transmission electron microscope. Bar = 100 nm. Reproduced with permission from Benga ef al. (l986b).

306

GHEORGHE BENGA TABLE V INTRAMEMBRANEPARTICLE SIZEA N D NUMBERFOR ERYTHROCYTES EXPOSEDTO SH REAGENTS" IMP parameter

Sample RBC, pH 7.4 Control N EM NEM + PCMBS RBC, pH 5.5 Control N EM N E M + PCMBS

Particle density (no. of particles per square micron)

Particle diameter

3311 -t 115 3147 112 3395 t 106

95 t 6 92 t 5 9629

3146 3095 2995

97 95 93

* 2

?

2

120 115 116

(A,

2

5

2 5 2

7

"Preparation of samples was as described in the legend to Fig. 1 I . For each sample -10 hcture faces have been evaluated. Membrane particles were counted for 0.25-pn2areas, and mean particle diameters were determined from measurements of ,300 particles per sample. The results represent means f SE. No significant differences have been found (p > 0.05, by the Student's r-test).

FIG. 12. Scanning electron micrographs of human RBC. Washed RBC were incubated and washed as described in the legend to Fig. I I. They were then fixed with 1% glutaraldehyde in 150 mM NaCI-5 m M sodium phosphate buffer. pH 7.5, postfixed in 1% osmium tetroxide, dehydrated and critical point-dried in CO,. Following mounting and sputter-coating with gold. samples were examined and photographed in a Philips EM 501B scanning electron microscope. f a ) No addition (control sample). (b) Sample incubated with NEM. (c) Sample incubated with NEM and PCMBS. Bar = 2.5 km. Reproduced with permission from Benga rl

nl. (1986b).

WATER EXCHANGE THROUGH T H E ERYTHROCYTE MEMBRANE

307

to be no changes in the shape of RBC or resealed ghosts under the same conditions (Fig. 12). These results indicate that major morphological changes do not take place in RBC membranes after treatment with the SH reagents used, under the present conditions.

VIII. Alterations of Water Permeability of Human RBC in Disease Processes It seems that a number of diseases are associated with a decreased water permeability of RBC, although the molecular defect is not yet known. In the abnormal RBC of the McLeod phenotype a defect in band 3 association has been suggested to play an important role (Glaubensklee ef al., 1982). An abnormally low permeability of RBC membranes in epileptic children has been suggested (Benga and Morariu, 1977) to reflect a generalized membrane defect affecting water permeability, responsible for the disturbances of water metabolism in human epilepsy (I. Benga et al., 1985). We have obtained interesting results on RBC water permeability in DMD subjects. DMD is a progressive degenerative muscle disorder inherited as an X-linked recessive trait, associated with proximal muscle weakness and atrophy, as well as with involvement of different organs (Radu, 1978; Walton and Gardner-Melwin, 1979). Elevated levels of muscle enzyme activities in the serum and their depletion in the muscle tissue implied an abnormality in the plasma membrane as the probable site of the genetic defect, although the primary inherited metabolic defect is unknown. The elucidation of the primary biochemical defect by studies of muscle tissue obtained by biopsy sample is made extremely difficult by the presence of atrophied, fibrous tissue, by the change in innervation that may cause multiple secondary biochemical changes, and by small yield (Rowland, 1980; Sato et al., 1978). All of these shortcomings in investigations of muscle directly prompted the search for a tissue which was readily accessible, could be sampled for biopsy repeatedly, and would yield enriched plasma membrane fractions. The human RBC appeared to be an obvious choice (Plishker and Appel, 1980). Since the publication of the original reports (Brown et al., 1967) of abnormalities in RBC from patients with muscular dystrophy, many biochemical, morphological, and biophysical alterations of RBC from DMD patients have been described (Plishker and Appel, 1980; Roses et al., 1980; Jones and Witkowski, 1983). Despite the disagreement in many of the findings, the overall results do support the concept that the RBC membrane is altered and that membrane defects are widespread in DMD.

308

GHEORCHE BENGA

The previously reported NMR measurements of water diffusion through membranes in DMD patients have given conflicting results. Conlon and Outhred (1972) could not find any significant difference in the water exchange time values for one patient and two carriers compared to controls. On the contrary. Ashley and Goldstein (1981b) reported significant differences in water exchange through RBC membranes of DMD patients compared to controls. In a first series of investigations we have compared the water permeability of RBC from 16 DMD patients (3 to 25 years old) with control males matched for age. The NMR measurements were performed by pairing one DMD patient with an age-matched control (Serbu et al., 1986). Higher T2L values were noted for the DMD erythrocytes, either in the presence or in the absence of PCMBS. The differences between the T2i values in the DMD patients and controls was statistically highly significant ( p < 0.001) using the paired Student's t-test. In later work we have randomly performed the N M R measurements and compared the water permeability of RBC from 39 DMD patients with 3 1 controls (Table VI). The difference was still statisticalfy highly significant (p < 0.001, using the unpaired Student's r-test). There were also

TABLE VI PARAMETERS C H A R A ~ ~ E K ITHE Z I NWATER G DIFFUSION ACROSS RBC MEMBRANE IN DMD PATIENTS COMPARED TO CONTROLS"

Age B O U P

Parameter

All subjects

T& T,. P

4-7 years

T:, T,, P

8-16 years

T&

Controls 6.55

2

(n

=

0.68 31) 6.85 6.69 6.95 rt_ 0.52 (n = 8 ) 7.29 6.28 6.59 2 0.50 (n = 35) 6.89 6.64

Statistical significance (controls versus DMD patients)

DMD patients 7.50 t 0.95 (n = 39) 7.89 5.81 8.26 2 1.43 (n = 6 )

p

< 0.001

p

< 0.01

8.74

5.24 7.12 rt_ 0.66 ( n = 17) 1.47 6.13

p < 0.005

~

"Experimentalconditions are described in Section VIII. Measurements have been performed at 37°C and values are expressed as mean ? SD: n is the number of patients investigated. For each patient the measurements were performed in duplicate. The p values were obtained using the unpaired Student's rtest. T; and T,. are expressed in milliseconds and P in c d s e c x lo'.

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

309

significant differences between the DMD patients and controls when two groups were formed according to the age: one group ranging from 4 to 7 years and the other ranging from 8 to 16 years (Table VI). A decreased water permeability of RBC appears to be associated with DMD. Our findings are of interest for several reasons. We have performed the NMR measurements using the method of Conlon and Outhred and still found significantly higher values of T2i in DMD patients compared to controls. This indicates that (1) DMD is associated with a decreased water permeability of RBC, and (2) differences in water permeability between DMD patients and controls can be detected by NMR using either low manganese concentrations, as employed by Ashley and Goldstein (1981b), or higher concentrations, as described in this article. Our data definitely indicate a decreased water permeability of RBC in DMD patients compared to controls. As described previously, we have identified the proteins in bands 3 and 4.5 as being associated with water channels in RBC. It is interesting, therefore, that freeze-fracture studies of DMD erythrocyte membranes indicate a decrease in the number of intramembranous particles (Wakayama e? al., 1979), which consists mainly of integral membrane band 3 protein and associated lipids. Taking into account these findings, Ashley and Goldstein (1981 b) suggested that the decreased water permeability may reflect a decrease in the amount of band 3 protein in the DMD erythrocyte membranes; this could reflect either a smaller amount of band 3 protein available to the membrane or a less favorable lipid environment for its incorporation. IX. Conclusions on the Mechanisms of Water Exchange in RBC Taking into account our present knowledge on plasma membranes as consisting of integral membrane proteins embedded in a lipid bilayer, two parallel pathways could contribute to the water permeability. One of them involves the membrane lipid phase and the other is related to proteins. As pointed out by Fettiplace and Haydon (1980), as yet there is no conclusive evidence for the mechanisms by which water crosses lipid bilayers. There are several possibilities: ( I ) Water may be aggregated in the bilayer such that it forms more or less continuous files (or pores) from one aqueous phase to the other (Huang and Thompson, 1%6); (2) water may be completely dispersed, as in dilute solution (Hanai and Haydon, 1966); or (3) water may cross the bilayer by transient holes, pockets of free volume in the lipid bilayer, or mobile structural defects (known as “kinks”), which occur because of conformational changes in the hydro-

3 10

GHEORGHE BENGA

carbon chains due to thermal fluctuations in membrane lipid (Trauble, 1971).

Although we do not know in detail the mechanisms of water movement across lipid bilayers, a comparison of their permeabilities with those of RBC membranes is of great interest: It has already been mentioned that the activation energy of the water diffusion across the RBC membrane is much lower than that of water diffusion across fluid bilayers. This suggests that a major role in RBC water permeation must be played by hydrophilic channels accommodated in proteins. Moreover, the osmotic permeability of RBC is one order of magnitude higher than that of the lipid bilayer, while the diffusional permeability of RBC is -3-Fold higher. Thus the POSIP,ratio is larger than one would expect for comparable artificial membranes. This again points to some aqueous pores in the RBC membrane proteins as being responsible for the additional permeability. Finally, the inhibitory effect of mercurials on water permeability is considered as an argument for the presence of aqueous pores located in red cell membrane proteins (Sha'afi, 1981; Macey, 1984). After treatment of RBC with PCMBS, the water permeability is reduced to that expected for the equivalent artificial membrane, the ratio of osmotic to diffusional permeability approaches unity, and the activation energy is increased to values corresponding to the bilayer (Macey and Farmer, 1970; Macey et al., 1972; Fettiplace and Haydon, 1980). An important issue is connected with the proportion of water permeating across the lipid and protein counterparts in RBC membranes. There are widely different views on this. Lawaczek (1987) claims that water permeates predominantly through the lipid part, along lipid-protein boundaries and through dynamic or static defects within the membrane; the author questions the existence of specific channels and further claims that, even if they exist, they are not important for the water translocation across RBC membrane. A contrasting view is that of Macey (1984), who for many years has maintained that there are channels in membrane proteins that transport only water and nothing else; that is, they are specific water channels. This author claims that such channels are responsible for 90% of water permeability, while only 10% of water is crossing through the lipid bilayer. These proportions are deduced from the 90% inhibition of osmotic permeability produced by PCMBS. As mentioned earlier, PCMBS inhibits only -50% of the diffusional water permeability. This would suggest that different mechanisms operate in case of water diffusion compared to water flow under an osmotic gradient, or that membrane alterations are induced by the osmotic stress.

WATER EXCHANGE THROUGH THE ERYTHROCYTE MEMBRANE

31 I

It is interesting how the concept of aqueous channels or pores in RBC membranes has developed and changed over the years. One of the earliest models (Stein and Danielli, 1956) consisted of a pore lined with a latticework of polar sites; the possibility of water entering and moving through such pores was explicitly pointed out. Koefoed-Johnsen and Ussing and Pappenheimer et al. (cited by Solomon, 1968) have introduced the concept of the equivalent pore as a description of the path taken by lipid-insoluble molecules. These authors have pointed out that the ratio of the hydraulic conductivity (measured under either an osmotic or a hydraulic pressure gradient) to the water diffusion coefticient (as measured by tracers) can be used to calculate an equivalent pore radius for channels in the membrane. Based on measurements of Po,(Side1 and Solomon, 1957) and Pd (Paganelli and Solomon, 1957), an equivalent radius of 4.5 A for human red cells, 5.9 A for dog, and 4.1 A for cow (Rich et al., 1968) were calculated. However, these calculations have been questioned by Galey and Brahm (1985).

Solomon et al. (1983) proposed a molecular model for an aqueous pore in RBC membranes. It consists of a channel 9 A in diameter that passes between the two halves of the band 3 dimer allowing regulated fluxes of water, cations, anions, and nonelectrolytes. However, such a common pathway for a variety of small molecules appears quite improbable. Although Solomon and co-workers reported some data suggesting a relation between red cell anion exchange and water transport (Yoon et al., 1984), or between nonelectrolytes and water transport (Chasan et al., 1984; Toon et al., 1985), such links are still debatable (Dix et al., 1985). In fact, Toon and Solomon (1986) have admitted that urea and water inhibition sites may be located on different proteins. These authors claim that the fluxes of water, urea, and anions are linked in the modulation sense: a single inhibitor is modulating the flux of more than a single permeating species by allosteric action on a single protein or a protein complex. Comparative physiological arguments have also been used (Brahm and Wieth, 1977; Macey, 1984) to suggest that water, urea, and anion transport do not enter the cell through the same aqueous channel. Human RBC have a high water and high urea permeability, chicken cells a low water and low urea permeability, duck cells a high water and low urea permeability, and amphiuma cells a low water and high urea permeability. Despite the different and sometimes contradictory views of various authors regarding the channels for water transport in RBC, one thing is obvious: the membrane proteins accommodating the water channels can be identified if the reaction with PCMBS is performed under conditions of maximal inhibition of water transport after the nonspecific SH groups are

312

GHEORGHE BENGA

blocked by NEM. Under these circumstances, our data (described earlier) indicate that band 3 and band 4.5 proteins accommodate the water channels in human RBC. A proper understanding of the molecular architecture and function of such channels will be obtained after further progress has been made in deciphering the structure of RBC membrane proteins. Bands 3 and 4.5 have been sequenced (Kopito and Lodish, 1985; Mueckler et al., 1985); however, their three-dimensional structure is still not known. Moreover, the band 3 seems to exist in sifu in dimeric and tetrameric form (Weinstein et al.. 1980: Schubert, 1987). While some evidence exists that the dimer is the functional form for the anion exchange (Cuppoletti et al., 1985). the tetrameric form may be the pore-forming unit for water transport (Benz et d.,1984). Since an aqueous channel might have a rather complicated structure (Ruff, 1986), it is clear that a lot of work is still necessary to elicit a clear picture of the mechanisms of water permeation across cell membranes. This review may be concluded with the following remarks. First, the principal reason for postulating pores involved in water (and solute) transport in red cells was to explain permeabilities which appeared to be too high to be accounted for by permeation through the lipid counterpart of the membrane (Macey, 1979). Second, the inhibitory effect of mercurials on water permeability offered the possibility of “marking” the SH groups associated with such pores by using radioactive mercurials. However, it was difficult to recognize the specific transport sites against a large background of nonspecific sites. For this purpose we have designed appropriate procedures (Benga et a / . , 1986b,c) which enabled u s to find that under conditions of maximal inhibition of water diffusion the inhibitor is bound to polypeptides migrating as bands 3 and 4.5. These findings point to an association of the proteins in these bands with water channels in RBC. Third, the NMR method is a very useful tool for studying water diffusion in RBC, having the advantages of relative technical simplicity, speed of data collection, and reproducibility of results. As such, the NMR method is very appropriate for further studies on changes in water diffusion following various chemical manipulations of the membranes, with the aim of understanding the molecular mechanisms of this transport process. In addition, the method is of great value for investigating alterations of water diffusion in disease processes, as documented by our studies on epileptic (Benga and Morariu, 1977) or DMD subjects (Serbu et a / . , 1986).

ACKNOWLEDGMENTS

I am grateful to many colleagues who participated in the experiments reported here and to the Ministry of Education and Teaching. the Academy of Medical Sciences. and the

WATER EXCHANGE THROUGH T H E ERYTHROCYTE MEMBRANE

3 13

National Council for Science and Technology (Romania), the Wellcome Trust (United Kingdom), and the National Science Foundation (United States) for financial support.

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Index

A Actin Caenorhabditis elegans and, 107, 108, 116 DNA methylation and, 212 microtubules and, 50, 65 Actinomycin D, UDG and, 137, 156 Adenine DNA uracil and, 125, 130 UDG and, 136 S-Adenosylmethionine, UDG and, 153, 162 Age-related changes, DNA methylation and, see DNA methylation Agglutination, Rmmecium and, 5, 6 Albumin, DNA methylation and aging, 210 development, 208, 209 a-Amanitin, Caenorhabditis elegans and, 89, 94 Amino acids erythrocyte membrane, water exchange and, 290 microtubules and, 55, 58, 62 UDG and, 135 Amphidinium klebsii, dinoflagellate sexuality and, 263 Anlagen, &ramecium and, 17 nuclear differentiation, 9-13 nuclear reorganization, 4, 15, 16 Antibodies, see also Monoclonal antibodies Caenorhabditis eleguns and, 88, 89, 103, 120 DNA uracil and, 141, 142 microtubules and bipolar neuroepithelial cell, 29 microtubule-associated proteins, 68 tubulin, 52, 53, 56

Antigens Caenorhabditis elegans and, 89, 100 DNA methylation and, 185, 210, 211 DNA uracil and, 142 Aphidicolon, Caenorhabditis elegans and, 94 Apyramidinic/apurinic (AP) sites DNA uracil and, 150, 170 UDG and, 140, 143-147, 156 Asymmetry, zygotic, Caenorhabditis elegans and, 85, 99, 110-112 cell division axes, 109 first cleavage, 100, 101 microfilaments, 104-108 oocyte, 99, 100 P granules, 101-104 unequal cleavage, 108, 109 Ataxia telangiectasia, DNA uracil and, 142, 143, 147, 148 ATP, microtubules and, 34 5-Azacytidine DNA methylation and, 182, 211 genome, 186, 187, 189 globin genes, 203, 204 proviral sequences, 194 X-linked genes, 197, 199, 200 DNA uracil and, 167, 169 UDG and, 162

B Bacillus stearothermophilus, UDG and, 138 Bacillus subtilus, UDG and, 134, 136, 137, 149 Bacteria DNA uracil and, 132, 164 UDG and, 126, 164

317

318

INDEX

Basal bodies dinoflagellate sexuality and, 256 microtubules and, 23, 28, 29 Basement membranes, eye, epitheliumcapillary interactions and, 235, 237, 239-241 Bipolar neuroepithelial cell, microtubules and, 28 interphase, 29-35 mitotic cells, 35, 36, 38, 41, 42 morphology, 28, 29 neuorogenesis, 25-27 Bisulfite, DNA uracil and, 129, I30 Blastomeres, Caenorhabditis elegans and, 87, 119 cell fate determination, 90-99 future perspectives, 119 genetic approaches, 114, 116, 117 zygotic asymmetry, 99-1 12 Bloom’s syndrome cells, DNA uracil and, 142, 157 Brain DNA methylation and, 185, 202, 212, 213 eye, epithelium-capillary interactions and, 222 microtubules and, 70, 71 microtubule-associated proteins, 62-64, 66 tubulin, 56, 59-61 5-Bromodeoxyuridine, DNA methylation and, 204 5-Bromouracil DNA uracil and, 125, 126, 130, 165 UDG and, 135 Bruch’s membrane, eye, epithelium-capillary interactions and, 242

C

Caenorhabditis elegans, embryogenesis in, 78, 85, 87, 88, 118, 119 body plan, 82-84 cell fate determination, 89, 90 ablation, 90, 91 blastomeres, 90-92, 97-99 DNA, 94, 95 early interactions, %, 97 fusion, 92, 93 induction, 95, 96 cell lineage, 84-86

future perspectives, 119-121 genetic approaches, 116-118 mutants, 112-116 life cycle, 81, 82 tissue specificity, 88, 89 zygotic asymmetry, 99, 110-112 cell division axes, 109 first cleavage, 100, 101 microfilaments, 104-108 oocyte, 99, 100 P granules, 101-104 unequal cleavage, 108, 109 Calcium microtubules and, 36, 58, 64, 65 hrarneciurn and, 5 , 11, 12 Calmodulin, microtubules and, 63-65, 68 Cancer, DNA uracil and, 142 Capillary, see Eye, epithelium-capillary interactions and Carcinogen treatment DNA methylation and, 210 UDG and, 155-159, 164 Catastrophe, microtubules and, 24, 38 Cuthamnthus, see Vinca cDNA Caenorhabditiselegans and, 120 DNA methylation and, 186, 209 Cell ablation, Caenorhabdifiselegans and, 90,91, 95, 119 Central nervous system, microtubules and, 70 migratory young neuron, 42, 43 neuorogenesis, 21, 22, 25 neuronal development, 50, 51 tubulin, 53 Centrioles Caenorhabditiselegans and, 109 microtubules and, 23 bipolar neuroepithelial cell, 28, 29 migratory young neuron, 42, 43 mitotic cells, 36, 39 Centromeres, DNA methylation and, 189, 197 Centrosomes Caenorhabditiselegans and, 85, 109 microtubules and, 38, 43, 46, 62 Cemtiurn comu&rn, dinoflagellate sexuality and, 254, 261, 263, 268 Chicken vitellogenin I1 gene, DNA methylation and, 213, 214 aging, 206-208 development, 205, 206 Chloramphenicol, UDG and, 137, 149

319

INDEX

p-Chloromercuribenzenesulfonate (PCMBS), water exchange and, 296, 310, 311 diffusional permeability, 280 disease processes, 308 electron microscopy, 304 inhibition of diffusion, 291-293, 295, 296 membrane proteins, 299-304 p-Chloromercuribenzoate (PCMB), water exchange and, 290, 294 Chloroplasts, dinoflagellate sexuality and, 263 Choriocapillaris, see Eye, epitheliumcapillary interactions and Chorionic gonadotropin, DNA methylation and, 212 Chromatids, DNA uracil and, 126. 160 Chromatin Caenorhabditis elegans and, 101 DNA methylation and, 204, 205, 210, 213 DNA uracil and, 160 UDG and, 140, 159 Chromosomes Caenorhabditis elegans and, 82,94, 114, 118 dinoflagellate sexuality and, 249, 257, 258 function, 269, 270 nuclear phenomena, 263, 264 DNA methylation and, 182, 183 X-linked genes, 197-201 DNA uracil and, 126, 171 repair, 167, 169, 170 sources, 129 dUTP and, 160 microtubules and, 36, 38, 39 Fbramecium and, 13, 14 UDG and, 153, 157 AP endonucleases, 147 deficient mutants, 147, 149 eukaryotes, 142 Chymotrypsin erythrocyte membrane, water exchange and, 296 microtubules and, 63 Cingulum, dinoflagellate sexuality and, 250, 256

Cleavage Caenorhabditis elegans and cell fate determination, 90-92, 94, 97, 98 genetic approaches, 113, 115-117 zygotic asymmetry, 100, 101, 105, 107-111

DNA uracil and, 125, 126, 130, 132 dUTPase and, 150

microtubules and, 35 UDG and, 134, 145 Clones Caenorhabditis elegans and, 84, 87, 120 cell fate determination, 90, 94, 98 genetic approaches, 117 dinoflagellate sexuality and, 261 DNA methylation and, 212, 213 a-fetoprotein, 209 genome, 186 globin genes, 203 highly repetitive sequences, 189, 191 X-linked genes, 199 hramecium and, 12 Colcemid Caenorhabditis elegans and, 104 microtubules and, 45 Colchicine, microtubules and bipolar neuroepithelial cell, 31, 33, 34 mitotic cells, 41 postmigratory young neuron, 45, 49 tubulin, 52 Cold treatment, microtubules and bipolar neuroepithelial cell, 31, 34 microtubule-associated proteins, 62, ,63 migratory young neuron, 36, 39-42 neuronal development, 50 postmigratory young neuron, 46, 50 tubulin, 53, 55, 56, 58, 60, 61 Colobomata, eye, epithelium-capillary interactions and, 226 Conjugation, hmmecium and, 1, 16 early micronuclear migration, 5 gametic nuclei exchange, 8 nuclear differentiation, 11, 12, 14 nuclear reorganization, 15 processes, 2-4 Crypthecodonium, dinoflagellate sexuality and, 254 Cryprhecodonium cohnii, dinoflagellate sexuality and, 253, 254, 257, 258 Cyclic AMP, microtubules and, 64 Cycloheximide, UDG and, 156 Cyclosis dinoflagellate sexuality and, 262, 263 hmmecium and, 5, 7, 11 Cyst formers, dinoflagellate sexuality and, 253, 267, 268

environmental control, 269 life cycles, 256-258 nuclear phenomena, 264

320

INDEX

Cysteine erythrocyte membrane, water exchange and, 290, 292, 293, 295 UDG and, 135 Cytidine, DNA uracil and, 127 Cytochalasin Caenorhabditis elegans and, 104, 109 microtubules and, 34. 45 Cytoplasm Caenorhabditis elegans and, 89, 119 cell fate determination, 92-95, 98 future perspectives, 120 genetic approaches, 113. 114, 117 zygotic asymmetry, 101, 103, 107-111 dinoflagellate sexuality and, 253, 260, 264 DNA methylation and, 205 DNA uracil and, 140 erythrocyte membrane, water exchange and, 303 eye, epithelium-capillary interactions and, 234, 235 microtubules and, 21, 23 bipolar neuroepithelial cell, 29 migratory young neuron, 42 tubulin, 56 Cytosines DNA methylation and, 184, 188 DNA uracil and, 125, 126, 171 repair, 165 sources, 127-131 UDG and, 153, 154, 157. 162, 163 deficient mutants, 148, 149 prokaryotes, 135, 136, 138 D

dAMP, DNA uracil and. 146 dCMP DNA uracil and, 132 UDG and, 153 dCTP, DNA uracil and, 132 Deamination DNA methylation and, 188 DNA uracil and, 126, 171 repair, 164 sources, 127-132 UDG and, 153, 154, 157 AP endonucleases. 148 prokaryotes, 135, 138 Dendrites, microtubules and, 21 microtubule-associated proteins, 61, 65, 66, 68

neuronal development, 50, 51 postmigratory young neuron, 43-45, 48-50 tubulin, 58, 60, 61 Deoxy-5-methylcytosine (dSmC), DNA methylation and, 187, 188 Depolymerization Caenorhabditiselegans and, 104 microtubules and, 24 bipolar neuroepithelial cell, 31, 33-35 microtubule-associated proteins, 62 migratory young neuron, 42 mitotic cells, 38, 39, 41 postmigratory young neuron, 46, 49 tubulin, 55, 57, 58 Determinators, DNA methylation and, 182 Diffusion, erythrocyte membrane, water exchange and, 279, 284, 289, 310, 312 disease processes, 308 electron microscopy, 304 ghosts, 282, 283 inhibition, 290-296 linear, 276 membrane proteins, 299, 300, 302, 303 methodology, 280-282 PCMBS, 299 permeability measurement, 274 pH, 282, 285 temperature, 285-288, 290 Dinoflagellate sexuality, 249, 250 cyst formers, 267, 268 environmental control, 268, 269 function, 269, 270 future research, 270 life cycles, 254 cemtium cornutam, 261 Crypthecodonium cohnii, 257, 258 Gloeodinium montanum, 261, 262 Nmtiluca xintillans, 260, 261 kridinium cinctum, 255 -25 7 kridinium inconspicuum, 258-260 nuclear phenomena, 262-267 patterns, 253, 254 reproduction history, 250-253 5,5’-Dithiobis(2-nitrobenzoicacid) (M’NB), erythrocyte membrane, water exchange and, 290, 293, 299, 300 DNA, see also DNA methylation; DNA polymerase; DNA uracil; Uracil-DNA glycosylases Caenorhabditd elegans and cell fate determination, 94, 95, 98 future perspectives, 120

INDEX zygotic asymmetry, 110

kramecium and, 16, 17 meiosis, 6 nuclear differentiation, 10, 13, 14 nuclear reorganization, 4, 15, 16 DNA methylation, age-related changes and, 181, 213, 214 aging, 183, 184

albumin aging, 210 development, 208, 209 chicken vitellogenin I1 gene aging, 206-208 development, 205, 206 chorionic gonadotropin, 212 a-fetoprotein aging, 210 development, 208, 209 genome, 184 aging, 187-189 development, 184-187 globin genes aging, 204, 205 development, 201-204 a2 U-globulin, 210 highly repetitive sequences, 189 aging, 190, 191 development, 189, 190 MHC class I genes, 210, 211 oncogenes, 212 proviral sequences, 191 aging, 195, 196 development, 191-195 roles, 181-183 X-linked genes aging, 200, 201 development, 197-200 DNA polymerase DNA uracil and, 125 sources, 131, 132 dUTP and, 161 dUTPase and, 150-152 UDG and, 155-157 AP endonucleases, 144-146 eukaryotes, 142 DNA uracil, 125, 126, 170, 171 repair, 164-170 sources dUMP, 131-134 nucleotides, 127-130 DNase Caenorhabditis elegans and, 104

321

DNA methylation and, 204, 206, 213 UDG and, 135 dNTP. DNA uracil and, 141

Dmsophila Caenorhabdiris elegans and, 111 DNA uracil and, 170

Ihmmecium and, 11 UDG and, 134, 148 Drugs, microtubules and bipolar neuroepithelial cell, 31, 33-35 mitotic cells, 36, 39, 41, 42 postmigratory young neuron, 46, 49 tubulin, 58, 61 dTTP DNA uracil and, 171 repair, 167, 170 sources, 131, 132, 134 methotrexate and, 161 UDG and, 161 Duchenne muscular dystrophy (DMD), erythrocyte membrane, water exchange and, 304, 307-309, 312 dUMP DNA uracil and, 125, 170, 171 repair, 165, 167, 169, 170 sources, 127, 131-134 dUTP and, 159, 160 dUTPase and, 149, 150 UDG and, 126, 159, 161, 162 AP endonucleases, 146, 147 deficient mutants, 148, 149 eukaryotes, 141 prokaryotes, 135, 137, 138 dUTP, 159-161 DNA uracil and, 131, 132, 134, 167, 171 dUTPase and, 149 methotrexate and, 159-161 UDG and, 134, 138, 141, 161, 162 dUTPase, 149-153 activity, 156, 158 DNA uracil and, 125, 126, 132, 169. 170 dUTP and, 159 mutants, 149-153 UDG and, 136, 144 Dynamic instability, microtubules and, 24

E Early micronuclear migration, Ihmmecium and, 4, 5, 16 EDTA, UDG and, 135, 136, 138, 141

322

INDEX

Electron microscopy Caenorhabditis elegans and, 83, 88, 89, 103, 108 erythrocyte membrane, water exchange and, 304-307 microtubules and, 34, 44 hramecium and, 8 Embryogenesis, Caenorhabditis elegans and, see Caenorhabditis elegans, embryogenesis in Endocytosis, eye, epithelium-capillary interactions and, 235 Endonucleases DNA methylation and, 183, 210, 212 chicken vitellogenin I1 gene, 205 genome, 185 globin genes, 201, 202, 204 highly repetitive sequences, 189-191 DNA uracil and, 170 dUTPase and, 150 UDG and, 134, I56 apyramidinic/apurinic, 143-147 eukaryotes. 140 Endoplasm, hramecium and, 11 Endoplasmic reticulum DNA methylation and, 1% microtubules and, 44 Enzymes Caenorhabditis elegans and, 118 DNA methylation and, 1% DNA uracil and, 125, 126, 170 repair, 165, 170 sources, 127, 132 erythrocyte membrane, water exchange and, 2%, 307 microtubules and, 55, 63, 64 UDG and, 134, 135, 153-159, 162 AP endonucleases, 143-146 eukaryotes, 138, 140-142 prokaryotes, 136, 138 Epidermal growth factor, DNA methylation and, 185 Epilepsy, erythrocyte membrane, water exchange and, 307 Epithelium-capillary interactions in eye, see Eye, epithelium-capillary interactions and Epitopes, microtubules and, 68 Equilibrium, erythrocyte membrane, water exchange and. 273, 275 Erythrocyte membrane, water exchange and, 273, 309-312 diffusional permeability, 279, 284, 289

ghosts, 282, 283 methodology, 280-282 pH, 282, 285 temperature, 285-288, 290 disease processes, 307-309 electron microscopy, 304-307 inhibition of diffusion, 290-296 membrane proteins, 299-304 PCMBS, 296-299 permeability measurement nonstationary methods, 274, 275 steady-state conditions, 275 Escherichia coli Caenorhabditis elegans and, 81 DNA uracil and, 125 repair, 164-166, 170 sources, 127, 131 dUTPase and, 149-152 UDG and, 126, 134, 153, 162 AP endonucleases, 143-147 deficient mutants, 147-149 eukaryotes, 141 prokaryotes, 135-137 Estradiol, DNA methylation and, 205, 206 Estrogen DNA methylation and, 208, 213 eye, epithelium-capillary interactions and, 224 N-Ethylmaleimide (NEM) erythrocyte membrane, water exchange and, 312 electron microscopy, 304 inhibition of diffusion, 290, 293, 295, 296 membrane proteins, 299, 300, 302, 303 PCMBS, 296, 298 UDG and, 135, 136, 138 Eukaryotes dinoflagellate sexuality and, 249, 258, 264 DNA uracil and, 164, 166, 170 UDG and. 126, 134, 138-143, 153-159 AP endonucleases, 143, 145, 146 Extracellular matrix (ECM), eye, epithelium-capillary interactions and, 239-241, 244 animal experimentation, 237 histologic widence, 224 Eye, epithelium-capillary interactions and, 221, 224, 225, 244 animal experimentation, 237 histologic evidence, 221 correlation, 222

323

INDEX explanations, 224 polarization, 222-224 human histopathology, 226, 227 intra-RPE in rats, 234-238 RPE-choriocapillaris extracellular matrix, 240, 241 interface, 225, 226 mechanisms, 239, 240 soluble factors, 241-244 sodium iodate retinopathy in rabbits, 227-236

F a-Fetoprotein, DNA methylation and, 198, 214 aging, 210 development, 208, 209 Fibroblast growth factor, eye, epitheliumcapillary interactions and, 239-241, 243 Fibroblasts DNA methylation and, 214 genome, 186, 188 globin genes, 204 highly repetitive sequences, 190 proviral sequences, 194 X-linked genes, 198-200 DNA uracil and, 134 UDG and, 156-158, 163 AP endonucleases, 145 eukaryotes, 140, 142 Fluoresceinmercuric acetate (FMA), erythrocyte membrane, water exchange and, 290, 292, 293 Fluorescence Caenorhabditis elegans and, 89 eye, epithelium-capillary interactions and, 229, 234 microtubules and, 31, 36, 38, 53 Founder cells, Caenorhabditi%elegam and, 84 Fusion Caenorhabditis elegans and, 92, 93 hramecium and, 8, 13

G

Gamete formation, dinoflagellate sexuality and, 255, 256 Gametic nuclei exchange, Rzramecium and, 2, 7-9

Gametogenesis, DNA methylation and genome, 184, 185 globin genes, 201 highly repetitive sequences, 189, 190 proviral sequences, 191 Gastrulation, Caenorhabditis eregans and, 87 cell fate determination, 97 genetic approaches, 112, 113 zygotic asymmetry, 106 Genome, DNA methylation and, 183, 184 aging, 187-189 chicken vitellogenin I1 gene, 205 development, 184-187 a-fetoprotein, 209 proviral sequences, 192 Ghosts, erythrocyte membrane, water exchange and diffusional permeability, 280-282, 285, 288-290 inhibition of diffusion, 293, 295, 296 membrane proteins, 300, 302 PCMBS, 299 Glial cells, microtubules and, 50, 59, 68 Globin genes, DNA methylation and aging, 204, 205 development, 201-204 genome, 186 a2 U-Globulin, DNA methylation and, 210 Gloeodinium montanum, dinoflagellate sexuality and, 254, 261, 262 Glucocorticoids, DNA methylation and, 206 Glucose-6-phosphate dehydrogenase (G6PD), DNA methylation and, 198, 199 Griseofulvin, Caenorhnbditis elegans and, 104

Growth cones, microtubules and, 44-46, 62 GTP, microtubules and, 23-25

H Heart, eye, epithelium-capillary interactions and, 222 Hematopoietic growth factors, eye, epithelium-capillary interactions and, 239 Hemin, DNA methylation and, 203 Hemoglobin DNA methylation and, 202, 204 erythrocyte membrane, water exchange and diffusional permeability, 280, 282, 285

324

INDEX

inhibition of diffusion, 295 membrane proteins, 302 permeability measurement, 274 Hemolysis, erythrocyte membrane, water exchange and, 274, 280, 293 Hermaphrodites, Caenorhubdi!is elegans and, 82-85 future perspectives, 120 genetic approaches, 113, 118 zygotic asymmetry, 99, 103 Herpes simplex virus (HSV), UDG and, 158 Heterochromatin, DNA methylation and, 184, 185, 189 Heterogeneity microtubules and, 21, 70 microtubule-associated proteins, 62, 63 tubulin, 59-61 UDG and, 141, 143 High molecular weight proteins, microtubules and, 62, 67, 68 High-performance liquid chromatography (HPLC), DNA methylation and, 185, 187. 191 High-resolution isoelectric focusing (HIEF), microtubules and, 5 5 , 59 Homology, Ifitamecium and, 17 Hormones DNA methylation and, 206 erythrocyte membrane, water exchange and, 273 microtubules and, 49, 66 Horse radish peroxidase, eye, epitheliumcapillary interactions and, 229, 234, 235 5-Hydroxyrnethylcytosine, DNA uracil and, 128, 129 5-Hydroxymethyluracil, UDG and, 135, 136 Hyperpolarization, fhmmecium and, 11 Hypnozygotic phase, dinoflagellate sexuality and cyst formers, 267, 268 function, 269 life cycles, 256, 257, 260. 261 nuclear phenomena, 262 Hypoxanthine-guanine phosphoribosyltransferase (HGPm), DNA methylation and, 200 Hypoxanthine phosphoribosyltransferase (HPRT), DNA methylation and, 197-200

I

lmmunofluorescence

Caenorhabditis elegans and, 99, 100, 115 microtubules and, 29, 36 Immunoprecipitation, UDG and, 141, 142 Inhibition of diffusion, erythrocyte membrane, water exchange and, 290-296, 311 Insulin, microtubules and, 57, 64 Interferons, DNA methylation and, 210 Intermediate-filament protein, Rzmmecium and, 8 Interphase Caenorhabditis elegans and, 96 dinoflagellate sexuality and, 249 microtubules and, 23 bipolar neuroepithelial cell, 34 mitotic cells, 36, 41, 42 lntracisternal A particle (IAP), DNA methylation and, 213, 214 proviral sequences, 192, 194-196 lntramembrane particles (IMP),erythrocyte membrane, water exchange and, 304. 306 Iodoacetamide, erythrocyte membrane, water exchange and, 290, 299, 300

K Kinetochores, microtubules and, 23 microtubule-associated proteins, 62 mitotic cells, 36, 38, 39, 41

L Leukemia DNA methylation and, 186, 192, 194 UDG and, 141, 155 Light microscopy eye, epithelium-capillary interactions and, 229 microtubules and, 44 Lipids, erythrocyte membrane, water exchange and, 287, 288, 309, 310 Liver DNA methylation and, 210-214 chicken vitellogenin I1 gene, 206, 208 a-fetoprotein, 208-210

325

INDEX genome, 187, 188 globin genes, 202, 205 highly repetitive sequences, 191 proviral sequences, 196 X-linked genes, 200 DNA uracil and, 167, 169, 170 UDG and, 155, 156, 162-164 AP endonucleases, 144 eukaryotes, 140, 141 Localization, hmmecium and, 9-13 Long terminal repeat (LTR), DNA methylation and, 183, 192, 194, 195

M Macronucleus, fhmmecium and, 12, 13 anlagen, 12-14 differentiation, 9-12 regeneration, 4, 14-16 Macular degeneration, eye, epitheliumcapillary interactions and, 221, 225, 226

Major histocompatibility complex, DNA methylation and, 210, 211 Manganese, erythrocyte membrane, water exchange and disease processes, 309 inhibition of diffusion, 295 permeability measurement, 276, 278 Meiosis Caenorhabditis elegans and, 82, 84, 85 cell fate determination, 96 zygotic asymmetry, 99-101 dinoflagellate sexuality and life cycles, 257, 258, 260-262 nuclear phenomena, 262-264 patterns, 254 DNA uracil and, 170 fhramecium and early micronuclear migration, 5 gametic nuclei exchange, 8 induction, 5, 6 nuclear differentiation, 10 nuclear reorganization, 2 products, 6, 7 Melanomas, eye, epithelium-capillary interactions and, 224 Mercury, erythrocyte membrane, water exchange and, 310 inhibition of diffusion, 290-293, 295

membrane proteins, 299-302 PCMBS, 299 Messenger RNA (mRNA) DNA methylation and, 181, 212 genome, 203, 204 proviral sequences, 196 microtubules and, 53, 59, 69 Methionine, UDG and, 162 Methotrexate, DNA uracil and, 159-161 Methylation, see also DNA methylation DNA uracil and, 164-167, 170 dUTPase and, 152 UDG and, 142, 154, 162 5-Methylcytosine DNA methylation and, 182 DNA uracil and, 164 UDG and, 142, 153, 154 Metoprine, DNA uracil and, 160

Micrococcus luteus DNA uracil and, 125 UDG and, 137, 143-146 Microfilaments Caenorhabditis elegans and, 119 genetic approaches, 116 zygotic asymmetry, 100, 104-108, 110, 111

microtubules and microtubule-associated proteins, 63 migratory young neuron, 42 mitotic cells, 41 neuronal development, 50 postmigratory young neuron, 45 Microtubule-associated proteins, 21, 24, 25, 61-70

bipolar neuroepithelial cell, 33, 34 migratory young neuron, 43 mitotic cells, 38 neuronal development, 50 postmigratory young neuron, 48, 50 tubulin, 51, 55, 58, 61 Microtubule-organizing centers, 23 bipolar neuroepithelial cell, 31 Caenorhabditis elegans and, 101 microtubule-associated proteins, 62 migratory young neuron, 42, 43 postmigratory young neuron, 45, 46, 50 Microtubules, 21-25, 69-71 bipolar neuroepithelial cell, 28 interphase, 29-35 morphology, 28, 29 Caenorhubdifiseregans and, 100, 103, 104

326

INDEX

migratory young neuron, 42, 43 mitotic cells cold treatment, 39-41 drugs, 41, 42 mitotic spindle, 36-39 morphology, 35 neuorogenesis, 21, 22, 25-28, 51 neuronal development, 50, 51 fhramecium and. 8-10, 16 postmigratory young neuron, 43, 44 assembly, 45-48 caliber, 48, 49 dendritic tree, 49, 50 elongation, 44, 45 tubulin, 51 cold treatment, 53, 55 heterogeneity, 59-61 subunits, 55-58 synthesis, 51-54 Migration Caenorhabditis elegans and, 84, 85 cell fate determination, 96 zygotic asymmetry, 103, 104, 107 microtubules and bipolar neuroepithelial cell, 28 migratory young neuron, 42, 43 neuorogenesis, 22, 26 neuronal development, 50 tubulin, 52 Migratory young neuron, microtubules and,

Moloney murine leukemia virus, DNA methylation and, 192, 194 Monoclonal antibodies Caenorhabditk elegans and, 89, 120 microtubules and. 38, 56, 62 UDG and, 141, 142, 159 Mouse mammary tumor virus, DNA methylation and, 193-196, 214 Mutants Caenorhabditis elegans and, 119 cell fate determination, 95, 96, 99 future perspectives, 119, 120 genetic approaches, 112-118 zygotic asymmetry, 103 dinoflagellate sexuality and, 258 DNA methylation and, 199, 200, 213 DNA uracil and. 126, 171 repair, 164-167, 170 sources, 127, 129, 130 dUTPase and, 149-153 fhmmecium and, 10, 12 UDG and, 153, 154, 156, 157, 163 AP endonucleases, 143, 146 deficient mutants, 147-149 prokaryotes, 137 Myosin, Cknorhabditk elegans and, 89, 107, 108

N

26, 42, 43

Mitochondria, UDG and, 138, 141, 157 Mitogen, eyc epithelium-capillary interactions and, 241, 243 Mitosis Caenorhabdifiselegans and, 85 cell fate determination, 95 zygotic asymmetry, 99, 101, 107, 110 dinoflagellate sexuality and, 259, 260 DNA uracil and, 160 eye, epithelium-capillary interactions and, 241

microtubules and, 23, 24, 70 bipolar neuroepithelial cell, 28, 29, 34, 35

cold treatment, 39-41 drugs, 41, 42 microtubule-associated proteins, 67 mitotic spindle, 36-39 morphology, 35 neuorogenesis, 22, 26 fbfamecium and, 2, 3, 8, 10

Neovascularization, eye, epitheliumcapillary interactions and, 241-244 Nerve growth factor, microtubules and, 48,58 Neuorogenesis, microtubules and, 70 microtubule-associated proteins, 61-69 migratory young neuron, 42, 43 mitotic cells, 35 postmigratory young neuron, 43, 49 tubulin, 51-61 Neural tubes, microtubules and, 34-36, 41 Neurites, microtubules and microtubule-associated proteins, 66, 69 neuronal development, 50 postmigratory young neuron, 44-46, 48,49 tubulin, 58 Neuroblastoma cells, microtubules and, 34, 58, 59

Neuroepithelial cells, microtubules and, 34, 35, 39

Neurofilaments, microtubules and, 44, 68 Neuronal cells, Caenorhabditis elegans and, 95

327

INDEX Nitrogen dinoflagellate sexuality and, 267, 268 DNA uracil and, 127 Nitrous acid DNA uracil and, 129, 130 UDG and, 148 Nocodazole Caenorhabditis elegans and, 103 microtubules and, 34, 49, 58 Noctiluca scintillaas, dinoflagellate sexuality and, 254, 260, 261, 268 Nuclear cyclosis, dinoflagellate sexuality and, 262, 263 Nuclear envelope, dinoflagellate sexuality and, 249, 263, 264 Nuclear magnetic resonance (NMR), erythrocyte membrane, water exchange and, 290, 312 diffusion, 280-282, 285-288 disease processes, 308, 309 measurement, 276-279 Nuclear reorganization, Ibramecium and, 2-4, 14-16 Nucleic acids, DNA uracil and, 129 Nucleosomes, DNA methylation and, 182 Nucleotides DNA methylation and, 206, 208 DNA uracil and, 125 repair, 165-167 sources, 127-130, 132 dUTP and, 160 dUTPase and, 150 UDG and, 157, 161, 162 AP endonucleases, 145-147 deficient mutants, 148, 149 eukaryotes, 141 prokaryotes, 137 Nucleus Caenorhabditis elegans and, 85 cell fate determination, 93, 95, 96, 98 zygotic asymmetry, 100, 109 dinoflagellate sexuality and life cycles, 257, 260, 261 phenomena, 262-267 microtubules and, 38, 41-43 UDG and, 138

0

Oligonucleotides, DNA uracil and, 151-153 Oncogenes, DNA methylation and, 212

Ornithine carbamoyltransferase (OCT), DNA methylation and, 200 Osmotic permeability, erythrocyte membrane, water exchange and, 274, 310 diffusion, 286 inhibition of diffusion, 290, 293, 294 membrane proteins, 300, 302

P P granules, Caenorhabditis elegans and, 88 future perspectives, 120 genetic approaches, 114, 116 zygotic asymmetry, 103, 105-107, 109, 111, 112 Ftrramecium, fertilization in, 1, 16, 17 early micronuclear migration, 4, 5 gametic nuclei exchange, 7-9 macronuclear anlagen, 12-14 macronuclear differentiation, 9-12 meiosis, 5-7 nuclear reorganization, 2-4, 14-16 Paramyosin, Caenorhabditk elegans and, 94 Peptides, microtubules and, 56, 62, 64, 68 Peridinium, dinoflagellate sexuality and, 254 Bridinium balticum, dinoflagellate sexuality and, 264 Peridinium cinctum, dinoflagellate sexuality and, 253-258, 269 Peridinium inconspicuum, dinoflagellate sexuality and, 254, 258-260, 263, 267 Peridinium tmhoideum, dinoflagellate sexuality and, 268, 269 Permeability, erythrocyte membrane and, see Erythrocyte membrane, water exchange and PH DNA uracil and, 125, 127, 129 erythrocyte membrane, water exchange and, 282, 285, 293 UDG and, 135, 136, 145 P halloidin, Caenorhabditis elegans and, 104, 107 Phenotype Caenorhabditis elegans and, 112-117 erythrocyte membrane, water exchange and, 304, 307 eye, epithelium-capillary interactions and, 244 hramecium and, 14

aa Phosphate, microtubules and, 56, 64 Phosphorylation, microtubules and microtubule-associated proteins, 62-65, 68, 69 migratory young neuron, 36, 38, 43 postmigratory young neuron, 48 tubulin, 57, 58 Photoreceptors, eye, epithelium-capillary interactions and, 244 intra-RPE in rats, 234, 235 RPE-choriocapillaris interactions, 225, 242, 243 sodium iodate retinopathy in rabbits, 227, 229, 234 Photosynthesis, dinoflagellate sexuality and, 249, 264, 267, 269 Phytohemagglutinin, UDG and, 141, 156 Pituitary, DNA methylation and, 211 Planozygotes, dinoflagellate sexuality and life cycles, 256-258, 261 nuclear phenomena, 263, 264 patterns, 254 Plasma membrane eye, epithelium-capillary interactions and, 222-224, 235, 237 microtubules and, 31, 44 water exchange and, 273, 307, 309 Plasmogamy, dinoflagellate sexuality and, 264 Polar body formation, Azramecium and, 17 Polarization, eye, epithelium-capillary interactions and, 222-224, 235, 237 Polyethylene glycol (PEG), microtubules and. 29, 34. 36 Polyinnervation, microtubules and, 51 Polymerization microtubules and, 21, 23-25 bipolar neuroepithelial cell, 31, 34 microtubule-associated proteins, 61, 62, 68, 69 postmigratory young neuron, 45, 46,48 tubulin, 53, 56-58 UDG and, 134 Polypeptides erythrocyte membrane, water exchange and, 301, 303, 312 microtubules and microtubule-associated proteins, 62, 65, 68, 69 tubulin, 53. 59 UDG and, 142 Polyribosomes, microtubules and, 53, 65

Postmigratory young neuron, microtubules and, 43, 44 assembly, 45-48 caliber, 48, 49 dendritic tree, 49, 50 elongation, 44, 45 microtubule-associated proteins, 66 neuorogenesis, 26 tubulin, 52 Posttranslational modification, microtubules and, 57 Potassium, Ibmmecium and, 11, 12 Prokaryotes, UDG and, 134-138, 145, 146 Protein dinoflagellate sexuality and, 249 DNA methylation and, 182, 183, 210-212 globin genes, 205 proviral sequences, 196 DNA uracil and, 129, ,170 dUTP and, 160 erythrocyte membrane, water exchange and, 299-304, 309-312 diffusional permeability, 286, 287 electron microscopy, 304 inhibition of diffusion, 296 PCMBS, 298, 299 microtubules and, 70, 77 microtubule-associated proteins, 61-63, 65, 66, 68, 69 migratory young neuron, 43 mitotic cells, 38, 41 neuroiial development, 50 tubulin, 51, 53, 55-58, 60 Azmmecium and, 8 UDC and, 134 AP endonucleases, 143 eukaryotes, 141, 142 prokaryotes, 135-138 Protein kinases, microtubules and, 64,65 r-Proteins, microtubules and, 62, 65, 68, 69 tubulin, 57, 60 Proteolysis erythrocyte membrane, water exchange and, 296 microtubules and, 62, 63, 68 Provirus, DNA methylation and, 181, 191, 213, 214 aging, 195, 196 development, 191-195 Pseudocleavage, Caenorhabditiselegans and, 101, 103, 104, 107

329

INDEX

R Rabbits, sodium iodate retinopathy in, 227-234 Rats, eye, epithelium-capillary interactions and, 234-238 Red blood cells, water exchange and, see Erythrocyte membrane, water exchange and Relaxation time, erythrocyte membrane, water exchange and, 276-278, 280 Replication, Pommecium and, 14 Rescue, microtubules and, 24, 38 Retinal pigment epithelium (RPE), see Eye, epithelium-capillary interactions and Retinitis pigmentosa, eye, epitheliumcapillary interactions and, 221, 225 Retinoic acid, DNA methylation and, 211 a-fetoprotein, 209 genome, 185, 187 proviral sequences, 195 X-linked genes, 200 Retrovirus, DNA methylation and, 192, 195 Reverse transcriptase, DNA methylation and, 192 Rhodamine, Caenorhabditis elegam and, 107 Ribosomes, microtubules and, 53 RNA, see also Messenger RNA; RNA polymerase; Transfer RNA Caenorhabditis elegans and, 120 DNA methylation and, 183, 194, 196, 213 DNA uracil and, 125 microtubules and, 53 Ihramecium and, 15 UDG and, 135, 136, 142 RNA polymerase, Caenorhabditis elegans and, 89 Rous sarcoma virus, DNA methylation and, 183

S

Saccharomyces cerevisiae DNA uracil and, 130 UDG and, 138 Scanning electron microscopy dinoflagellate sexuality and, 255, 256 erythrocyte membrane, water exchange and, 304 eye, epithelium-capillary interactions and, 227

Serine, UDG and, 135 Skein, firamecium and, 3, 14, 15 Sodium bisulfite DNA uracil and, 129, 130 UDG and, 148. 155, 156 Sodium iodate retinopathy in rabbits, eye, epithelium-capillary interactions and, 227-234

Soluble factors, eye, epithelum-capillary interactions and, 241-245 Steroids DNA methylation and, 206 eye, epithelium-capillary interactions and, 224

Sulcus, dinoflagellate sexuality and, 249, 256

Sulfhydryl reagents, erythrocyte membrane, water exchange and, 311, 312 electron microscopy, 304, 306, 307 inhibition of diffusion, 290-294, 296 membrane proteins, 299, 301-303 Synaptogenesis, microtubules and, 50, 51 Synkaryon, Ruamecium and, 7-9 nuclear differentiation, 9, 10, 12 nuclear reorganization, 2

T Taxol, microtubules and, 45, 63 Temperature, erythrocyte membrane, water exchange and diffusional permeability, 281, 282, 285-288, 290 inhibition of diffusion, 291, 295 membrane proteins, 300 PCMBS, 299 Tetradecanoyl phorbol acetate (TPA), UDG and, 155 Tetrahydrofolate, UDG and, 153 Etrahymena, htnmecium and, 8, 9, 14, 17 Thin-layer chromatography (TLC), DNA methylation and, 187, 188 Thymidine DNA methylation and, 188 DNA uracil and, 160 UDG and, 136, 147, 149 Thymidine kinase, Ibmmecium and, 6 Thymidylate synthase, DNA uracil and, 159, 160, 169, 170

330

INDEX

Thymine DNA uracil and, 125 repair, 164, 165, 167 sources, 131, 132 dUTP and, 160 UDG and, 134-136, 148, 149 Thymus DNA methylation and, 1%. 211 DNA uracil and. 131 UDG and, 140, 148 Thyroid, microtubules and, 49 Tissue specificity, DNA methylation and, 184, 214

genome, 185-187 globin genes, 204 proviral sequences, 192 Trans-acting factors, DNA methylation and, 182, 213

Transcription

cold treatment, 53, 55 heterogeneity, 59-61 microtubule-associated proteins, 63, 64, 67, 69

migratory young neuron, 38, 39, 41 postmigratory young neuron, 44-46, 48 subunits, 55-58 synthesis, 51-54 a-Tubulin, microtubules and, 55-58 P-Tubulin, microtubules and, 5 5 , 58 microtubule-associated proteins, 63, 66 neuronal development, 50 lhmors DNA methylation and, 195, 210 DNA uracil and, 169 eye, epithelium-capillary interactions and, 224

Srosine, microtubules and, 55-57 Tyrosine ligase, microtubules and, 56

Caenorhabditis elegans and, 89 cell fate determination, 94, 99 future perspectives, 120 genetic approaches, 118 DNA methylation and, 181, 182, 210-213 chicken vitellogenin I1 gene, 205, 206 a-fetoprotein, 208, 209 genome, 186 globin genes, 203, 205 proviral sequences, 194, 196 DNA uracil and, 127 microtubules and, 69 hmmecium and, 14 UDG and, 155. 156 Transfer RNA (tRNA), DNA methylation and, 183, 203 Translation Caenorhabditis elegans and, 120 microtubules and, 62, 69 UDG and, 134, 156 Translocation DNA methylation and, 199, 201, 210 microtubules and, 42, 45 Transmission electron microscopy dinoflagellate sexuality and, 255-257, 264 eye, epithelum-capillary interactions and,229 Trypsin erythrocyte membrane, water exchange and, 296 microtubules and, 63 Tubulin, microtubules and, 23, 24, 51, 70 bipolar neuroepithelial cell, 29, 31. 34

U Uracil, see DNA uracil; Uracil-DNA glycosylases Uracil-DNA glycosylases (UDG), 126, 134, 135, 139

activity variation, 154-159 AP endonucleases, 143-147 deamination, 153, 154 deficient mutants, 147-149 DNA uracil sources, 128, 130-132 dUTPase and, 150-153 eukaryotes, 138, 140-143 liver activity, 162-164 methylation inhibitors, 161, 162 prokaryotes, 135-138 UTEX 1336, see kridiniurn cinctum

V

Valinomycine, hramecium and, 12 Vinblastine (VBL) Caenorhabditiselegans and, 104 microtubules and, 33-35, 41 Vinca, microtubules and bipolar neuroepithelial cell, 31, 33, 34 mitotic cells, 41, 42, 58 Vincristine (VCR), microtubules and, 33, 34, 41

331

INDEX Vitamin A, eye, epithelium-capillary interactions and, 225, 235 Vitellogenin, see Chicken vitellogenin 11 gene

development, 197-200

Xenopus, Cuenorhabditis ereguns and, 111 Xeroderm pigmentosum, UDG and, 142, 143

W

Water exchange, erythrocyte membrane and, see Erythrocyte membrane, water exchange and

X

Y Yeast, UDG and, 138

Z 2 u mays, UDG and, 138

X-linked genes, DNA methylation and aging, 200, 201

Zygotic asymmetry, Cuenorhabditis eleguns and, see Asymmetry, zygotic

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