International Review of Cytology: A Survey of Cell Biology, Volume 152

VOLUME 152

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

1949-1 988 1949-1 984 19671984-1 992 1993-

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

Audrey Muggleton-Harris Andreas Oksche Muriel J. Ord Vladirnir R. Panti6 M. V. Parthesarathy Lionel 1. Rebhun L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred Stein Ralph M. Steinrnan M. Tazawa Yoshio Watanabe Robin Wright Alexander L. Yudin

Edited by

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

Jonathan Jarvik Department of Biological Sciences Carnegie Mellon University Pittsburgh, Pennsylvania

VOLUME 152

ACADEMIC PRESS A Division of Harcourt Brace & Company San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

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CONTENTS

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

IX

The Unique Structure of Lepidopteran Spindles I. lntroduction ..

IV. Spindle Architecture . . V. Lepidoptera as Experim

Klaus Werner Wolf ............................. ......................... ........................ ems .........................

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32

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36

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Cellular Mechanisms for the Activation of Blood Coagulation Carolyn L. Geczy I. lntroduction ...................... ..................................... II. Tissue Factor ..................... ..................................

V

49 50

CONTENTS

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Function and Modulation of Expression of Auxin-Regulated Genes Yohsuke Takahashi, Sarahmi Ishida, and Toshiyuki Nagata I. II. 111. IV. V.

Introduction ....... Cells Affected by A Prokaryotic Genes ..... Genes Downregulated by Auxin Future Prospects ......

.........

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

135

Regulation of Mitochondria1 Gene Expression in Saccharomyces cerevisiae Carol L. Dieckmann and Robin R. Staples I. II. 111. IV. V. VI. VII.

Introduction ............ Transcription ........... RNA Processing and Tu Translation ............. Post-translationalFactors ....... Regulation .............. Concluding Remarks ... References .............

145 148 150

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

159 162 164 167 170

Dynamics of the Calcium Signal That Triggers Mammalian Egg Activation Karl Swann and Jean-Pierre Ozil I. Introduction ................................................................................ 11. A Calcium Signal in Eggs at Fertilization .................................................. Ill. Generation of the Calcium Signal .......................................................... IV. Effects of the Calcium Signal on the Egg .................................................

200

V. Summary and Future Directions ........................................................... References .................................................................................

213 215

183 185 189

CONTENTS

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Regeneration of Mammalian Retinal Pigment Epithelium Gary E. Korte, Jay I. Perlman, and Ayala Pollack I. Introduction ................................. ......................................... II. RPE Regeneration in the Mammalian Eye ................................................ 111. Concluding Remarks .......................................................................

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223 224 252 256

Habituation as a Tumorous State That Is Interchangeable with a Normal State in Plant Cells Kunihiko SyGno and Tomomichi Fujita I. II. 111. IV. V.

Introduction .................................................................... Habituated Cells and Reversal of the Habituated Phenotype ................. Properties of Habituated Cells ............................................................. Genetic Tumor as an Example of Exaggerated Habituation ........ Concluding Remarks ........... .................................................... References ......... ..............................

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

275 292

301

This Page Intentionally Left Blank

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

Carol L. Dieckmann (145), Department of Biochemistry, University of Arizona, Tucson, Arizona 8572 1 Tomomichi Fujita (265), DepartmentofPureandAppliedSciences, Universityof Tokyo, Tokyo 153, Japan Carolyn L. Geczy (49), Heart Research Institute, Camperdown, New South Wales 2050, Australia Sarahrni lshida (109),Department of Biology, Faculty of Science, University of Tokyo, Tokyo 113, Japan Gary E. Korte (223), Departmentsof Ophthalmologyand VisualSciences,andAnatomy and Structural Biology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 10467 Toshiyuki Nagata (109),Department of Biology, Faculty of Science, University of Tokyo, Tokyo 113, Japan Jean-PierreOzil(l83), lnstitut Nationalde la RechdrcheAgronomique, Unite de Biologie de la Fecondation, 78352 Jouy-en-Josas, France Jay I. Perlrnan (223), Department of Ophthalmology, Loyola UniversityMedical Center, Chicago, Illinois 60626 Ayala Pollack (223), Kaplan Hospital, Rehovot, Israel, and Hadassah Medical School, Hebrew University, Jerusalem, Israel Robin R. Staples (145), Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 Karl Swann (183), MRC Experimental Embryology and Teratology Unit, St. George's Hospital Medical School, London SWl7 ORE, United Kingdom ix

X

CONTRIBUTORS

Kunihiko SyGno (265),Department of Pure and AppliedSciences, University of Tokyo, Tokyo 153, Japan Yohsuke Takahashi (109),Department of Bio/ogy, Faculty of Science, Universily of Tokyo, Tokyo 113, Japan Klaus Werner Wolf (1), Medizinische Universitaf zu Lubeck, lnstituf fur Bio/ogie, D23538 Lubeck, Germany

The Unique Structure of Lepidopteran Spindles Klaus Werner Wolf Medizinische Universitat zu Lubeck, Institut fur Biologie, D-23538 Lubeck, Germany

I. Introduction

The spindle apparatus plays a central part in the reproduction of cells: it ensures the faithful transfer of one copy of the genome into each daughter cell prior to completion of cytokinesis. Defects in spindle function result in aneuploid cells, which, in turn, may fail to develop any further. If the germ line is affected, the propagation of the species is endangered. Therefore, spindle function is under rigid selection pressure and represents an example of a highly optimized element. This does not mean, however, that spindle structure is unaltered from protists to mammals. Instead, we encounter a great variability among different groups of organisms. These “evolutionary experiments” (Wise, 1988) can be exploited to advance our understanding of cell division. Spindle structure is rather homogeneous among vertebrates. The spindle apparatus is bipolar, with microtubules (MTs) focused toward the spindle poles. These usually consist of a pair of centrioles embedded in a mass of dense material, the pericentriolar material (PCM). The nuclear membrane dissolves at the onset of nuclear division and re-forms around the daughter nuclei in telophase. The mass of membranes in the spindle area is moderate. The chromosomes are consistently monokinetic and the homologs are kept together by chiasmata prior to disjunction in anaphase I of meiosis. Only in a few species do sex chromosomes fail to form chiasmata (Raman and Nanda, 1986). Somatic vertebrate cells are routinely held in culture and are thus amenable to modern experimental approaches such as microinjection and laser microbeam manipulation. Therefore, much work on spindle structure and function is currently carried out using vertebrate cells (see Paweletz and Schroeter, 1987). In new lung cells, chromosomes are more than 30 pm long and the interpolar length of the International Review of Cyfology, Y o / . I52

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Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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spindle is more than 40 pm. Therefore, cultured cells of this type provide a favorable system for light microscopic analyses of mitosis (Rieder and Hard, 1990; Rieder, 1990). When we look at spindles beyond vertebrates, we are faced with a baffling diversity with respect to behavior, form, size, membrane content, and pole structures. Spindles in dinoflagellates are extranuclear and diatoms have a massive central spindle. Many fungi and algae show closed mitosis, that is, the nuclear envelope persists throughout mitosis. A survey of these various forms is given by Amos and Amos (19911, and reviews have been devoted to spindle organization in individual systematic units (Baskin and Cande, 1990; Pickett-Heaps, 1991). Achiasmatic meiosis is not uncommon among lower eukaryotes and chiasma substitutes have evolved, Kinetochores may also extend over considerable portions of the poleward chromosome surface; that is, we are dealing with holokinetic chromosomes. Insects have traditionally provided material for researchers concerned with spindle structure. Diptera (Fuge, 1973; Lin et al., 1981) and Orthoptera species have been favored. Because of their large chromosome size, spermatocytes of Orthoptera have often been selected for micromanipulation studies (Nicklas and Kubai, 1985; Nicklas and Staehly, 1967; Arana and Nicklas, 1992). Work on spindle structure in other insect orders, including Lepidoptera, has advanced at a slow pace. I developed an interest in Lepidoptera spindles some years ago because of the controversy regarding centromere size in this order (discussed in Section 111). What I did not expect at that stage was the wide variability of spindle structure within one species of this order, depending on the cell type. It soon turned out that meiosis in male Lepidoptera shows a set of characters that distinguishes it from nuclear division in other groups. A new term, “sheathed nuclear division,” has been coined to account for the unusual combination of features. This chapter focuses on spindle architecture in Lepidoptera. The variability in spindle structure will be illustrated by describing meiosis as well as mitosis in both sexes. In addition, Lepidoptera show a feature, double spermatogenesis, which is briefly described because it is not that widespread. Eukaryotes usually reproduce sexually. To this end, they form gametes of different sizes: the macro or female gamete and the micro or male gamete. Their fusion gives rise to the zygote. However, there are a few systematic groups where two types of microgametes are regularly produced. This phenomenon is known as double spermatogenesis (FainMaurel, 1966). Although not directly aimed at double spermatogenesis, Jamieson’s book (1987) is also a valuable source of information on this issue. Spermatogenesis in Lepidoptera is in fact a dichotomous process. Bipotent spermatogonia give rise to two different types of spermatocytes.

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These develop into two types of sperm. Eupyrene sperm are fertile, whereas apyrene sperm are anucleated and therefore sterile. The chromatin is lost at the onset of apyrene spermiogenesis. Eupyrene and apyrene spindles differ in structure and behavior. As a rule, chromosome segregation is highly irregular in apyrene spermatocytes. Apyrene meiosis, therefore, adds an intriguing facet to the spindle issue in Lepidoptera. Finally, meiosis in female Lepidoptera is achiasmatic. Although some of these aspects, such as achiasmatic meiosis in females (see Section IV,B), pecularities in the kinetic organization of the chromosomes (see Section HI), and double spermatogenesis (see Sections IV,C and D) have been known for quite some time, it is only recently that more detailed analyses have begun. For the time being, Lepidoptera are not broadly used for research among cell biologists. Therefore, it seems appropriate to provide some basic information on rearing the insects and handling the tissues used for work on spindle structure in order to assist newcomers in the field.

II. Technical Aspects Lepidoptera are a relatively large order with more than 150,000 species described thus far. About 3000 species occur in central Europe and more than 11,000 in the United States and Canada (Borror et al., 1989; Jacobs and Renner, 1988). For the evolution and classification of Lepidoptera, see Common (1975). There are several Lepidoptera species that are not very demanding in their rearing requirements. My favorite organism, the Mediterranean meal moth Ephestiu kuehniella (Pyralidae) formerly Anagasta kuehniella, is kept on rolled oats at 20-21°C in a 12-hr light, 12-hr dark regimen (Traut et al., 1986).For work aimed at sperm, synchronization is probably necessary, since sperm release appears to be controlled by light (Riemann et al., 1974; LaChance et al., 1977; Giebultowicz et al., 1989). For work on spermatogonial and meiotic spindles, sychronization can be dispensed with. I did not detect differences in the number of spindles within testes when animals reared at room temperature under natural conditions were compared with moths taken from a synchronous culture. Continuous light, however, should be avoided because it interferes with the reproductive capacity of the moth (Riemann and Ruud, 1974). E. kuehniella is not diapausing and, although egg deposition decreases somewhat during winter, it is consistently available for experimentation. For starting a mass culture, about 10 insects of each sex are transferred into a plastic container (20 x 20 x 6 cm) in which the bottom is covered with rolled oats. The number of deposited eggs varies and the container

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may become overpopulated. This is definitely the case when early instar larvae start leaving the food layer and are found on the walls of the container. Subdividing the material into two containers is recommended in such cases. Otherwise the metabolism of the numerous animals will generate enough humidity to promote the growth of fungi in the container. The generation time of the mealmoth is about 3 months. Young larvae of other Lepidoptera species may be collected in the field and kept in the laboratory on their natural diet for some time (for a review of Lepidoptera and their food plants, see Hargreaves and Carter, 1986). Synthetic and semisynthetic media have been developed for many Lepidoptera species. It is not surprising that these are most often species of economic importance. One of the best-known species in this order, the silk moth Bombyx mori (Bombycidae), is reared on an artificial diet of “silk mate” (Osanai et al., 1991). An artificial diet has also been described for the tobacco hornworm, Manduca sexta (Sphingidae), the pink bollworm, Pectinophora gossypiella (Gelechiidae) (Bell and Joachim, 1976), and the naval orangeworm, Parumyelois trunsitellu (Pyralidae) (Finney and Brinkman, 1967). A semisynthetic diet has been developed for the codling moth, Cydiu pomonella [ = Laspeyresiu pomonella] (Tortricidae) (Huber et al., 1972). Media for additional species, and handling are described in Smith (1966) and Singh and Moore (1985). How can one obtain Lepidoptera species? Besides approaching colleagues working with specific species or collecting them in the field, one can contact dealers through popular entomological journals. These offer exotic or less common local species. Also, biological supply houses usually provide Lepidoptera larvae. Finally, garden centers occasionally run exhibitions of live insects, including Lepidoptera; these may be approached as a source for experimental material. Before starting an analysis, a check for parasites, symbionts, or viruses within the insects is advisable. An infection need not necessarily rule out further work with the insects, but one should be aware of it. Gottlieb (1972) reported the presence of mycoplasma-like symbionts within the testis sheath of an E. kuehniella laboratory strain and fertility was not affected. In contrast, silkworm lavae infected with mycoplasms showed symptoms of disease (Kawakita et al., 1969). An infection can be also informative. Virus-like particles were found in the testes of the large white Pieris brassicae (Pieridae) captured in the field (Wolf, 1988).The particles, previously located in the cytoplasm, became associated with the chromatin in prometaphase I when the nuclear envelope opened. They remained associated with the chromosomes until completion of meiosis. Thus, the virus-like particles were included in the sperm nuclei and transferred through the male germ line into the next generation (Wolf, 1988). This butterfly was, however, an exception. In a series of other Lepidop-

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tera species caught in the field or fed with plants collected outdoorsDeilephila elpenor (Sphingidae), Znachis io (Nymphalidae), Lepidea amurensis (Pieridae), Orgyia antiqua, 0. thyellina, 0. recens (Lymantriidae), Phragmatobia fuliginosa (Arctiidae), and Yponomeuta spec. (Ypon0meutidae)-I did not detect signs of infections within testicular tissue. Cell lines of somatic tissue of a few Lepidoptera species have been established (Grace, 1962; Hink, 1970; McIntosh and Ignoffo, 1983; Lynn and Oberlander, 1983; Tsang et al., 1985; Hoffmann et al., 1990) and serum-free media for Lepidoptera cell culture are available (LCry and Fbdibre, 1990). Tissue culture cells offer themselves for the study of mitosis in insects using diverse light microscopy techniques and transmission electron microscopy (TEM) (Rieder et al., 1990), but pertinent work in Lepidoptera is still missing. For the more interesting meiotic spindles, one has to work with gonads and eggs. Usually, eupyrene meiotic spindles are to be found in male last instar larvae. Apyrene spindles abound in pupae (Machida, 1929; Virkki, 1963). A precise timetable of dichotomous spermatogenesis has been worked out for the codling moth (Friedlander and HauschteckJungen, 1986) and the tobacco hornworm (Friedlander and Reynolds, 1988). Testes transplantations in the former species showed that an apyrene spermatogenesis-inducingfactor becomes active toward pupation (Jans et al., 1984).However, there are also species such as Papilio rutulus (Papilionidae) (Munson, 1906) and Charaxesjasius (Nymphalidae)(Trentini and Marini, 1986) where eupyrene meiosis persists into pupal and adult stages. A surprising finding has been reported for the European corn borer, Ostrinia nubilalis (Pyralidae). The normal sequence of spermatogenesis, with eupyrene meiosis preceding apyrene meiosis, was followed in facultative diapausing strains of the species. The timing of the formation of the two sperm types was reversed, however, in an obligate diapausing strain (Keil et al., 1990). Spermatogonial divisions are present in all larval instars of male Lepidoptera. Larvae contain two testes which fuse upon pupation (Nowock, 1973). For information on the location, gross morphology, and handling of testes in Lepidoptera, see Emmel(l968). It should be added that intact testes can be cultured for several days after isolation in modified Grace’s medium (Friedlander and Benz, 1981) or in blood plasma of Lepidoptera pupae (Kambysellis and Williams, 1972). These techniques have been developed in order to study the effects of hormones on spermatogenesis. Insect eggs are arrested in the first maturation division until release from the ovary (Zissler, 1992). In E. kuehniella, newly deposited eggs contain metaphase I spindles. Egg deposition can been induced by decapitation of the adult female (Wolf, 1987, 1993). Eggs produced in this way

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have been followed for up to 10 hr after deposition and appear to develop properly. Various fixation schemes have been described for preparing Lepidoptera tissue for TEM. Tolbert and Hildebrand (1981) and Osanai and Kasuga (1990) have used perjodate-lysine-paraformaldehyde fixative. A mixture of glutaraldehyde and paraformaldehyde (Tolbert and Hildebrand, 198 1 ) and phosphate- or cacodylate-buffered glutaraldehyde alone (Gottlieb, 1972; Rasmussen, 1977; Mandelbaum, 1980) has also been used with good success for fixation. Although the quality of preservation varies slightly from species to species and from tissue to tissue, the following scheme for preparing ovaries, testes, and imaginal disks of Lepidoptera species for TEM works well for me. The electron micrographs shown in this chapter are taken from these preparations. For dissection, 1use a Ringer’s solution according to Glaser (1917, cited in Lokwood, 1961). The composition per liter of distilled water is 9 g NaCl, 0.42 g KCI, 0.25 g CaCl,, and 0.2 g NaHCO,. The tissues are then transferred in Ringer’s solution (see above) containing 2.5% glutaraldehyde. After 5 min, 3 volumes of 8% tannic acid (Merck) in phosphate buffer (0.067 M, pH 6.8) are added. The specimens are postfixed in phosphatebuffered OsO, (l%), dehydrated in ethanol, and embedded in Epon 812 (Wolf, 1987). Ultrathin sections are stained with uranyl acetate and lead citrate. Under these conditions, MTs have a straight appearance when viewed with the TEM (Wolf and Bastmeyer, 1991a; Wolf, 1992) and do not show the wavy paths which are considered characteristic of poor fixation (McDonald et al., 1992). Also, membranes are well defined. The eggs of Lepidoptera species possess a resistant shell. When eggs are prepared for electron microscopy, they have to be incised while immersed in fixative. Probably on account of insufficient penetration of the fixative under these conditions, this approach does not preserve ultrastructural details as well as those obtained in testes of E . kuehniella (Wolf, 1987). Neither is incision of eggs always possible. Using microscissors or tungsten needles, I was unable to open eggs dissected out from the moth Orgyia antiqua; the egg shell was too resistant. Indirect immunofluorescence is used to study the overall morphology of the spindle. To label Lepidoptera spindles with antitubulin antibodies, gonads or eggs are minced in a microtubule-stabilizing buffer (100 rnM PIPES, pH6.8, 1 mM MgSO,, 1 mM EGTA, 1% Triton X-100). Cytospin preparations of the lysed spindles are then fixed, stabilized by immersion in 100% methanol ( - 20°C) and incubated with the antibodies. The chromatin is visualized by a DNA-specific fluorescent dye (Wolf and Bastmeyer, 1991 a). Gonial cell divisions in higher insects are incomplete. The germ cells form clones of adhering cells. In males, they are surrounded by a layer

7 of somatic cells. In testes of E . kuehniella, seven or eight sheath cells have been counted around one clone (Garbini and Imberski, 1977; Wolf et a/., 1988). The entire complex is referred to as a cyst. In males and females of higher insects, the individual germ cells are interconnected by so-called polyfusomes (Telfer, 1975). This applies also to testes of Lepidoptera. The presence of polyfusomes in this order has been demonstrated in ultrathin sections and TEM (King and Akai, 1971; Mandelbaum, 1980). Recently, a microspreading technique has been used to display the polyfusomes in both sexes of E . kuehniella (Marec et al., 1993). In ovaries, clusters consisting of eight cells are formed through oogonial divisions; only one completes meiosis and gives rise to an oocyte, whereas the others transform into nurse cells. In male meiosis of most Lepidoptera species, six successive spermatogonial divisions take place. Thus, we meet with 64 primary spermatocytes and, upon completion of meiosis, with 256 spermatids (Virkki, 1969). The germ cells, grouped in a cyst, develop almost synchronously and one can capitalize on this arrangement. Using light microscopy and semithin sections, cysts at the desired stages can be located within the testis follicles. The semithin section may be stained for ease of inspection (Jeon, 1965). Then, the block is selectively trimmed. The meiotic cysts are not tightly packed within the testis follicle and can be recognized individually when the block face is viewed under the dissecting microscope. I usually try to produce serial sections through the remainder of the cyst and a larger number of cells at the desired stage is then available for analysis. More sophisticated methods such as flat embedding of cells previously viewed with the light microscope have not yet been tried in Lepidoptera. These techniques have been developed in order to determine the movements of individual chromosomes or to survey chromosome positions prior to fixation for TEM (Nicklas et al., 1979). It is questionable whether this attempt makes much sense in light of the fact that Lepidoptera chromosomes are unusually small. Goodpasture (1975) has measured the lengths of mitotic chromsomes in a series of Lepidoptera species and they range from 1 to 3 pm. In addition, most Lepidoptera have relatively high chromosome numbers of around n = 30 (Suomalainen, 1969a). These two factors combine to render Lepidoptera spindles, in contrast to those of Diptera (Dietz, 1956) and Orthoptera (Arana and Nicklas, 1992), a less promising insect system for live observations. Nevertheless, in order to obtain information on the time course of the meiotic divisions of a Lepidoptera species, testes smears of E . kuehniella were prepared in paraffin oil and observed with phase-contrast microscopy. Eupyrene and apyrene spermatocytes I were observed for about 2 hr. Meiosis proceeded under these conditions in both developmental lines (Wolf and Bastmeyer, 1991a,b). Although there is no experience available in this respect, testes smears prepared in UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

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paraffin oil may also serve as a substrate for microinjection experiments. The diameter of the roughly spherical metaphase I spermatocyte in E. kuehniella is about 25 pm.

111. Kinetic Organization of Lepidoptera Chromosomes

The centromeres, that is, the sites of interaction between the chromosome and the spindle, differ from the remainder of the chromosomes in containing blocks of specific repetitive DNA (Willard, 1990),in their association with specific nonhistone proteins (Earnshaw and Rothfied, 1989, and in undercondensation (Rattner and Lin, 1987). I refer to the kinetic organization of a chromosome when the size and the position of the centromeres relative to the chromosome are addressed. There are two extreme cases: the monocentric and the holocentric chromosome. In the former type, the centromere occupies a small portion of the chromosome. According to modern terminology of monokinetic chromosomes (Earnshaw, 1991), the expression centromere refers to a chromosome segment, the primary constriction. The term kinetochore designates a proteinaceous element located at the poleward surface of the chromosome within the primary constriction, which is visible only by electron microscopy. There, spindle MTs are inserted. In holokinetic chromosomes, attachment of spindle MTs occurs over a wide area of the poleward chromosomal surface, primary constriction is not detectable, and kinetochore plates may be missing. Polykinetic chromosomes are characterized by the presence of multiple independent attachment sites for spindle MTs. So far, the opinion prevails that Lepidoptera chromosomes are holokinetic. This notion is based on indirect evidence. The average haploid chromosome number in Lepidoptera is around 30 (Suomalainen, 1969a). Females have heteromorphic sex chromosomes (Traut and Mosbacher, 1968).Most Lepidoptera contain a sex bivalent, which, according to modern terminology, is termed WZ (Traut et al., 1986).Only few Lepidoptera species show a sex univalent (Traut and Mosbacher, 1968)or a sex trivalent in females (Suomalainen, 1969b). Chromosome numbers in Lepidoptera are extremely variable. The lowest number, counted in Erebia aethiopellus (Satyrinae), is n = 7 (de Lesse, 1964) and the highest is around 220 in a species of the genus Lysundru. For compilations of chromosome numbers in Lepidoptera, see Robinson (1971) and White (1973). Differences in chromosome number among species of one genus are frequent. In Orthosia grucilis (Noctuidae),for instance, the haploid genome contains 14 chromosomes. Orthosia rorida possesses 120 chromosomes (Werner, 1975). Even more informative in this context is the observation that the chromosome number varies within populations of one species. In Agrodiuetus transcas-

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pica (Lycaenidae), 16, 17, 19,20,33 through 37,52, and 53 chromosomes have been counted (de Lesse, 1963). In the South American butterfly Philaetria dido (Nymphalidae), the chromosome number varies from 12 to 88 (Suomalainen and Brown, 1984; Brown el al., 1992). Polyploidy probably does not account for this variability since the DNA content does not change accordingly.This has been determined in some Cidaria species (Geometridae) (Suomalainen, 1965). Also, the observation that the size of individual chromosomes increases with diminishing chromosome number per complement (Werner, 1975; Suomalainen and Brown, 1984;Brown et al., 1992) indicates that most probably chromosome rearrangements have occurred which did not affect the DNA content dramatically. A high rate of both chromosome fusion and fission is more compatible with a holokinetic than with a monokinetic organization of the chromosomes. Lepidoptera tolerate levels of radiation which are expected to induce chromosome fragmentation and reciprocal translocations. This has been shown in Ostrinia nubilalis (Barry et al., 1967), Pieris brassicae (Bauer, 1967), Trichoplusia ni (Noctuidae) (North and Holt, 1968), and Bombyx mori (Murakami and Imai, 1974). As a rule, Lepidoptera resist unusually high doses of radiation (Lachance et al., 1967). Fragments of holokinetic chromosomes have a better chance to survive experimentally induced karyotype rearrangements than monokinetic chromosomes. Therefore, the resistance against radiation-induced sterilization has been taken as an argument in favor of a holokinetic organization of Lepidoptera chromosomes. In holokinetic chromosomes, a primary constriction is missing. Cytogenetic analyses of Lepidoptera chromosomes have produced inconsistent results. In some studies (Traut and Mosbacher, 1968;Murakami and Imai, 1974; Trentini and Marini, 1986), primary constrictions in Lepidoptera chromosomes were not recognized. However, there is a series of reports describing primary constrictions in mitotic chromosomes of Lepidoptera species (Bigger, 1975, 1976; Rishi and Rishi, 1978,1979, 1981, 1990; Gupta and Narang, 1981; Gus et al., 1983). The conflicting findings may be explained by the small size of Lepidoptera chromosomes, which renders analysis difficult and thus ambiguous. Electron microscopy could solve the question of centromere structure in Lepidoptera. In fact, the analysis of ultrathin sections through gonial chromosomes of E. kuehniella and Trichoplusia ni revealed that 30 to 45% of the poleward side of each chromosome is covered by a kinetochore (Gassner and Klemetson, 1974). Personal observations revealed a vaguely defined kinetochore plate at the poleward surface of mitotic chromosomes (Fig. 1) and confirmed the previous findings in E. kuehniella. This species has a high chromosome number (n = 30) and it seemed worthwhile to look at the chromosomes of a species with fewer chromosomes. In Orgyia antiqua ( n = 14), 60 to 70% of the poleward surface of mitotic chromo-

FIGS. 1-4 Electron micrographs of chromosomes in longitudinally sectioned metaphase spindles of diverse Lepidoptera species. If not indicated otherwise, the micrographs are the author’s unpublished material. FIG. 1 Ephestia kuehniella spermatogonium. The cross section through the chromosome shows kinetochore material connected with microtubules (arrows). Bar = 400 nm. FIG. 2 Orgyia antiqua somatic cell. The longitudinal section through the periphery of the equatorial plate shows that kinetochore material (arrowheads) covers a larger portion of the chromosome surface. On the left side, this chromosome is in contact with another chromosome. Bar = 400 nm.

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

11

somes was covered by indistinct kinetochore plates (Fig. 2). This points at an intermediate size of Lepidoptera centromeres and an increase of absolute centromere size in mitosis with decreasing chromosome number. Caution is advisable, however, as to whether this correlation applies generally in Lepidoptera. So far, the number of species examined is low and the fine structure of chromosomes in species with haploid chromosome numbers well above 30 is not known at all. An intermediate centromere size is, however, compatible with the relatively high rate of fission of specific radiation-induced fusion chromosomes in E. kuehniellu (Traut, 1986). The fusion chromosome may contain two distinct centromeres with the ability to orient toward opposite spindle poles. Upon anaphase, the chromosome would be torn apart by spindle forces. A kinetic organization of Lepidoptera chromosomes between the monokinetic and the holokinetic form is difficult to reconcile with the high tolerance of Lepidoptera for radiation. We must assume that other factors in addition to the kinetic organization of the chromosomes influence radiation tolerance in an organism. The structure of meiotic chromosomes in Lepidoptera differs in both sexes from that of mitotic chromosomes. Female meiosis is difficult to study with the EM, since the tiny spindle is located in the voluminous cytoplasm. So far I have managed to obtain serial sections of only a portion of a metaphase I spindle. The analysis showed that the kinetochores of the sister chromatids were not fused. Instead, there were two elongate kinetochore plates separated by a chromatin ridge at each poleward surface of the bivalent. The kinetochores covered 50 to 70% of the chromosome length (K. W. Wolf, unpublished observations). The elimination chromatin, modified synaptonemal complexes considered to be the glue keeping homologs together (see Section IV,B), is located between them. Thus, a prereductional meiosis I, that is, separation of the homologous centromeres (for terminology, see Sybenga, 1981), is highly probable for female Lepidoptera. This is in contrast to previous interpretations of segregation behavior in female meiosis in this order (Suomalainen, 1953). A prereductional meiosis I for male Lepidoptera was inferred from the behavior of a supernumerary chromosome fragment in Graphiurn surpedon (Papillionidae) (Maeki and Hayashi, 1979). Also, in male meiosis the kinetochores of the sister chromatids are not fused. In Bornbyx rnori (Holm and Rasmussen, 1980) and in E . kuehniella (Wolf and Traut, 1991), FIG. 3 Ephestia kuehniella spermatocyte I. Two microtubule bundles (arrowheads) are attached to each poleward surface of the bivalent. Bar = 400 nm. (From Wolf and Traut, 1991.) FIG. 4 Orgyia rhyellina spermatocyte I. Microtubules are inserted into the chromatin at various sites (arrows). Bar = 400 nm.

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species with a haploid set of 28 and 30 chromosomes respectively, two separate kinetochore plates connected with MT bundles are found at both poleward surfaces of the bivalents (Fig. 3). Since Trichoptera are closely related to Lepidoptera (Kristensen, 1975; Friedlander, 1983), it should be added that an analysis of mitotic and meiotic chromosome structure of a Trichoptera species, Anabolia furcata (Limnephilidae), revealed close similarity to the situation in these moths (Wolf et al., 1992). However, in a Lepidoptera species with a lower chromosome number, Orgyia thyellina (n = 12), distinct kinetochore plates could not be readily detected in metaphase I bivalents. Kinetochore microtubules (kMTs) were inserted into shallow depressions throughout the chromosome surface (Fig. 4). This form was interpreted to represent a polykinetic organization (Wolf et al., 1987).

IV. Spindle Architecture A. Mitosis

The spindles of gonial and somatic tissue of Lepidoptera are structurally similar to mammalian spindles. In both systems, the two centrioles situated at each spindle pole are embedded in a small cloud of PCM. In metaphase, the MT mass of each half-spindle is cone shaped; that is, the MTs are focused toward the the spindle poles (Fig. 5a, b). These are up to 8 p.m apart in mitotic metaphase of Lepidoptera. The lengths of mammalian metaphase spindles vary from 5 to 9 pm, depending on the cell type (Brinkley and Cartwright, 1971). As regards the MT mass, however, mammalian spindles are consistently larger than mitotic spindles of Lepidoptera. The highest number of MT profiles counted per half-spindle is about 500 in Chinese hamster cells, around 1500 in PTK, cells (Brinkley and Cartwright, 1971), and up to 1700 in specific human cell lines (Mclntosh and Landis, 1971). Only slightly more than 300 MTs were counted per half-spindle in a serially cross-sectioned spermatogonium of E. kuehnieffa (Wolf, 1990b). Some cisternae and vesicles excepted, the spindle matrix is devoid of membranes in mitosis of Lepidoptera (Wolf, 1990b). In contrast, mammalian spindles contain membranous elements (Zatsepina e f al., 1977; Moll and Paweletz, 1980; Paweletz and Finze, 1981).The insect spindles show, however, a spindle envelope (Fig. 6). This consists of up to three membrane layers, which are missing only at the spindle poles (Wolf, 1990b). Gonial cells usually possess a better-developed spindle envelope than somatic cells. In some Lepidoptera species, small dense elements are scattered

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

13

FIG. 5(a, b) Epifluorescent micrographs of antitubulin-stained (a) and DAPI-stained (b) spindles in metaphase spennatogonia of the peacock butterfly Inachis io. DAPI is a DNAspecific fluorescent dye. The microtubules converge toward the spindle poles (arrowheads). Bar = 5 pm. (From Wolf, 1992.) FIG. 6 Electron micrograph of a longitudinally sectioned metaphase spindle in an oogonium of Orgyia antiqua. A relatively regular spindle envelope (SE) is visible. Nucleolar remnants are scattered throughout the spindle area. C, centriole. Bar = 2 Wm. (From Wolf, 199Ob.)

throughout the matrix of the mitotic spindle. These most probably represent persisting nucleolar remnants (Wolf, 1990b, 1992). In metaphase, the chromosomes have numerous lateral contacts (Fig. 6). There are gaps in the equatorial plate, but individual chromosomes are difficult to distinguish. Early anaphase of mitosis in Lepidoptera is characterized by the presence of chromatin threads between the separating chromatids (Fig. 3 in Wolf, 1990b). This phenomenon has also been observed in mitosis of Hemiptera species (Rieder et al., 1990, and references therein). The coincidence may point at particular properties of chromosomes in both groups, which have in common the presence of relatively large centromeres (Section I11 and Hughes-Schrader and Schrader, 1961).This feature excepted, the development of the mitotic spindle in Lepidoptera follows the usual course-an interzone spindle forms between the two separating chromatin masses and the diameter of the interzone spindle decreases throughout telophase (Wolf, 1992).

6. Female Meiosis In female meiosis of Lepidoptera, chiasmata between the homologous chromosomes are missing. Meiosis is of the achiasmatic type (Suoma-

14

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lainen, 1969a; Turner and Shepard, 1975; Traut, 1977; Fisk, 1989). The development of oocytes in Lepidoptera follows the common pattern until pachytene. The chromosomes pair and synaptonemal complexes (SCs) with the usual tripartite shape build up (Rasmussen, 1976; Traut, 1977). Toward the end of prophase I , the SCs disassemble in the nurse cells but persist in a modified form in the oocytes. The lateral elements thicken, fuse, and form a layer between the homologous chromosomes. The layer may be continuous, as reported for Bombyx mori (Rasmussen, 1977), or perforated, as in E . kuehniella (own unpublished observations). At the onset of anaphase I, the homologous chromosomes detach from the modified SCs, which remain at the spindle equator. Therefore, the term “elimination chromatin” has been coined for the discarded material (Fogg, 1930; Wagner, 1931). The term is still customary although it was recognized very early that the elimination chromatin differs from the chromosomes in its staining properties (Fogg, 1930). In fact, the elimination chromatin is Feulgen-negative (Bauer, 1933; Schaffer, 1944; Ris and Kleinfeld, 1952). The basophily of the elimination chromatin is removed through ribonuclease treatment (Ris and Kleinfeld, 1952). Therefore, it may contain ribonucleic acid. At the onset of anaphase I, an unusual chromosome movement has been observed in oocytes of E. kuehniella (Wolf, 1993). Prior to the start of anaphase A, the homologous chromosomes move centripetally, that is, toward the central spindle axis (Figs. 7-9). This migration differs from conventional movements of chromosomes within the spindle. Prometaphase movements, anaphase A, and anaphase B occur parallel to the preferential orientation of the spindle MTs. In contrast, the centripetal shift of homologous chromosomes takes place at right angles to the spindle MTs. Nevertheless, a MT-based mechanism could account for the centripetal movement of homologs. The chromosomes possess kMTs in metaphase and early anaphase I of E . kuehniella oocytes. The formation of MT bundles involving the kMTs of several chromosomes would drag these toward the center of the spindle. Specific MT-associated proteins could bring about the bundling of MTs (cf. Scott et al., 1992). The centripetal movement of the chromosomes takes place close to and parallel to the poleward surfaces of the elimination chromatin. Therefore, the possibility of an interaction between the two components aimed at translocating the chromosomes toward the spindle center is conceivable as well. Movement of chromosomes along stationary material such as the inner face of the nuclear envelope has been observed (Rickards, 1975; LaFountain, 1982, 1983, 1985). In this case, however, a role for cytoplasmic MTs is likely. The elimination chromatin persists beyond meiosis I of Lepidoptera. It is located between the two spindles in meiosis I1 and may serve in keeping the two chromosomes sets separated (Seiler, 1964; Wolf, 1987). Microtu-

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15

FIGS. 7-9 Isolated spindles of Ephestia kuehniella primary oocytes at different stages. The first micrograph in each line (a) represents a phase-contrast image, the second (b) an epifluorescent micrograph of the antitubulin-stained spindle, and in the third micrograph (c), the chromatin is visualized using DAPI. Bar = 5 pm. (From Wolf, 1993.) FIG. 7(a-c) Metaphase I. Individual bivalents with an unstained equatorial zone (arrows in c) are visible in the periphery of the metaphase plate. FIG. B(a-c) Early anaphase I. The homologs have moved toward the center of the spindle, while the elimination chromatin (arrowheads in a) extends throughout the entire diameter of the spindle. FIG. 9(a-c) Midanaphase. The elimination chromatin (arrowheads in a) still occupies the spindle equator, while the chromosomes move toward the spindle poles.

bular remnants of the first division are embedded in the elimination chromatin. The analysis of serial cross sections through these MTs showed C-shaped forms and oversized circular profiles. These were interpreted as transition stages in the disassembly of MTs. The widening of the MT diameter may indicate rearrangements in the monomer lattice before disassembly (Tilney and Porter, 1967; Wolf, 1987).

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C. Male Eupyrene Meiosis This section deals with nuclear division in eupyrene spermatocytes of Lepidoptera. Male meiosis is usual in that homologous chromosomes pair until pachytene and chiasmata are formed until metaphase I (Traut, 1977; Holm and Rasmussen, 1980). Recombination nodules, elements wich most probably play a role in meiotic recombination (Carpenter, 1987), have been found in association with the synaptonemal complexes in prophase I spermatocytes of Bombyx mori (Holm and Rasmussen, 1980)and Galleria mellonella (Pyralidae) (Wang et al., 1993). As in meiosis of many other organisms, chromatin decondenses transiently in late prophase I spermatocytes of Lepidoptera species (Wolf and Traut, 1991). Eupyrene spindles, however, deviate in several aspects from what we know of higher eukaryotes. Spindle MTs behave in an unusual way and the spindles of most species examined to date possess an abundant membrane inventory. The two meiotic divisions in the eupyrene line differ quantitatively. Spindles in meiosis I1 are smaller. There are also some minor qualitative aspects such as the origin of the spindle membranes and the structure of late telophase cells, which distinguish both meiotic divisions. The eupyrene divisions have been observed in E. kuehniella and Znachis io (Nymphalidae) (Wolf and Bastmeyer, 1991a; Wolf, 1992)by using an antibody against P-tubulin and indirect fluorescence. Significant differences between the two species were not detected and the major events in the restructuring of the microtubular cytoskeleton in eupyrene spermatocytes of Lepidoptera will be restated here. A central feature of eupyrene spindles in Lepidoptera, the termination of most spindle MTs halfway between the equatorial plate and the spindle poles, has been confirmed in Orgyia antiqua (Wolf and Bastmeyer, 1991a). The onset of spindle development in eupyrene meiosis is signaled by the formation two small aster-like MT arrays close to the spherical nucleus. During late prophase I, the MT arrays migrate toward opposite poles of the nuclear area (Fig. 10a, b). MT density is high around the spindle poles. Throughout prometaphase, however, a zone largely devoid of MTs develops underneath the spindle poles (Fig. l l a , b). In metaphase, the majority of the MTs end abruptly about halfway between the equatorial plate and the spindle poles. Whereas this applies also to early anaphase, MTs re-form between the migrating chromosome plates and the spindle poles in late anaphase (Fig. 12 a, b). Concomitantly, an interzone spindle is formed and the pole-to-pole distance of the spindle increases. Eupyrene midtelophase spindles are the largest in comparison with those of all other stages and cell types described here. MTs projecting from the spindle poles are rare beyond midtelophase, when the spindle elongates significantly and often assumes a curved shape. The first step in the formation of the second

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

17

FIGS. 10-12 Isolated spindles of Ephesria kuehniella eupyrene spermatocytes I at different stages. The first micrographs (a) represent epifluorescent images of the antitubulin-stained spindles, and the adjacent micrographs (b) show the chromatin of the same cell, visualized using DAPI. Bar = 5 pm. (From Wolf and Bastmeyer, 1991a.) FIG. 10(a, b) Early prometaphase. The chromosomes form an irregular clump. Most microtubules are focused toward the spindle poles. FIG. ll(a, b) Metaphase. The bivalents are aligned at the spindle equator. At the spindle poles (arrows), small microtubular foci are visible. Most spindle microtubules end halfway between the equatorial plate and the spindle poles. FIG.12(a, b) Late anaphase. The chromosomes are to be seen halfway between the spindle equator and the spindle poles. Underneath the spindle poles numerous microtubules are visible. Also, the interzone contains microtubules. FIG. 13 Electron micrograph of a longitudinally sectioned eupyrene anaphase I1 spindle of Phragrnatobia fuliginosa. The spindle envelope (asterisks) is still intact. The spindle poles are marked by the presence of basal bodies (BB). The spindle area contains membranous vesicles. These are more numerous in the half spindles than in the interzone. Bar = 4 pm.

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meiotic spindle overlaps the gradual disassembly of the interzone spindle of the first division. Two small MT foci appear at opposite poles of the daughter nuclei of the first division (for a scheme, see Fig. 14). Interkinesis, that is, a distinct interphase-like stage between the two meiotic divisions, appears to be missing. The further restructuring of secondary spermatocytes is very similar to that described above. In late telophase I1 spindles, however, MT foci close to the daughter nuclei are missing since the daughter cells of the second division give rise to spermatids instead of preparing for another division. Lepidoptera spermatids show some remarkable features in terms of odd extracellular appendages (Zylberberg, 1969; Riemann, 1970; Riemann and Thorson, 1971; Phillips, 1971; Friedlander and Gitay, 1972; Riemann and Gassner, 1973; Friedlander, 1976; LaChance and Olstad, 1988), but spermiogenesis is beyond the scope of this chapter. In order to estimate the MT mass of eupyrene meiotic spindles in Lepidoptera, ultrathin serial cross-sections through metaphase I spindles of two species were analyzed. In Orgyiu thyellinu, the maximum number of MTs per half-spindle was 760 and in E . kuehniella, 1770. In the latter species two different spindles were studied, which differed only slightly (Wolf, 1990b). The MT numbers fall into the same range as those determined in mammalian spindles (Brinkley and Cartwright, 1971; McIntosh and Landis, 1971). In other systems, where spindles have been examined quantitativeley, such as male meiosis of a Diptera species, Pules ferruginea, and endosperm cells of a higher plant, Huemanthus katherinae, more

FIG. 14 Diagrammatic representation of the structural changes in the microtubule cytoskeleton in eupyrene meiosis of Lepidoptera. The interval from late prophase I to prophase I1 is depicted. (a) Late prophase I. (b) Early prometaphase I. (c) Metaphase I. (d) Early anaphase I . (e) Late anaphase I. (f) Midtelophase I . (9) Late telophase I . (h) Transition from meiosis I to meiosis 11. (i) Prophase 11. (From Wolf and Bastmeyer, 1991a.)

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

19

MTs have been counted (Fuge, 1973; Jensen and Bajer, 1973). The larger size of the spindle in these systems is corroborated by determining the MT content of the entire spindle. The total MT length in the spindle area, that is, aster MTs excluded, was about 1100 pm in Orgyia thyellina and about 9800 pm in the larger of two spindles of E. kuehniella (Wolf, 1990b). A metaphase I spindle in male meiosis of Palesferruginea, by comparison, contained 23,000 pm (Fuge, 1974). Thus, with respect to MT content, Lepidoptera spindles occupy the lower end of the range in higher eukaryotes. Before discussing the implications of the immunofluorescence findings in eupyrene spermatocytes of Lepidoptera, it is necessary to learn the fine structure of the spindles in these cells and their development. The restructuring of eupyrene spermatocytes has been studied in more or less detail in a series of species using electron microscopy. These were Ostrinia nubilalis (Roth et al., 19661, Euproctis chrysorrhea (Lymantriidae) (Leclerq-Smekens, 1978), Bombyx mori (Holm and Rasmussen, 1980; Friedlander and Wahrman, 1970), Calpodes ethlius (Hesperiidae) (LaiFook, 1982), Orgyia thyellina (Wolf et al., 1987), Pieris brassicae (Wolf, 1988), Orgyia antiqua (Wolf, 1990a), and lnachis io (Wolf, 1992). In E. kuehniella, late meiotic prophase I spermatocytes (Wolf and Traut, 1991) and the structure of the metaphase I spindle were examined extensively (Wolf and Bastmeyer, 1991a). Additional studies of mine showed the presence of a thick spindle envelope in Deilephila elpenor (Sphingidae), Phragmatobia fuliginosa, and Yponomeuta spec. (Yponomeutidae), but these findings have not yet been published. There are species-dependent differences. For instance, the membrane inventory of spermatocytes in Bombyx mori and Pieris brassicae is lower than that of the other species. Another Pierid species, Lepidea amurensis, however, showed a rich membrane inventory again (K. W. Wolf, unpublished observations) and, thus, there is no clear pattern detectable in the differences at the ultrastuctural level. The picture which emerged from these studies is consistent with the following description. First of all, the structure of the spindle poles deserves attention, since the PCM located there is considered important for organizing the MT cytoskeleton (Gould and Borisy, 1977). The restructuring of the spindle poles was analyzed in Bombyx mori (Friedlander and Wahrman, 1970; Holm and Rasmussen, 1980) and in E. kuehniella (Wolf and Traut, 1987; Wolf and Kyburg, 1989). In brief, the spindle poles were marked by the presence of basal bodies continuous with flagella. Two basal bodies are found in meiosis I and only one in meiosis 11. This is in keeping with the situation generally found in insects (Friedlander and Wahrman, 1971). The basal bodies are embedded in a cloud of PCM. This complex will henceforth be referred to as the centrosome (cf. Mazia, 1984). Taken together, the structure of the spindle poles in eupyrene spermatocytes of

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Lepidoptera looks conventional. Development of flagella starts in diplotene spermatocytes. At that stage, the cysts represent hollow spheres with the germ cells situated at the periphery. The nuclei, and in later stages the spindles, occupy the adluminal portions of the cells and the flagella project into the lumen of the cysts. Flagella are retained throughout the meiotic divisions. It is not known whether they are motile during this period. The most prominent feature of eupyrene spindles in Lepidoptera is their membranous envelope (Fig. 13). Its development starts in prophase I. In early pachytene spermatocytes, the cytoplasmic face of the nuclei is devoid of additional membrane layers. In late pachytene cells, however, when there are still long SCs detectable in the nucleoplasm, layers of smooth endoplasmic reticulum begin to form around the nuclei. More membrane layers are added throughout diplotene, when synaptonemal complexes disassemble, chromatin highly decondenses transiently, and indirect evidence indicates high transcriptional activity in the nucleus. In diakinesis there is a multilayered membranous envelope around the nucleus, but portions next to the centrosomes, the presumptive spindle poles, are less studded with membranes. Throughout all these stages, stacks of Golgi cisternae are visible in the lateral cytoplasm and the membranous sheets around the nucleus may be derived from the Golgi complex. The membranous sheath around the nuclei may contain lacunae. Amorphous material that is interspersed between the individual membrane layers is clearly visible in lnachis io (Wolf, 1992), but is barely detectable in other species. Diakinesis nuclei of some species contain small dense elements comparable to those detected in mitotic spindles (see Section IV,A). The particles possibly represent nucleolar remnants. The nuclear lumen is completely devoid of membranes at that stage. In prometaphase I, the membranous sheath around the nuclei develops polar fenestrae close to the centrosomes. MTs originating from the centrosomes project into the spindle area. Concomitantly, membranous elements become visible there. Since the original nuclear envelope is still intact, these membranes enter the spindle area through the polar fenestrae. Possibly they are translocated there by the MTs. This correlation was also assumed to exist in mammalian spindles (Paweletz and Fehst, 1984). In some species, for example, Orgyia antiqua (Wolf, 1990a), the nuclear envelope dissolves late in metaphase. Consequently, the membrane content of the spindle area remains relatively low in comparison with other species such as lnachis io (Wolf, 1992). There, the nuclear membrane dissolves early in metaphase I . Membrane stacks detach from the multilayered spindle envelope and contribute to the formation of the intraspindle membrane system. In most species, the intraspindle membranes are irregular cisternae and vesicles (Wolf, 1990b),which renders detailed analysis difficult. However,

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

21

one species examined by the author, Phragmatobia fuliginosa, deviated in that relatively regular membranous tubules arranged parallel to the spindle axis form within the spindle area until metaphase (Wolf, 1990b). The regular tubules are lost again in anaphase. The reconstruction of portions of the intraspindle membrane system of a metaphase I1 spermatocyte of Phramatobia fuliginosa showed numerous connections between individual membranous tubules (Wolf, 1994). Therefore, we must assume that the intraspindle membrane system represents a continuum, which-and this was also evident from the reconstructions-possesses numerous junctions with the spindle envelope. All Lepidoptera species examined in this respect using electron microscopy have in common a very low MT density in polar areas of the spindles in metaphase. This is in keeping with the immunofluorescence observations (Wolf and Bastmeyer, 1991a; Wolf, 1992) and has been confirmed by a quantitative approach. A count of the MT profiles in a serially crosssectioned metaphase I spindle of E. kuehnieffarevealed that less than 10% of the MTs present in the half-spindle extend to the centrosomes (Wolf and Bastmeyer, 1991a). This amount includes some aster MTs, which cannot be distingushed from spindle MTs proper. In metaphase, membranes accumulate again in polar areas of the spindle. These are often less regular than the membranes forming the remainder of the spindle envelope. As a consequnce, the centrosomes are separated from the spindle area proper. This applies also to anaphase. As in mitotic anaphase, chromatin threads are also observed in meiosis between the separating half-bivalents (Guthrie et al., 1965). However, the spindle envelope ruptures in telophase. Telophase spindles possess mitochondria1 threads aligned parallel to the spindle. This seems to be characteristic of insect spermatogenesis (Krishan and Buck, 1965). Stacks of membranes persist close to the daughter nuclei. Most probably these are recycled and form the spindle envelope of the second division. The spindle in eupyrene spermatocytes of Lepidoptera is clearly different from vertebrate spindles since a prominent spindle envelope is missing in the latter. Also the term “closed nuclear division,” used mostly for protist species in which the nuclear envelope persists throughout mitosis (Heath, 1980), does not account for what we see in eupyrene meiosis of Lepidoptera. There, the nuclear envelope dissolves sooner or later. Therefore, the term “sheathed nuclear division” has been coined for eupyrene meiosis in Lepidoptera (Wolf, 1990a). The major characteristics of this type of division are summarized in Fig. 14. Although the details are far from clear, spindle membranes are interpreted to play a part in the regulation of the MT cytoskeleton through accumulating CaZCions (Hepler, 1989). Since experimental data on the sequestering of Ca2+ions in Lepidoptera spindles are missing, this issue is not touched upon any futher here.

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The number of spindle MTs and, in particular, the lengths of kMTs, vary among the organisms studied, A recent analysis of PTK cells revealed that most kMTs run from their kinetochores to the vicinity of the pole (McDonald et al., 1992). Thus, a strong mechanical connection between the chromosome and the PCM exists. This seems to apply also to spermatocytes of a crane fly, Nephrotoma suturalis, where kinetochore fibers have been studied using serial cross sections through the spindle (Scarcello et al., 1986). However, using longitudinal sections instead of cross-sections for reconstruction, Fuge (1984) found very short kMTs in another crane fly, Palesferruginea. It does not come as a surprise that in organisms lacking defined spindle poles such as the alga Oedogonium cardiacum (Schibler and Pickett-Heaps, 1980,1987), the protozoa Tetrahymena pyriformis (Davidson and LaFountain, 1975; LaFountain and Davidson, 1980), and a higher plant, Haemanthus katherinae (Jensen, 1982), kMTs are shorter. Thus, spindles in eupyrene spermatocytes of Lepidoptera show the combination of canonical centrosomes present but most spindle MTs end far before them in metaphase. Only some aster MTs radiate off both spindle poles. MTs are polar elements. The majority of MTs in plant and animal spindles have their fast-growing or plus endings distant from the poles (Euteneuer and McIntosh, 1980,1981; Telzer and Haimo, 1981; Euteneuer er al., 1982). Eupyrene spermatocytes of Lepidoptera show a high MT density around the spindle poles in early prometaphase. Clearly, centrosomes are active in nucleating MTs when the spindle forms. Centrosomal initiation and subsequent detachment of MTs from the centrosomes have been suggested (Vorobjev and Chentsov, 1983) and could account for the initial formation of the metaphase spindle in eupyrene spermatocytes of Lepidoptera. Most probably, these MTs have their plus endings distant from the poles as well. However, MT detachment from the centrosomes has been considered a key event in MT disassembly (McBeath and Fuijwara, 1990). MTs are highly dynamic structures. The growth of an individual MT may abruptly end and the MT may disassemble completely. The most prominent elements to nucleate regrowth of MTs are the centrosomes. The phenomenon is known as dynamic instability of MTs (Gelfand and Bershadsky, 1991). Therefore, the question has to be raised of how spindle MTs are organized in metaphase of eupyrene meiosis without contact with the centrosomes. Three proposals are discussed here: (1) membranes have an MT-nucleating capacity, (2) a high concentration of monomeric tubulin is maintained within the spindle area and leads to the formation of MTs, and (3) free minus endings of MTs may be stabilized by a cellular factor. 1. Membranous elements are abundant in the region where spindle MTs end. Therefore, a role for membranes in nucleating MTs during the sheathed nuclear division of eupyrene spermatocytes in Lepidoptera has

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

23

to be considered. Membranes may have the potential to nucleate the growth of MTs. This has been suggested for cytoplasmic (Dickinson and Sheldon, 1984; Mogensen and Tucker, 1987; Vantard et al., 1990) and spindle MTs (Davidson and LaFountain, 1975; Eichenlaub-Ritter and Ruthmann, 1982; Kuck and Ruthmann, 1985; Tucker et al., 1985). A clearcut correlation between membranes and MT endings is not obvious in the apical portions of eupyrene spindles of Lepidoptera (Wolf and Bastmeyer, 1991a). The situation is, however, difficult to analyze owing to the small size of the elements involved and the irregular shape of the membranous vesicles. Therefore, the direct involvement of spindle membranes of eupyrene spermatocytes of Lepidoptera in the nucleation of MTs cannot be definitely excluded. 2. Another possibility that could account for the existence of spindle MTs during sheathed nuclear division also relies on the presence of spindle membranes. From late prometaphase until early anaphase, the spindle area is a closed compartment. There is a multilayered spindle envelope and the polar portions of the spindle area are sealed as well. Quantitative studies on the MT and membrane content of eupyrene spindles in three Lepidoptera species showed that the MT mass increases with the volume occupied by intraspindle membranes (Wolf, 1990b). Membranous compartments reduce the free volume within the spindle. As a consequence-and this is an unconventional idea on the role of intraspindle membranes-monomeric tubulin may be concentrated and the formation of tubulin polymer may be favored. Tubulin monomer and MTs form an equilibrium (Walker et al., 1988). 3. The pole-proximal endings of spindle MT may be stabilized through a cellular factor. Cytoplasmic (Tao et al., 1988; Walker et al., 1989) and spindle MTs (Leslie and Pickett-Heaps, 1984; Steffen et al., 1986; Hiramoto and Nakano, 1988; Wilson and Forer, 1988; Nicklas, 1989; Nicklas et al., 1989; Spurck et al., 1990) with free minus endings have been created experimentally. Surprisingly, the MTs remained stable in these cases. The situation is reminiscent of what we see under natural conditions in Lepidoptera spermatocytes. It has been suggested that the stability of newly exposed minus ends is a genuine feature of MT dynamics (Nicklas et al., 1989). The stabilization of noncentrosomal MTs through association with cellular factors such as MT-associated proteins (Bre et al., 1987; McNiven and Porter, 1988) is also conceivable. In eupyrene spermatocytes of Lepidoptera, most MTs, including kMTs, end halfway between the centrosomes and the equatorial plate from late prometaphase until early anaphase. It is not possible to distinguish through antitubulin immunofluorescence whether all kMTs end there or whether a small number of them establish a link between the PCM and the kinetochores. A reconstruction of the kMTs from ultrathin serial sections has yet to be done, and the use of an antibody against acetylated MTs, which

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was expected to stain kinetochore fibers only, was not informative in this respect (see Section IV,E). Therefore, the implications of the unusual spindle organization for chromosome movement are difficult to assess. A very low number of MTs, as few as one, may be sufficient to sustain chromosome movement (King and Hyams, 1982; Hayden et al., 1990). D. Male Apyrene Meiosis

This section deals with structural aspects of apyrene meiosis in Lepidoptera. As a rule, the apyrene spindles contain far fewer MTs than the eupyrene spindles. This has been shown in antitubulin immunofluorescence studies (Wolf and Bastmeyer, 1991b; Wolf, 1992) and through MT counts using serial sections and electron microscopy (Wolf et al., 1987). The membrane content of the apyrene spindles is lower as well, and a spindle envelope is missing (Friedlander and Wahrman, 1970; Wolf et al., 1987; Wolf, 1992). The gross morphology of the MT cytoskeleton of apyrene spindles is, compared with that of eupyrene spindles, more conventional in that most MTs have contact with the spindle poles throughout the course of division. However, the behavior of the chromatin is highly aberrant. The analysis of two geographically different strains of E . kuehniella, L and Sbr, revealed two modes of apyrene meiosis, termed type L and type Sbr (Wolf and Bastmeyer, 1991b). A survey of the literature on apyrene meiosis in other Lepidoptera species showed that this meiosis can be interpreted to follow either type L or Sbr (Table I). Taking into account these light and electron microscopic observations, the following general description of apyrene meiosis can be given. The terminology customary for nuclear division cannot be used for apyrene meiosis, since, for instance, a regular metaphase and anaphase are missing in this developmental line. The first detailed fine structure study of apyrene spermatocytes was carried out in Orgyia thyellina. The terminology introduced at that stage was based on ultrastructural criteria such as the behavior of membranes (Wolf et al., 1987). A new terminology which relies on light microscopic observations and therefore has broader application was developed later (Wolf and Bastmeyer, 1991b) and is used here. Apyrene meiosis is subdivided into apyrene prophase, predisjunction phase, disjunction phase, and apyrene telophase. The structure of the spindle poles in apyrene spermatocytes is comparable with that in the eupyrene line in that there is the usual number of basal bodies embedded in PCM. However, the close and regular end-to-end association of the basal bodies found in eupyrene spermatocytes I is occasionally lost (Wolf et al., 1987). Type L apyrene meiosis is characterized by numerous chromatin clumps

25

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES TABLE I Lepidoptera Species and Type of Apyrene Meiosis

Species Bombyx mori Bombyx mori Calpodes ethlius Ectomyelois ceraioniae Ephesiia cautella Ephestia kuehniella strain L Ephesiia kuehniella strain Sbr Galleria melonella Inachis io Laspyresia pomonella Orgyia aniiqua Orgyia ihyellina Orgyia recens Ostrinia nubilalis Phragmaiobia fuliginosa Pieris brassicae Pygaera anachoreta Pygaera bucephala Pygaera pigra Yponomeuia spec.

Type

Reference

Sbr L L L L L Sbr L Sbr Sbr L L L Sbr L Sbr L L L L

Friedlander and Wahrman (1970) Katsuno (1987) Lai-Fook (1982) Leviatan and Friedlander (1979) Friedlander and Miesel (1977) Wolf and Bastmeyer (1991b) Wolf and Bastmeyer (1991b) von Kemnitz (1914) Wolf (1992) Friedlander and Hauschteck-Jungen (1986) Cretschmar (1928) Cretschmar (1928); Wolf et al. (1987) own unpublished observations Keil e f al. (1990) own unpublished observations Zylberberg (1963, 1969) Federley (1913) Meves (1903) Federley (1913) Wolf, unpublished observations

within the nuclei throughout prophase I (Fig. 15a, b). Pachytene cells, defined by the presence of SCs, have not been found in apyrene development. In fact, apyrene spindles of Orgyia thyellina, a species with n = 12, were found to contain 22 chromatin clumps (Cretschmar, 1928; Wolf et al., 1987). Thus, univalents segregate in type L apyrene meiosis. There are small MT asters at opposite poles of the chromatin mass during prophase. In disjunction phase I, the nuclear envelope is lost, MTs enter the nuclear area, and chromatin clumps are scattered throughout an elongate area between the two spindle poles (Fig. 16a, b). The chromatin clumps segregate in disjunction phase I without congression in terms of a conventional metaphase plate (Fig. 17a, b). The univalents are partially surrounded with membranes and connections with MTs may exist. In Orgyia thyellina, MTs were found to penetrate deeply into some chromatin clumps, whereas others of the same spindle were apparently not connected with MTs (Wolf et al., 1987). Conventional kMTs, ending at the poleward

FIGS. 15-17 Isolated spindles of apyrene spermatocytes I at different stages for different strains of Ephesfia kuehniella. The first micrograph (a) represents epifluorescent images of the antitubulin-stained spindles and the adjacent micrograph (b) shows the chromatin of the same cell, visualized using DAPI. Bar = 5 pm. (From Wolf and Bastmeyer, 1991b.)

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

27

surface of the chromatin, were missing in this species, but were found in E. kuehniellu in addition to MTs perforating the chromatin (Wolf and Bastmeyer, 1990). Observation of living apyrene spermatocytes showed that the segregation of the chromatin clumps in type L apyrene meiosis of E. kuehniellu needs much more time than anaphase of eupyrene meiosis in the same species (Wolf and Bastmeyer, 1991a,b). The course of mitosis is also delayed in the budding yeast, when centromeres are experimentally altered (Spencer and Hieter, 1992). Taken together, this may indicate that the apyrene chromosomes are defective in centromere structure and function. Finally, however, the chromatin clumps reach the spindle poles in apyrene telophase I type L but they do not fuse. The chromatin clumps then possess a complete nuclear envelope and are referred to as micronuclei. The micronuclei are scattered throughout the polar portions of the spindles. In type L apyrene meiosis, the distribution of the chromatin into the daughter cells is roughly even. There is no apyrene prophase 11. Instead, predisjunction phase I1 follows telophase I. Spindles of the second meiosis are, though smaller, very similar to those of the first apyrene division. In type Sbr apyrene meiosis, larger chromatin clumps are found within the prophase I nuclei. For a scheme of the key features of type L and type Sbr apyrene meiosis, see Fig. 20. In predisjunction phase I of apyrene meiosis type Sbr, the chromatin clumps align along the equator of the spindle (Fig. 19). With respect to the chromatin, the appearance of apyrene spermatocytes type Sbr in predisjunction phase I is reminiscent of a eupyrene metaphase spermatocyte. However, a closer inspection reveals numerous differences. The apyrene cells have a sparse membrane inventory

FIG. 15(a,b) Apyrene prophase I (type L). The nucleus appears intact. Two microtubular foci are situated at opposite poles of the nucleus. FIG. 16(a,b) Predisjunction phase I (type L). Small chromatin clumps are scattered throughout the spindle area. An elongate bipolar spindle has formed. FIG. 17(a,b) Disjunction phase I (type L). Chromatin clumps are clustered around the spindle poles. There, the microtubule density is high when compared with equatorial portions of the spindle. FIG. 18(a, b) Disjunction phase I (type Sbr). Larger chromatin clumps are to be seen at various stages of segregation. In cell 1, a curved chromatin thread bridges the space between the spindle poles. In cells 2 and 3, large chromatin clumps are located close to the spindle poles. Cell 4 contains uneven amounts of chromatin at opposite spindle poles. Bar = 5 pm. (From Wolf and Bastmeyer, 1991b.) FIG. 19 Electron micrograph of a longitudinally sectioned apyrene spermatocyte I of Inachis io in predisjunction phase (type Sbr). Large chromatin clumps are aligned at the spindle equator, mimicking a metaphase plate. The membrane content of the spindle is low. A basal body (BB) is encountered at one spindle pole. Bar = 2 pm. (From Wolf, 1992.)

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and the chromatin clumps are very irregular and extend further toward the spindle poles than bivalents in eupyrene meiosis (Wolf and Bastmeyer, 1990; Wolf, 1992). Clearly, we are not dealing with bivalents in apyrene meiosis type Sbr, either, but with larger chromatin blocks. MTs end at both poleward surfaces of the chromatin clumps (Wolf and Bastmeyer, 1990). These elongate in disjunction phase I. Instead of a regular bipartition, the chromatin threads break irregularly (Fig. Ma, b). As a consequence, the chromatin mass per spindle pole of many apyrene telophase I spermatocytes of type Sbr is highly uneven. Lobed daughter nuclei of different sizes are present. In immunofluorescence studies, the observer is confronted with a wide range of meiosis I1 spindles differing in chromatin and MT content. The analysis revealed that the MT mass of the spindles increases with the amount of chromatin (Wolf and Bastmeyer, 1991b). A conservative interpretation of this phenomenon involves the stabilization of MTs through association with kinetochores (Huitorel and Kirschner, 1988).More recent observations using Xenopus egg extracts point to a general role for chromatin in spindle organization (Sawin and Mitchison, 1991).According to this view, the kinetochores do not appear to be decisive for the stabilization of spindle MTs (Hyman and Mitchison, 1990). Both in type L and type Sbr apyrene meiosis, the nuclei are lost from the cell during early spermiogenesis. Transparent clefts develop around the micronuclei. The micronuclei are released through fusion of the clefts with the intercellular space and degenerate since they are missing in sperm

FIG. 20 Diagram illustrating the major differences in chromatin behavior between type Sbr (a) and type L (b) of apyrene meiosis. In type Sbr, large chromatin clumps, located at the equator of the spindle, separate in a highly unequal fashion. In type L apyrene spermatocytes, small chromatin clumps are scattered throughout the spindle area in predisjunction phase. Apyrene telophase spermatocytes of this type contain roughly the same quantity ofchromatin per spindle pole. (From Wolf and Bastmeyer, 1991b.)

UNIQUE STRUCTURE

OF LEPIDOPTERAN SPINDLES

29 bundles containing mature spermatozoa ( Friedlander and Miesel, 1977; Wolf et al., 1988). Apyrene sperm lack complex extracellular appendages (Zylberberg, 1969; Riemann, 1970; Riemann and Thorson, 1971 ; Phillips, 1971; Friedlander and Gitay, 1972; Riemann and Gassner, 1973; Friedlander, 1976; LaChance and Olstad, 1988). They are also shorter than eupyrene bundles (Thibout, 1980). Basically, apyrene sperm may be de-

scribed as cytoplasmic rods containing an axoneme besides mitochondria. Suggested functions include clearing the way for eupyrene sperm in the posttesticular ducts of the male genital tract (Katsuno, 1977), assisting the migration of the eupyrene sperm in the female (Friedlander and Gitay, 19721, hindering sperm present in females from previous matings and delaying further matings by the female (Silberglied et al., 1984), and causing the dissociation of eupyrene sperm bundles by vigorous rolling of the apyrene sperm in the bursa copulatrix of the female (Osanai et al., 1987). Irrespective of which idea on the role of apyrene sperm is correct, apyrene sperm are sterile and regular segregation of chromosomes is not needed during the preceding meiotic divisions. The spindle apparatus has lost its function in the apyrene line. Loss of biological function of a structure and non-use are the causes of regressive evolution (Sewertzoff, 1931; Wilkens et al., 1979). Examples of this phenomenon are abundant in the macroscopic world. Cave-dwelling animals tend to lose their eyes and body pigmentation. Birds on predator-free remote islands lose the wing muscles necessary for flight. In fact, we find that the criteria used to define cases of regressive development (Wilkens et al., 1979) also apply to apyrene meiosis of Lepidoptera. The apyrene spindles are smaller than the euyprene spindles; the regression in the apyrene line is genotypically based; apyrene development shows great variance; and apyrene meiosis is not influenced by selectional forces. Thus, apyrene meiosis of Lepidoptera has been interpreted as an example of degenerative evolution at the cellular level (Wolf et al., 1987, 1988). In contrast to the spindle apparatus, cytokinesis is carried out properly in the apyrene line. Thus, the production of a great number of individual apyrene sperm is guaranteed. It may be significant that we find the combinationof an unusual eupyrene meiosis, sheathed nuclear division, and apyrene meiosis, a cellular model of regression, in Lepidoptera. Could it be that the two traits evolved together? If, possibly owing to the poor connection between chromosomes and centromeres, sheathed nuclear division was error-prone in the early Lepidoptera, then a larger number of aneuploid spermatocytes and finally sperm existed in the testes. These, in turn, could have acquired a function independent of fertilization. The presence of these sperm may have represented an advantage for the carrier and therefore spread throughout the population. What we see today in the form of apyrene meiosis are the degenerate remainders of the spindle apparatuses leading to the sterile sperm type.

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Lepidoptera have close relatives, the Trichoptera (Kristensen, 1975). Some karyological traits such as mitotic and meiotic chromosome structure are very similar in both orders. In both orders, female meiosis is achiasmatic (Wolf et af., 1992, and references therein). Unfortunately, the structure of the male meiotic spindle is not known in Trichoptera. The fine structure study designed to close this gap failed because membranes were generally poorly preserved (Wolf et al., 1992). It is clear that Lepidoptera and Trichoptera have common ancestors, but apyrene sperm are missing in the latter. Double spermatogenesis is believed to be an evolutionary novelty in Lepidoptera and is present in the most primitive groups of this order (Friedlander, 1983). This points to a small population which separated from an evolutionary branch common with the Trichoptera and developed double spermatogenesis prior to the wider radiation of the order Lepidoptera. E. Post-translational Modifications of Microtubules in Lepidopteran Spindles

MTs may be post-translationall y modified. The best-known modifications are acetylation of the &-aminogroup of a lysine residue at position 40 of a-tubulin (L’Hernault and Rosenbaum, 1985) and the removal of a tyrosine residue from the carboxy-terminal domain of a-tubulin (Hallak et al., 1977). The modifications preferentially affect polymerized MTs and are reversed on monomeric a-tubulin (Piperno et af., 1987; Kumar and Flavin, 198 1). Antibodies detecting specific tubulin isoforms are available. The 6-1 1B-1 antibody recognizes the acetylated form of a-tubulin decribed above (Piperno and Fuller, 1985), the YL1/2 antibody detects the tyrosinated form of a-tubulin (Kilmanin et af., 1982), and antibodies against the detyrosinated form of a-tubulin have been generated (Gundersen et al., 1984; Wehland and Weber, 1987). There is a general consensus that acetylated and detyrosinated MTs are more stable than the unmodified forms (Bulinski and Gundersen, 1991; Webster and Borisy, 1989). Though the differences may be small in absolute terms, the pertinent antibodies can be used as analytical tools in order to identify stable and labile MTs of a given system. In order to obtain a better understanding of spindle organization in Lepidoptera, cytoskeletons isolated from testes of E . kuehnieffa larvae were probed with antibodies against acetylated and tyrosinated MTs. Though the results have yet to be published, a brief description of the findings appears justified here. MTs of spermatogonial spindles were both tyrosinated and acetylated (Fig. 21a, b). The spindles cannot be distinguished from those stained with an antibody against P-tubulin (Fig. 5a, b). In eupyrene spermatocytes, by contrast, only the flagella were stained

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

31

FIG. 21(a, b) Isolated spindles of Ephestiu kuehniella spermatogonia. In the first micrograph (a) an epifluorescent image of the spindle stained with an antibody against acetylated atubulin, 6-11B-I, is shown. The entire spindle is stained. The second micrograph (b) shows the chromatin of this cell, visualized using DAPI. Bar = 5 pm. FIG. 22(a-c) Isolated spindle of Ephesriu kuehniellu eupyrene spermatocyte I1 in anaphase. In the first micrograph (a) an epifluorescent image of the spindle stained with an antibody against acetylated a-tubulin, 6-IIB-I, is shown. Only the flagella are stained. Their basal bodies (arrows) appear unstained. The second micrograph (b) is a phase-contrast image of the same cell. Arrows indicate the position of the centrosomes. In ( c ) ,the chromatin plates of this cell, visualized using DAPI, are depicted. Bar = 5 pm.

from prophase until anaphase using the 6-1 1B-1 antibody (Fig. 22a-c). Beyond anaphase, the spindles increasingly reacted with the 6-1 1B-1 antibody. MTs of the eupyrene spindles were tyrosinated throughout all these stages. At first sight, the presence of large amounts of tyrosinated and acetylated MTs in sperniatogonial spindles is confusing in light of the introductory remarks on the possible meaning of the post-translational modifications. However, the enzyme mediating the removal of a tyrosine residue from the carboxy-terminal domain of a-tubulin may be missing in E. kuehniella and consequently detyrosinated MTs do not form. If this is true, the presence of tyrosinated MTs does not indicate labile MTs in this system. The lack of detyrosinated MTs is not without precedent. Mitosis of the fission yeast also fails to show detyrosinated MTs (Alfa and Hyams, 1991). The absence of detyrosinated MTs from eupyrene spindles is understandable. Detyrosinated a-tubulin easily incorporates into membranes (Nath and Flavin, 1978). Membranes are abundant in eupyrene spindles of Lepidoptera, and tubulin would be withdrawn from use for spindle assembly. More informative are the findings on MT acetylation in eupyrene spermatocytes of E . kuehniella. In contrast to observations in more conventional spindles (Wilson and Forer, 1989), kMTs are not acetylated in metaphase and anaphase spindles. These observations are compatible with the view that we are dealing with labile MTs, and the question of the

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organization of spindle MTs in eupyrene metaphase spermatocytes of Lepidoptera is all the more pressing (see Section IV,B).

V. Lepidoptera as Experimental Systems A few examples are listed here in order to show that Lepidoptera have consistently been used as experimental systems in cytology, genetics, and cytogenetics. Then I describe the advantages and limitations of Lepidoptera species for spindle research and try to define some modem research needs in the field. The presence of specific somatic cells within the gonads, which have turned out to be characteristic of insect reproduction and are known as apical or Verson’s cells (Carson, 1949, have been detected in the silk moth (Verson, 1889). For a recent study on this particular cell, see Wolf (1991). Experiments on intersexuality have been carried out using the gypsy moth (Goldschmidt, 1931) and early genetic work involved the mealmoth (Kuhn and Henke, 1930). More recently, questions concerning chromosome pairing and chromatin structure have been addressed in the silk moth (Rasmussen, 1976, 1977; Holm and Rasmussen, 1980), the meal moth (Weith and Traut, 1980, 1986; Marec and Traut, 1993), and the wax moth (Wang et al., 1993). This chapter has focused on a relatively narrow cytological aspect of Lepidoptera: chromosome and spindle structure. On account of the structural variability of the spindles depending on the cell type, and since Lepidoptera show double spermatogenesis, which, in turn, may vary among different isolates of one organism, species of this order offer themselves as a rich source of material for studies aimed at spindle structure and function. Mitotic spindles are, though smaller, comparable with mammalian spindles and may serve as a reference or control. Meiotic spindles in both sexes show a series of unique features. To date, only basic structural knowledge is available. Since some Lepidoptera species can be easily reared, they are handy systems, in particular for smaller, understaffed laboratories. It is surprising that the exploitation of the potential of Lepidoptera spindles started so late. There are limitations in our ability to manipulate Lepidoptera cells. Foremost among these is the small size and the high number of the chromosomes, which render Lepidoptera less attractive for light microscopic studies aimed at chromosome behavior. The small chromosome size may be advantageous at the ultrastructural level. The interesting spindle types, that is, those in euprene and apyrene spermatocytes, occur in cysts and are surrounded by a layer of somatic cells. As mentioned in Section 11, this represents an advantage for conventional light and electron microscopy, but causes problems when, for example, the effect of drugs on

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

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spindle structure has to be assessed. Straightforward drug interference studies, comparable to those in a monolayer of cultured cells, are not possible. Testes smears have to be prepared and treated when one wishes to avoid inconsistencies caused by uneven penetration of the agent (cf. Daub and Hauser, 1986, 1988). Metaphase I spindles can be easily isolated from oocytes and their further development may be studied using light microscopy (Wolf, 1993). However, owing to the large volume of the egg and the resistent egg shell, meiotic spindles of females are not easy experimental systems if one wishes to go beyond immunofluorescence (Wolf, 1987). Recent developments in the preparation of insect eggs for electron microscopy, however, appear to be promising (McDonald and Morphew, 1993). In the present context, future cytological and cell biological work involving Lepidoptera may be subdivided into three categories: chromosome structure, organization of eupyrene spindles, and organization of apyrene spindles.

A. Chromosome Structure Clearly, the kinetic organization of Lepidoptera chromosomes varies qualitatively and quantitatively both between mitosis and meiosis of one species and among species with different chromosome numbers, Reorientations in the kinetic activity from mitosis to meiosis have been described in Hemiptera species (Nokkala, 1985). It is interesting to note that Hemiptera show holokinetic chromosomes (Hughes-Schrader and Schrader, 1961) and that there is another case of variable kinetic organization in a species with holokinetic chromosomes: Ascaris uniualens. During the early embryonic divisions of this parasite, the euchromatic portions of the chromosomes show kinetic activity (Goday and Pimpinelli, 1986). MTs are attached with the entire chromosomal surface in gonial mitosis (Goday et a/., 1985). In male meiosis, however, only the telomeres show kinetic activity (Goday and Pimpinelli, 1989). Therefore, chromosomes with larger centromeres tend to be variable in the attachment of MTs. The centromeres of monokinetic chromosomes possess large amounts of tandemly repeated transcriptionally inactive sequences. Subsets of those are most probably responsible for binding centromere proteins (Willard, 1990). These, in turn, mediate the attachment of kMTs directly or through further proteins. In holokinetic chromosomes, we expect on theoretical grounds that centromere DNA will be interspersed with coding sequences. However, the analysis of the molecular architecture of holokinetic chromosomes in terms of DNA and protein composition is still in its infancy. In the Hemipteran species Oncopeltus fasciatus, repeated DNA sequences were believed to be short and scattered throughout the

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genome (Lagowski et al., 1973). This is in keeping with the expectation formulated above. A study on the distribution of ribosomal DNA in Luzula pilosa, a monocotyledonous plant with holokinetic chromosomes, showed that one chromosome contains most of the rDNA loci (Bowen et al., 1988). We have to assume that the rDNA is flanked or interspersed with centromeric DNA. A lower eukaryote, the fission yeast Schizosaccharomyces pombe, contains tRNA genes interspersed with centromeric DNA (Kuhn et al., 1991; Takahashi et al., 1991). Does the situation in this unicellular organism reflect the ancestral state of centromere architecture? If so, did it give rise on the one side to the monokinetic chromosomes, where centromeric DNA is tightly clustered and does not contain coding sequence, and on the other side to holokinetic chromosomes, with centromeric sequences dispersed throughout the chromosome and serially flanked by transcribed sequences? Lepidoptera chromosomes show a centromere size intermediate between that of monokinetic and holokinetic chromosomes. Did dispersal of centromeric DNA come to a halt earlier in Lepidoptera than in species with true holokinetic chromosomes, or is centromere evolution in Lepdidoptera still in progress? We do not know the answers to these questions. However, a molecular approach toward unravelling the kinetic organization of holokinetic chromosomes is due. On account of their intermediate position, Lepidoptera chromosomes clearly deserve attention as well. B. Organization of Eupyrene Spindles

What makes eupyrene spindles in Lepidoptera unusual is the presence of a rich spindle membrane system and the abrupt ending of the bulk of the spindle MTs halfway between the equatorial plate and the centrosomes in metaphase. To date the spermatocytes have been studied only by using conventional light and electron microscopy. To the author’s knowledge, freeze cleavage preparations of dividing cells aimed at spindle membranes have not been carried out. These preparations may offer new insights into the function of spindle membranes. Intramembrane structures, which could be correlated with Ca2+transport, may show up. Also, links with MTs, which may provide clues about the transport of membrane into the spindle area, might become more clearly visible than in ultrathin sections. There are Lepidoptera species, namely Phragmatobia fuliginosa, showing regular membranous tubules within the spindle area of eupyrene spermatocytes (see Section IV,C). This characteristic arrangement of the intraspindie membranes may facilitate a pertinent analysis, and testes of Phragmatobia fuliginosa may be the objects of choice for freeze cleavage

UNIQUE STRUCTURE OF LEPIDOPTERAN SPINDLES

35

preparations. Intracytoplasmic membranes can be visualized in freezefracture preparations (Kessel et af., 1985). With respect to MTs in Lepidoptera spindles, open questions include the protofilament number of the MTs. This number, which is usually 13 (Unger et af., 1990), may change when the centrosome is not involved in nucleation (Evans et af., 1985; Tucker et al., 1986). Knowledge of the protofilament number appears particularly important for the MTs which have their pole-proximal ends free in the spindle area in metaphase of eupyrene spermatocytes. The information should contribute to an understanding of how these MTs, which are possibly dynamic (Section IV,E), are organized. Technically, the issue seems resolvable, since the protofilament number of accessory tubules is routinely determined in insect spermatozoa, including Lepidoptera (Dallai and Afzelius 1990). C. Organization of Apyrene Spindles

Apyrene spindles of Lepidoptera deviate from functional spindles by showing highly irregular chromosome segregation under natural conditions. There are indications that the centromeres are defective in the apyrene line. The utility of these spindles is not confined to advancing our understanding of centromere function by, for example, allowing a comparison of the presence of various centromere proteins between eupyrene and apyrene chromosomes; human autoantibodies (Earnshaw and Rothfield, 1985) can also be used to this end. Also, factors influencing chromatin clumping may be assessed by comparing type L and type Sbr apyrene meiosis in Lepidoptera.

VI. Summary

Lepidoptera chromosomes are unusually small, and light microscopy has not contributed much to our knowledge of chromosome structure in this group. Although observations are limited to a few species, fine structure studies suggest that Lepidoptera chromosomes represent a type intermediate between monokinetic and holokinetic chromosomes. To date, four different spindle types have been identified in Lepidoptera. These are somatic and gonial mitoses, female meiosis I, and divisions in eupyrene and apyrene spermatocytes. Though smaller, the spindles of spermatogonia and somatic tissues of Lepidoptera resemble those of mammalian cells. Meiosis I in female Lepidoptera is achiasmatic. Modified synaptonemal complexes, known as elimination chromatin, are believed to serve as a

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glue in keeping the homologous chromosomes together until the onset of anaphase I. Before the homologous chromosomes separate clearly from the elimination chromatin, they move toward the spindle axis. This movement is unique to meiosis I spindles of female Lepidoptera. Eupyrene spindles of Lepidoptera, which give rise to fertile spermatozoa, usually show a prominent spindle envelope and a rich intraspindle membrane system. The fact that the majority of spindle MTs end abruptly halfway between the centrosomes and the spindle equator from late prometaphase to early anaphase represents an unusual feature. To account both for the presence of the spindle membranes and the strange behavior of the MTs, the eupyrene meiosis of Lepidoptera has been termed “sheathed nuclear divsion.” Apyrene spindles, which produce sterile sperm, possess fewer membranes and MTs than the spindles of the “sheathed nuclear division.” Segregation of the chromatin is highly irregular in the apyrene line. Differences in the behavior of chromatin were detected among different isolates of one species. Irrespective of this behavior, the chromatin is lost in young apyrene spermatids. Therefore, the apyrene spindles have lost their function and apyrene development is interpreted as an example of degenerative evolution at the cellular level. The idea is advanced that the evolution of the sheathed nuclear division contributed to the development of double spermatogenesis in Lepidoptera. Finally, the acetylation status of MTs in mitotic and eupyrene meiotic spindles and its implications for MT behavior are described. The last section deals with Lepidoptera as experimental systems. Research needs in the context of chromosome and spindle structure are listed. Acknowledgments I am grateful to Professor Dr. W. Traut (Liibeck) and Dr. F. Marec (Ceskk Budtjovice) for their critical comments on the manuscript and to Mr. K . Fernandes (Ottawa, Canada) for linguistic advice.

References Alfa, C. A., and Hyams, J. S.(1991). Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of a-tubulin. Cell Moiil. Cyioskel. 18, 86-93. Amos, L. A., and Amos, W. B. (1991). “Molecules of the Cytoskeleton.” Macmillan Education Ltd., London. Arana, P., and Nicklas, R. B. (1992). Orientation and segregation of a micromanipulated multivalent: Familiar principles, divergent outcomes. Chromosomn 101, 399-412. Barry, B. D . , Guthrie, W. D., and Dollinger, E. J. (1967). Evidence of a diffuse centromere

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Munson. J. P. (1906). Spermatogenesis of the buttertly, Papilio rutulus. Proc. Boston SOC. Nut. Hist. 33, 43-124. Murakami, A., and Imai, H. T. (1974). Cytological evidence for holocentric chromosomes of the silkworms, Bombyx mori and B . mandarina. Chromosoma 47, 167-178. Nath, J . , and Flavin, M. (1978). A structural difference between cytoplasmic and membranebound tubulin of brain. FEES Lett. 9, 335-338. Nicklas, R. B. (1989). The motor for poleward chromosome movement is in or near the kinetochore. J . Cell Biol. 109, 2245-2255. Nicklas, R. B., and Kubai, D. F. (1985). Microtubules, chromosome movement, and reorientation after chromosomes are detached from the spindle by micromanipulation. Chromosoma 92, 313-324. Nicklas, R. B., and Staehly, C. A. (1967). Chromosome micromanipulation. I. The mechanics of chromosome attachment to the spindle. Chromosoma 21, 1-16. Nicklas, B. R., Brinkley, B. R., Pepper, D. A., Kubai, D. F., and Rickards, G. K. (1979). Electron microscopy of spermatocytes previously studied in life: Methods and some observations on micromanipulated chromosomes. J . Cell Sci. 35, 87-104. Nicklas, R. B., Lee, G. M., Rieder, C. L., and Rupp, G. (1989). Mechanically cut mitotic spindles: Clean cuts and stable microtubules. J. Cell Sci. 94, 415-423. Nokkala, S. (1985). Restriction of kinetic activity of holokinetic chromosomes in meiotic cells and its structural basis. Hereditas 102, 85-88. North, D. T., and Holt, G. (1968). Inherited sterility in progeny of irradiated male cabbage loopers. J . Econ. Entomol. 61, 928-931. Nowock, J. (1973). Growth and metamorphosis in the testes of Ephestia kuehniella in vitro. J . Insect. Physiol. 19, 941-949. Osanai, M., and Kasuga, H. (1990). Sperm motility and micropore formation in the flagellar membrane caused by endopepdidase. Experientia 46, 261-264. Osanai, M., Kasuga, H., and Aigaki, T. (1987). Physiological role of apyrene spermatozoa of Bombyx mori. Experientia 43, 593-596. Osanai, M., Kasuga, H., and Aigaki, T. (1991). Motility-related ultrastructural changes in the flagellar membrane of apyrene spermatozoa of the silkworm, Bombyx mori,, induced by Arg-C endopepdidases. Invertebr. Reprod. Dev. 19, 193-201. Paweletz, N., and Fehst, M. (1984). Are membranes of the mitotic apparatus translocated by microtubules? Cell Biol. I n t . Rep. 8, 117-125. Paweletz, N., and Finze, E.-M. (1981). Membranes and microtubules of the mitotic apparatus of mammalian cells. J. Ultrastruct. Res. 76, 127-133. Paweletz, N., and Schroeter, D. (1987). On the ultrastructure of the mitotic apparatus. In “Aneuploidy, Pt A: Incidence and Etiology” (B. K. Vig and A. A. Sandberg, eds.), pp. 341-393. Alan R. Liss, New York. Phillips, D. M. (1971). Morphogenesis of the lacinate appendages of Lepidopteran spermatozoa. J . Ultrastruct. Res. 34, 567-585. Pickett-Heaps, J. (1991). Cell division in diatoms. Int. Rev. Cytol. 128, 63-108. Piperno, G., and Fuller, M. T. (1985). Monoclonal antibodies specific for an acetylated form of a-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J. Cell Biol. 101, 2085-2094. Piperno, G., LeDizet, M., and Chang, X. (1987). Microtubules containing acetylated atubulin in mammalian cells in culture. J . c e / / Biol. 104, 289-302. Raman, R., and Nanda, 1. (1986). Mammalian sex chromosomes. I. Cytological changes in the chiasmatic sex chromosomes of the male musk shrew. Chromosoma 93, 367-374. Rasmussen, S. W. (1976). The meiotic prophase in Bombyx mori females analyzed by threedimensional reconstructions of synaptonemal complexes. Chromosoma 54, 245-293. Rasmussen, S. W. (1977). The transformation ofthe synaptonemal complex into the “elimination chromatin” in Bombyx mori oocytes. Chromosoma 60,205-221.

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Cellular Mechanisms for the Activation of Blood Coagulation Carolyn L. Geczy Heart Research Institute, Camperdown, New South Wales 2050, Australia

1. Introduction

Coagulation is not only an essential defense against injury, but also an integral part of the host’s immune and inflammatory responses. Although normally nonthrombogenic, it is now clear that a number of cell types, principally including blood monocytes (Mo),inflammatory macrophages (Mac), and endothelial cells, can be stimulated to express surface-bound procoagulants. The major activity is attributed to tissue factor (TF), which activates the extrinsic coagulation pathway (Bach, 1988; Nemerson, 1988) although other inducible activators of this pathway are also involved. In addition to the effects of the cascade of coagulation enzymes, amplification also occurs at the level of membrane-substrate complexes (Mann et al., 1990).

This chapter describes the biology of the cellular activators of the extrinsic coagulation pathway. However, these procoagulants do not act in isolation. Upon stimulation of both Mos and endothelial cells, there is a concomitant upregulation of the antifibrinolytic system by decreased or unchanged plasminogen activator synthesis and increased production of inhibitors of plasminogen activator (Vassalli et nl., 1991). Downmodulation of anticoagulation via regulation of thrombomodulin (McCachren et al., 1991), the essential cofactor for the activation of protein C, the inhibitor of factors Va and VIIIa (Stern et al., 1988), also contributes to the outcome of the hemostatic balance. In addition, regulation of the activity of the TF-factor VIIa complex by the extrinsic pathway inhibitor (TFPI) (Rapaport, 1991), and other natural inhibitors of coagulation, including antithrombin, activated protein C and S, and heparin cofactor 11, can all modulate the thrombotic complications of diseases in which cellular procoagulants are involved.

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II. Tissue Factor A. General

Tissue thromboplastin “substance” was a clot-promoting activity of tissues described by Nolf in 1908. Early attempts to isolate thromboplastin implicated one protein (Chargaff et al., 1944) although attempts to identify a bleeding disorder due to defective thromboplastin cast doubt on the single-protein hypothesis. Purification of a protein with clot-promoting activity which is now known as “tissue factor” was achieved by Bach et al. in 1981. In contrast to other members of the coagulation cascade present in plasma, TF is located in a number of cell types within the blood and tissues. TF is now thought to play a pivotal role in the regulation of coagulation, hemostasis, and thrombogenesis. The pathway of TFmediated coagulation is shown in Fig. 1. TF is a transmembrane cell surface receptor for plasma factor VII. The homogeneous protein was purified from bovine brain and represented a polypeptide of 40-43 kDa (Bach et al., 1981, 1986; Carson er al., 1985). Subsequently, the human protein was purified by immunoaffinity using monoclonal antibodies (Carson et al., 1987; Spicer er al., 1987) and by ligand affinity to human factor VII (Broze et al., 1985; Guha et al., 1986) in quantities sufficient to obtain the N-terminal amino acid sequence. The

JINIRINSICJ Factor IX

Factor IXa

factor Vlla

(EXTRINSICI

factor Vlla Factor X

Xa-Va

-

prothrombinase complex Mo, lymphocytes [Gets.

prothrombin

fibrin

thrombin

fibrinogen

FIG. 1 Tissue-factor-mediated pathway of activation of coagulation.

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complete primary structure of the human TF gene, mRNA, and protein is now established. The cDNA was cloned at about the same time by four groups (Fisher et al., 1987; Momssey et al., 1987; Scarpati et al., 1987; Spicer et al., 1987). The TF gene is located on chromosome 1 (Scarpati et al., 1987) at 1~21-22(Kao et al., 1988) and spans 12.4 kbp, excluding the promoter and regulatory enhancer elements (Mackman et al., 1989).The single gene comprises six exons separated by five introns with the 5' region of exon 1 encoding the translational start point and the 3' region encoding the 32residue leader sequence. A number of potential sites for DNA binding and transcriptional regulatory protein binding have been identified and are discussed in detail by Edgington et ul. (1991). The open reading frame encodes a 295-amino-acid polypeptide which undergoes processing to remove a 32-residue leader sequence, yielding a mature protein of 263 residues. The primary sequence indicated a unique transmembrane protein of 30 kDa consisting of an extracellular domain (Serl-Glu2,g),a hydrophobic membrane-spanningregion (residues 220-242), and a cytoplasmic tail (residues 243-263). Two of the three potential Winked glycosylation sites within the extracellulardomain are sites of post-translationalmodification. Glycosylation is apparently not required for functional activity because removal of carbohydrate by endoglycosidase F does not affect the clotting capacity of the native protein (Bach, 1988)and recombinant TF produced in Escherichiu coli is functional (Paborsky et al., 1989). B. Tissue Factor as a Member of the Cytokine, Growth Factor Receptor Superfamily

Tissue factor is a member of a family of receptors for a diverse group of hematopoietic factors, growth hormones, and interferons with apparently unrelated sequences. In contrast, the family of cognate receptors reveals a striking resemblance of binding domains containing a distinctive conservation of four cysteine pairs in the N-terminal half and a WSXWS (one letter amino acid code, X is nonconserved)near the C-terminal end (Miyajima et al., 1992). TF is most closely related to the interferon (aand y) receptors (type 11 cytokine receptors) which contain characteristic cysteine pairs at both N- and C-terminals and which are evolutionarily related to the type I cytokine receptors. They include receptors for growth hormone, prolactin, erythropoietin, interleukin (IL) 2, IL-4, IL6. granulocytemacrophage-colony-stimulating factor (GM-CSF), IL3, G-CSF, IL7, and IL5 (Bazan, 1990a,b). Structural analysis of extracellular segments indicates a common globular protein fold constructed from seven conserved @-strandswith a topol-

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ogy analogous to an immunoglobulin (Ig) constant domain. The binding segment domains from this receptor family are related to a common 90amino-acid sequence known as the fibronectin type I11 domain. Bazan has postulated that this subclass of Ig-like proteins has evolved from primitive adhesion molecules to their present role in specific protein binding. A model of the TF surface domain based on the structure of PapD (a protein with P strand organization (Holmgren and Brandeur, 1989) and aligned according to the analysis of Bazan (1990b)is presented by Edgington et al. (1991). These authors propose a pair of seven-strand Ig-like modules for the TF surface domain, and the model predicts that the residues of T F would align in two sheets of three and four P-strands respectively, in each module. They assume that interactions between the two modules are critical for T F function, based on modeling of the Class 1 receptors, and suggest that this model is consistent with the multiple interactive binding sites on factor VII/VIIa. The four cysteines in the extracellular region of TF are covalently bonded to form two disulfide loops. The tertiary structure of the molecule is required for functional activity (Bach et al., 1981) and the carboxylterminal cysteines 186 and 209 are essential for fully functional ligand binding (Rehemtulla et al., 1991). Furthermore, the amino acid sequences of rabbit and murine TF are respectively 71 and 58% identical to human TF and are consistent with the relative functional activity of each in human plasma. The structural organization of the protein indicates a high degree of conservation of the extracellular domain and the relative positions of the cysteine residues in all three species (Andrews, 1991). The presence of three WKS repeats within human TF (only one of which is found in the mouse and two in the rabbit) (Andrews et al., 1991) and within several proteins involved in coagulation or in proteins that share some functional properties with the coagulation proteins, has been suggested to represent a functional sequence motif on the basis of its high affinity for human but not for murine factor VII (Andrews et al., 1991) although the structural significance of this repeat has been disputed (Bazan, 1991). There are several indications in the literature that TF may exist in dimeric form. Covalent homodimers may occur by self-association of Cys,,, within the cytoplasmic tail as a purification artifact (Bach, 1988) although this cysteine can also be acylated (Bach et al., 1988). In an attempt to draw functional analogies between activation of the large cofactor coagulation proteins V and VIII, which form multidomain complexes on the phospholipid surface, and the activation of factor X by factor VII/VIIa, Roy et al. (1991) used chemical cross-linking experiments to demonstrate that TF exhibits a tendency to self-associate on cell surfaces.

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Mutation experiments indicate that deletion of the cytoplasmic domain does not affect cross-linking and that the transmembrane sequence is necessary for interaction between TF molecules. Furthermore, a study of binding and activity of factor VII on a bladder carcinoma cell line 582, which expresses high levels of TF, indicated at least two sites on TF with positive cooperativity for ligand binding (Fair and MacDonald, 1987). Because the stoichiometry of the TF-factor VII complex is 1:1, Fair and MacDonald postulated that TF was expressed as a dimer and that association of factor VII with the first site may perturb the dimer enough to permit binding of the second ligand. Cross-linking studies have confirmed the presence of a dimer on these cells although high levels of the monomer were also present (Roy et al., 1991). A multisubunit structure is a common feature of a number of receptors and some, like the granulocyte-colony-stimulating factor (G-CSF) receptor, function as homodimers (Fukunaga et al., 1990). It has been suggested that the number of TF molecules correlates with the ability of cetls to initiate the coagulation cascade (Rodgers et af., 1984), but several studies (Fair and MacDonald, 1987; Ploplis et al., 1987; Walsh and Geczy, 1991; Walsh et al., 1992) do not support this view. Variations may occur between cell types and in the composition and integrity of the cell surface phospholipid environment (see later discussion). In addition, and although there has been no correlation with functional activity, TF dimerization, like that observed for other membrane receptors, may represent a magnification of functional activity and therefore a means of modulating coagulation. TF also forms a heterodimer with a 13-kDa polypeptide in some preparations (Carson, 1987; Morrissey et al., 1988a). The resultant 58-kDa form is a functionally active disulfidelinked heterodimer composed of TF and the a-chain of hemoglobin. The interactions between these two molecules appear to be relatively specific and are proposed to occur as a result of lysis of red cells (Momssey et al., 1988a).The physiological significanceof this heterodimer, which might appear following tissue injury or as a consequence of surface shedding of TF (Bona et al., 1987), is unclear.

C. Function Tissue factor is the high-affinity receptor for plasma factor VIIIVIIa. In contrast to factors V and VIII, which also bind their respective enzymes in a 1 :1 stoichiometric complex and which are activated by partial proteolysis, TF requires no further processing. In the presence of Cazc ions, binding of factor VII/VIIa to TF increases the proteolytic activity of factor VIIa for its primary substrate, factor X, to directly initiate the

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extrinsic coagulation cascade (Bach, 1988; Nemerson, 1988). Mapping of TF-binding sequences using monoclonal antibodies to a series of peptides indicates at least two linear sequences within the extracellular domain involved in factor VII binding. It is proposed that interaction within these sites might represent the structural counterparts of the two functional properties of TF, namely to serve as a receptor and as a cofactor for factor VIIa. Observations that two structural sites within factor VIIa-the y-carboxyglutamic acid domain (Sakai et al., 1990) and residues 195-206 in the catalytic domain (Wildgoose et al., 1990)-are involved in highaffinity binding to TF would support this view. TF activity is phospholipid dependent. T F is normally inserted within a membrane lipid bilayer which provides phospholipid cofactors; the pure molecule requires lipid (phosphatidyl choline/phosphatidyl serine) for activity (Bach, 1988; Nemerson, 1988). Recent experiments with a recombinant TF mutant (TFI-219) with membrane-spanning and intracellular domains removed, indicate that free factor VIIa, but not TFl-219or TF,,,,-VIIa complex, forms a stable association with phospholipid (Ruf et al., 1991). These authors suggest that the catalytic function of TFVIIa is independent of its assembly on phospholipid and that the primary protein:protein interactions of factor VIIa with the surface domains of T F are sufficient to markedly enhance the catalytic function of factor VIIa. On the other hand, factor X is recognized as a preferential substrate for the TF-VIIa complex when it is associated with phospholipid surfaces. A situation analogous to the phospholipid association of prothrombin via the Gla domain (Malhotra et al., 1985), which induces a conformational change essential for proper substrate presentation to the prothrombinase complex on cell surfaces, is proposed (Ruf et al., 1991). These studies cannot exclude the possibility that membrane-anchored TF-VIIa preferentially cleaves free factor X (Forman and Nemerson, 1986; Nemerson, 19881, although the decreased apparent Michaelis constant of TF in reconstituted phospholipid vesicles suggests that phospholipid interaction with factor X might facilitate its presentation to the TF-VIIa membrane complex. In an attempt to simulate conditions in uiuo, recent experiments were designed to study phospholipid-dependent coagulation under flow conditions (shear rates 25 sec-I to 1200 slc-I) using a capillary coated with stable phospholipid bilayers containing TF and perfusion with factors VIIa and X (Gemmell et al., 1991). Under these conditions there was apparent increased binding affinity of factor VIIa to TF, and generation of Xa was approximately the same whether factor VII or VIIa were perfused. Furthermore, the functional activity of the immobilized complex was not altered by excess prothrombin fragment I, a split product of prothrombin which displaces factor X from lipid vesicles (Forman and Nemerson,

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1986). This suggests that factor X-phospholipid interactions did not play a significant role under these circumstances although the amount of factor X binding to the lipid bilayer was not measured. TF binds both the active and zymogen forms of factor VII with equal affinity (Zur et al., 1982). The conversion of factor VII to VIIa is most rapidly catalyzed by factor Xa, and at a slower rate by factor IXa, and is more rapid in the presence of TF (Bom and Bertha, 1990; Nemerson and Repke, 1985; Radcliffe and Nemerson, 1976; Rao et al., 1985, 1986). When the active serine of factor VII is inactivated with diisopropylfluorophosphate, the procoagulant activity is lost (Bach et a!., 1981). Based on enzyme kinetic measurements, Nemerson and Gentry (1986) propose a model for TF-mediated activation of coagulation which involves two related ligand-enhanced conformationalactivations. In the absence of TF, factor VIIa is not significantly catalytic and binding to TF is thought to create sites within factor VIIa which allow interaction with its substrate, factor X. The latter in turn may result in a form of factor VIIa which binds more tightly to TF to form a “conformational cage” which precludes the dissociation of factor VIIa from TF while significant concentrations of factor X are present (Nemerson, 1986). The extrinsic clotting pathway can also be activated indirectly by TF via activation of factor IX by TF/VIIa (Osterud and Rapaport, 1977). Comparison of the kinetic parameters for the activation of factor IX and factor X suggests that at plasma concentrations of these substrates, the rate of factor Xa formation would be approximately 2.5 times that of factor IXa formation (Born et al., 1990; Osterud and Rapaport, 1977). The relative importance of the extrinsic factor IX activation to the overall levels of factor Xa generation in uiuo is unclear although when low levels of TF are present (e.g., when limited amounts of factor Xa and factor 1Xa are formed), the contribution of this pathway may be significant (Bom er al., 1990).

D. Localization Procoagulant activity associated with TF was initially described in a number of organs and cell types, including fibroblasts, smooth muscle cells, endothelial cells, Mo/Mac, and a variety of neoplastic cell types by virtue of the dependence of clotting capacity on factors VII and X,and in some cases sensitivity to phospholipase C. Furthermore, many studies were performed using cell lysates, and activity of lysed cells was often much greater than that of intact cells. Levels of intracellular or “cryptic” TF were reported (Leoni and Dean, 1985) and it was suggested that T F activity was mobilized from an intracellular compartment or occurred within the

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cell membrane in a latent form which became available after the cell surface was perturbed (Maynard et al., 1975; Schorer et al., 1985). In the past few years the availability of monoclonal antibodies to TF has made it possible to confirm the location and activity of TF within a variety of cells and tissues (Drake et al., 1989a,b; Faulk et al., 1990; Fleck et al., 1990; Morrissey et al., 1988b). Immunohistochemical studies of normal human tissue show that the majority of TF-reactive cells are not in contact with blood; TF is anatomically distributed in perivascular cells, in capsules surrounding organs, and in cells of epithelial surfaces. In contrast to earlier reports (Zeldis et al., 1972), endothelial cells do not normally express TF. Furthermore, normal peripheral blood cells are also TF-negative, ensuring that the intravascular compartment does not represent a procoagulant environment whereas the normal distribution of extravascular TF suggests that it could activate coagulation following vascular injury. Localization of TF in normal human tissue is described in detail by Drake et al. (1989a,b) and Fleck et al. (1990). It is expressed in high amounts in the gray matter of the brain and spinal cord, and in lesser amounts in the meninges. Recent experiments using fresh brain specimens from the baboon show a distinct pattern of TF antigen expression associated with the microvasculature (cortical gray matter > basal ganglia L cerebellum > cortical white matter) which correlated with functional activity (del Zoppo et al., 1992). To date, there has been no specific neuronal cell associated with the diffuse distribution of TF in gray matter. Del Zoppo and colleagues have made the interesting suggestion that normal migration of Mo/Mac into the cortical parenchyma may shed sufficient T F to account for its diffuse localization. The gray/white matter partition of TF may be accounted for by greater microvascular content and endothelial cell surface area, allowing more Mo/Mac transmigration into cortical gray matter than white matter. The bronchial mucosa, alveolar septae, epithelial cells, and macrophages of the lung are TF positive. Fibroblast-like cells and Mac, but not trophoblasts, in connective tissue of the villi of the placenta and in amnion encasing the placenta and umbilical cord are generally strongly positive (Faulk et al., 1990; Fleck et al., 1990). Epithelial cells delimiting body/ environment boundaries (e.g., squamous epithelium of the skin and cervix, gut mucosa, cuboidal epithelium of the bladder) strongly express TF. TF in the kidney is limited to the glomeruli in which epithelial and mesangial cells of the glomerular tuft react strongly, and epithelium of Bowman’s capsule is also positive whereas glomerular capillary endothelium is negative. Skeletal muscle cells do not express T F whereas cardiac myocytes possess a cytoplasmic distribution and only smooth muscle cells of the muscularis mucosa of the esophagus showed positive reactivity. In addi-

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tion, cells composing the fibrous caps of liver, spleen, kidney, and adrenals express varying levels of TF. Expression of TF by connective tissue fibroblasts occurs most prominently in the adventitia of blood vessels whereas its location within these cells at other sites is apparently variable (Drake et al., 1989a; Fleck et al., 1990; Wilcox et af., 1989). As expected from the widespread distribution of human TF described by immunohistochemistry, constitutive expression of TF mRNA has been found in many mouse tissues and is especially abundant in lung, brain, midterm placenta, testis, and kidney, with lower levels apparent in heart, spleen, and intestine (Hartzell et al., 1989). In addition, mRNA in human tissue is found in adipose tissue, placenta, adrenal gland, small intestine, kidney (Fisher et al., 1987), and brain (Scarpati et af., 1987), whereas it was not detected in pancreas, liver, and spleen (Fisher et al., 1987). In situ hybridization studies (Wilcox et a[., 1989) located strong TF mRNA expression in adventitial fibroblasts, which correlated well with antigen expression in samples of normal human saphenous vein and internal mammary arteries. Scattered smooth muscle-like cells within the tunica media had levels of mRNA similar to those of adventitial fibroblasts, but had lower levels of protein.

1. Monocyte-MacrophageTissue Factor Mo/Mac procoagulant activity (MPCA)is induced by a number of intrinsic and extrinsic stimulants and is the subject of a number of reviews (Dean er al., 1984; Edwards and Rickles, 1980a; Geczy, 1984; Ryan and Geczy, 1987). It is induced by a variety of infections, including both gram-negative and gram-positive bacteria, bacterial toxins, viruses, parasites [e.g., Plasmodulinfalciparum-infected erythrocytes (Pernod et af., 1992)],activated complement components (Osterud et al., 1984), immune complexes (Schwartz et al., 1982a), modified lipoproteins (Levy et al., 1981), free cholesterol (Lesnik et al., 1992), and cytokines which regulate cell-mediated immune reactions and inflammation (see later discussion). One of the best-studied inducers of MolMac, TF is bacterial lipopolysaccharide (LPS) (endotoxin), which in a clinical situation plays a pivotal role in the development of gram-negative septicemia. The hematological manifestations of this condition include activation of the coagulation, fibrinolytic, and complement systems (van Deventer ef al., 1990). Fibrin deposition and complement activation can cause extensive damage of vessel walls and may be associated with multiple organ failure. A role for TF in the disseminated intravascular coagulation (DIC) associated with administration of LPS is substantiated by the reduction of fibrin formation and DIC by pretreatment of rabbits with anti-TF antiserum and the promotion of DIC and pulmonary artery thrombosis by infusion with TF (Warr

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et al., 1990). Furthermore, a monoclonal antibody to TF administered to baboons prior to injection of E. coli attenuated the coagulopathy and protected against its lethal effects by reducing the degree of cardiovascular collapse and cell injury (Taylor et al., 1991). Although part of the procoagulant effect has been attributed to increased levels of IL1 and tumor necrosis factor-a (TNF-a) (Bauer et al., 1989; Conkling et al., 1988a; van der Poll et al., 1990) which have variable effects on Mo T F and which modulate TF expression by endothelial cells, markedly more TF is expressed by Mo/Mac in direct response to LPS. Furthermore, delayed treatment with anti-TNF antibodies did not reverse the consumption of fibrinogen in plasma of baboons given E. coli (Hinshaw et al., 1990), supporting the proposal that an additional pathway of procoagulant induction contributes to lethality. In addition, peritoneal Macs (Robinson et al., 1978), blood Mos, and spleenic cells (Rothberger et al., 1983) from animals injected with LPS express high levels of procoagulant activity. Actinomycin D, which sensitizes mice to the lethal effects of LPS, renders murine Macs sensitive to levels of LPS some 100,000-fold less than normal, whereas TNF production is only doubled (Wheeler et al., 1991). Human Mos are exquisitely sensitive to LPS, responding to levels as low as 0.1-1 pg/ml in our experiments. Blood Mos are not heterogeneous with respect to their responsiveness; our immunohistochemical studies show that >98% of these cells convert from surface TF-negative to TFpositive following incubation for 16 hr with I ng/ml LPS (Walsh and Geczy, 1991). Our studies have also consistently found that blood Mos from female donors are more sensitive to LPS than those from male donors ( J . D. Walsh and C. L. Geczy, unpublished). Although the reason for this observation is uncertain, the recent evidence that estrogen can regulate TF gene expression in the immature rat uterus (Jazin et al., 1990) suggests that TF levels in Mos may also be under hormonal control. TF transcripts are detectable in human Mos 0.5 hr postinduction with LPS and reach maximal levels within 4 hr, coinciding with expression of TF activity on viable cells (Gregory et al., 1989). Interestingly, LPS coordinately initiates the transcription of the IL- l p and TNF-a genes over the same time course as that of TF although the selective reduction of TF mRNA expression in LPS-stimulated Mos from patients with advanced AIDS indicates that these genes can segregate under pathological conditions (Lathey et al., 1990). Furthermore, reduction of TF in this condition may contribute to the diminished resistance to infection observed in AIDS patients. Experiments with the monocytoid cell line THP-1 indicate that after stimulation, TF gene transcription increases threefold and at 1 hr T F mRNA is stable over 60 min with a half-life of >120 min, whereas at 2 hr the half-life declines to 25 min, suggesting that both transcriptional and post-transcriptional mechanisms control its synthesis (Brand et al.,

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1991). Possible mechanisms of transcriptional control of the TF gene are described by Mackman et al. (1989) and reviewed by Edgington et al. ( 1991) .

2. Cytokine-Mediated TF Induction on Monocytes and Macrophages: Implications in Cell-Mediated Immune Reactions Fibrin deposition is a common feature of inflammation and of numerous diseases in which cell-mediated immunity (CMI) plays a role (Dunn and Willoughby, 1981; Edwards and Rickles, 1980a; Geczy, 1983; Geczy et al., 1984). The swelling typical of classical delayed-type hypersensitivity (DTH)reactions is caused by water trapped within fibrin (Colvin and Dvorak, 1975; Colvin et al., 1973). A role for extravascular fibrin in these reactions is confirmed by observations that afibrinogenemic patients fail to respond to skin test antigens (Colvin et al., 1979) and anticoagulants inhibit induration induced by skin test antigens (Edwards and Rickles, 1978; Nelson, 1965). Mo/Mac procoagulants, induced in response to cytokines released as a consequence of immune activation, are now considered important initiators of coagulation. In addition to the exogenous inflammatory stimuli which influence MCPA (see earlier discussion), our earlier studies (Geczy and Hopper, 1981) and those of Edwards and Rickles (1980b) and van Ginkel and colleagues (1981) implied a role for T lymphocytes and Tlymphocyte-derived products in this response. In addition, a number of other products of an active immune response, particularly autoantibodies (Tannenbaum et al., 1986), antigen-antibody complexes (Lyberg et al., 1982; Rothberger et al., 1977; Schwartz et al., 1982a),and activated complement components C5a and C3b (Muhlfelder et al., 1979; Prydz et al., 1977) all influence Mo/Mac procoagulant expression. Our earlier experiments showed that generation of MPCA following culture of mononuclear cells with microbial antigens is a close in v i m correlate of DTH skin test reactions in man (Geczy and Meyer, 1982) and there is a parallel between the capacity of mouse strains to develop DTH and the intensity of MPCA generation in uitro (Geczy et al., 1983). Although there may be a requirement for direct T-lymphocyte-Mac contact under some circumstances of TF activation (Edgington et a)., 1981;Helin et al., 1983; Levy and Edgington, 1982;Tsao et al., 1984; Fan and Edgington, 1988), a clear involvement of lymphocyte-derived cytokines is now well accepted although the identity of the cytokines involved is still somewhat unclear. This pathway of Mo/Mac procoagulant induction may contribute to the pathology of many immunologically mediated diseases such as multiple sclerosis [evidence from an animal model system-experimental autoim-

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mune encephalomyelitis (Geczy et al., 1984)l the Guillain-Barre syndrome (Geczy et al., 1985), influenza (Schiltknecht et al., 1984), coeliac disease (Devery et al., 1990), allograft rejection (Halloran et al., 1985; Zappala et al., 1989),glomerulonephritis (Cole et al., 1985),and malignancy (Lyberg, 1984b; Rickles and Edwards, 1983)and other diseases, such as atherosclerosis (Wilcox et al., 1989)in which a role for lymphocyte-derived cytokines has only recently been recognized (Serneri et al., 1992). Our studies indicate that murine Lyt 1'2- T, spleen cells in the presence of a major histocompatibility complex (MHC) class 11, antigenpositive, adherent accessory cell produce a factor which we called macrophage procoagulant-inducing factor (MPIF) (Geczy et al., 1983; Ryan and Geczy, 1986). Antigen presentation by monocytes in the context of MHC class I1 antigen was necessary for MPCA induction by protein antigens, confirming a classical immune response to antigen (Schwartz, 1985). Furthermore, in contrast to CD 8 + cells, alloantigen-activated murine CD4+T cells produce MPIF (Fan and Edgington, 1988)and the human alloantigen response is mediated by T cell clones of the CD3+,CD4+, CD8- phenotype (Gregory and Edgington, 1985). Moreover, cyclosporin A, an immunosuppressive drug which alters T cell function, inhibits MPIF induction in uitro (Chung et al., 1991; Thomson et al.. 1983a) and in vivo (Thomson et al., 1983b). Because much of the background work in this area has been the subject of a number of reviews (Geczy, 1983, 1984; Ryan and Geczy, 19871, only the more recent work will be presented here. Cytokines modulating MPCA are given in Table I. Several studies clearly implicate cytokines, in addition to MPIF, in MPCA induction although it is interesting that procoagulant responses of various cell types appear to be somewhat cytokine-specific. In contrast to their effects on cultured endothelial cells, ILl-a and -p induce weak activity on human blood Mos (Carlsen et al., 1988; Carlsen and Prydz, 1988). We find that these mediators have no effect on murine Macs. Although high levels of TF induction on U937 monocytoid cells and blood Mos by TNF has been described (Conkling et al., 1988b), we and others have been unable to demonstrate this effect (Carlsen et al., 1988; Gregory et al., 1986; Ryan and Geczy, 1986). Carlsen and Prydz (1988) demonstrated a 15-fold enhancement of TF activity on human Mos by IL2 whereas others (Gregory et al., 1986) found that 100-1000 U/ml IL2 induced low levels of TF compared with MPIF, and we failed to induce procoagulant on murine exudate Macs with IL2. Interferon-? (IFN-y), a product of both CD4+ and CD8+ lymphocytes, apparently plays a regulatory role in the procoagulant response. IFN-y is an important activator of many cell types, particularly Macs, which are produced as a consequence of infection, activated CMI, and inflammation (Adams and Hamilton, 1984), and is thought to play a significant role in

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TABLE I Modulation of MolMac Procoagulants by cytokines ~

Cytokine T-lyrnphocyte-derived IFN-y MPIF IL2 IL4 MolMac-derived TNF-CY ILI-a. p GM-CSF IL6

Tumor-derived VPF

Procoagulant effect Primes inflammatory Macs Induces TF expression on both Mos and Macs Induces TF on Mos Inhibits MPCA induction by some stimulants Some reports of TF induction on Mos Induces moderate levels of TF on Mos Primes inflammatory Macs to respond to LPS Weak potentiation of LPS response on Macs Induces weak TF activity on Mos

the pathology of septic shock (Heinzel, 1990). Our studies show that viable, thioglycollate-elicited murine peritoneal exudate (TG-PEC) Macs primed with IFN-y expressed 10-40-fold more MPCA in response to suboptimal levels of LPS (Moon and Geczy, 1988). The major procoagulant activity had characteristics of TF which were maximal after 24-hr culture and kinetics similar to those described for induction of tumoricidal or microbicidal activities exhibited by these cells in response to IFN-yI LPS (Adams and Hamilton, 1984). In addition to the synergy observed between IFN-y and LPS, we have investigated the possibility that, although some cytokines may not act alone, they may synergize with IFN-y to initiate procoagulant expression or TG-PEC. Of a number of cytokines tested, we found that IL6 synergizes weakly with IFN-y to induce TF levels approximately four times greater than basal levels. MPIF apparently also synergizes with IFN-y although further studies with pure MPIF are required to confirm this (A. Jones and C. L. Geczy, unpublished data). We also found that IFN-y synergized with PMA, but not LPS, to induce TF in a myelomonocytoid human cell line, RC2a (Geczy and Jones, 1988), although its potential interaction with other cytokines has not been tested in this system. There is one report that GM-CSF primes cultured (1-14 days) adherent TGelicited Macs to respond to LPS (Zuckerman and Surprenant, 1989) al-

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though we failed to demonstrate this activity using TG-PEC cultured with GM-CSF and LPS in nonadherent conditions. Although IFN-y alone failed to induce significant levels of procoagulant in viable TG-PEC, lysed cells express an activity similar to factor VIIa. There are a number of reports of factor VII/VIIa induction on Mo/Macs (Chapman et al., 1985; Tsao et al., 1984), and murine exudate Macs may constitutively express factor VII (Shands, 1984; Shands et al., 1988), which could combine with TF within disrupted cell membranes to provide active factor VIIa. Our recent experiments show that rIFN-y upregulates T F mRNA expression in murine TG-PEC to levels somewhat greater than those induced by LPS (Fig. 2), although we cannot detect increased amounts of TF antigen in lysates of cells cultured over 2-24 hr with IFNy in Western blotting experiments (Jovanovich and Geczy, 1994). These experiments confirm that IFN-y can modulate TF gene transcription although factors regulating expression of functional activity are still unclear. In contrast to exudate Macs, neither resident Macs nor blood Mos respond to IFN-y, either to express intracellular activity or extracellular T F when cultured with LPS (Moon and Geczy, 1988). Furthermore, IFNy does not induce TF on human blood Mos, and suppression of T F induction by allogenic lymphocytes or LPS occurs when these cells are cocultured with IFN-y (Carlsen et al., 1987; Carlsen and Prydz, 1988; Conkling et al., 1988b). By contrast, human Mo-derived Macs (blood Mos cultured in Teflon bags for 6-8 days) grown in suspension culture express procoagulant activity in response to IFN-y (Miserez and Jungi, 1992), and a murine monocytoid leukemic cell line (WEHI 265) is also directly responsive

FIG. 2 Northern blot analysis of mRNA extracted from murine TG-PEC incubated for 4 hr with (i) control media, (ii) LPS (Ipg/ml), (iii) IFN-y(100 U/ml) and probed with (A) a cDNA fragment probe to murine TF (kindly provided by Dr. D. Nathans) and (B) an oligonucleotide probe to rat 18s ribosomal RNA for comparative quantitation of total RNA.

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(Wheeler et al., 1991). These experiments add weight to the proposal that the maturation and differentiation states of Mo/Mac may determine their reactivity to different stimulants of TF expression (Geczy, 1984; Lyberg et al., 1983b). In contrast to the narrow range of activity induced by IFN-y, human and murine MPIF directly stimulate TF expression on human Mos (Gregory and Edgington, 1985) and murine TG-PEC respectively, and on some monocytoid cell lines (Farram et al., 1983). In contrast to the slow response elicited by IFN-y/LPS on TG-PEC, MPIF induces high levels of activity after 6-8 hr of culture which are maintained over 24 hr. MPIF has been purified to homogeneity in our laboratory but its complete amino acid sequence is still not determined (M. Lackmann and C. L. Geczy, unpublished). Our earlier studies (Ryan and Geczy, 1986) and those of Gregory et al. ( 1986) indicated that MPIF was a heparin-binding protein which displays heterogeneity with respect to size and charge. We isolated two active murine components with PIS of 8.5 (a)and 8.8-9.0 (p) and a third component (PI 5.5) which we now believe to be IFN-y (M. Lackmann and C. L. Geczy, unpublished data). The human and murine MPIFs are apparently novel proteins. A large panel of cytokines tested for MPIF activity was inactive and purified fractions are devoid of many of the cytokines, including CSFs, IL1, IL2, TNF-a, and $3, and IFN-y, and antibodies to a number of cytokines fail to alter activity. Furthermore, human MPIF enhances TF on RC2a cells whereas LPS fails to alter TF expression on those cells, confirming our suggestion that the activity is not merely due to contaminating LPS or to a synergistic response with LPS (Geczy and Jones, 1988). The relationship between MPIF, factors present in supernatants of a number of murine tumor cell lines which induce MPCA on TG-PEC (Inoue et al., 1983), and vascular permeability factor (VPF) produced by murine meth A fibrosarcoma cells (Clauss et al., 1990a,b) (which induces TF on endothelial cells and somewhat weaker activity on human Mos) is unknown, although the species specificity of murine MPIF is different than that of VPF. There have been relatively few studies to determine whether cytokines affecting MPCA induce fibrin deposition in uiuo. The presence of fibrin in DTH lesions is well established and recent enzyme and immunohistochemical studies confirm the presence of thrombin (Imamura and Kambara, 1992) and of TF-positive Macs at these sites (Imamura et al., 1992). Our studies showed that a highly enriched fraction containing MPIF induced an indurated lesion containing extravascular fibrin, an early influx of neutrophils, and a sustained influx of mononuclear cells over 24-48 hr (Ryan and Geczy, 1988) when injected intradermally into rat skin.

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We subsequently separated MPIF from a unique protein member of the family of SlOO Ca*+-bindingproteins (called CP-10; chemotactic protein, 10 kDa) which has potent chemotactic activity for neutrophils and Mos (Lackmann et al., 1993). This protein induces a strong neutrophil infiltrate 4-6 hr postinjection, with increased numbers of Mo/Mac evident after 24 hr (Lackmann et al., 1993; Devery et al., 1994). The time course and cellular characteristics of the inflammatory response are similar to a DTH response and in marked contrast to the more transient infiltration of neutrophils elicited by other proinflammatory cytokines such as ILl (Cybulsky et al., 1986), macrophage inflammatory protein (MIP) (Wolpe et al., 1987) and IL 8 (Foster ef al., 1989), and by C5a (Yancey et al., 1985). Interestingly, the classical chemotactic stimulants for phagocytic cells, C5a and bacterial cell wall peptide fMet Leu Phe (FMLP), induce T F on human Mos (Janco and Morris, 1985; Muhlfelder et al., 1979) and platelet activating factor (PAF), a chemotactic factor and potent inflammatory mediator produced by a variety of cells, including activated neutrophils, Mo/Mac, platelets, and endothelial cells, primes TG-elicited murine Macs to express 2- to 5-fold higher procoagulant levels (Kucey et al., 1991). In addition, PAF is a potent neutrophil stimulant which enhances LPSinduced blood Mo TF expression when these cells are cultured together with neutrophils and platelets. Osterud (1992) has suggested that this reaction is mediated by neutrophil-derived cathepsin G, which is a more potent platelet activator than PAF (Selak et al., 1988). On the other hand, we have been unable to influence procoagulant with CP-10 or a number of other chemotactic proteins, including MIPl and 2, macrophage chemotactic proteins (MCP) 1 and 2 (Yoshimura et al., 1989), platelet factor 4, and transforming growth factor-p (TGF-p). Chemotactic peptides may contribute to the activation of cogulation in uiuo by virtue of their ability to release enzymes which may indirectly modulate the response and/ or alter expression of adhesion receptors on infiltrating cells (see later discussion). These agents induce cellular migration into tissues, a process which causes changes in the vasculature. This event alone may be sufficient to promote fibrin deposition as proposed by Dvorak and colleagues (1985). 3. Regulation of Endothelial Cell TF

The endothelium was originally considered to be a passive barrier between blood plasma and cells and the interstitial matrix, referred to by Florey (1966) as the “cellophane wrapper” of the vascular tree. However, studies over the past 10 years suggest that the normally anticoagulant surface of endothelial cells may promote coagulation when inflammatory and cellmediated immune responses are activated. TF is not normally expressed

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by endothelial cells in tissues or by these cells from a variety of sources in vitro. Many studies indicate however, that TF can be induced in culture by intrinsic stimuli such as the proinflammatory cytokines IL1 and TNFa,by thrombin and platelets, by antibodies and immune complexes, by some components of normal plasma and serum, and by extrinsic agents such as LPS, phorbol esters, and allogenic lymphocytes (see Table 11). Despite extensive studies to characterize TF induction on endothelial cells in culture, there is little evidence of its expression by these cells in pathological situations in uiuo. Although only a small number of studies have been performed, sensitive in situ hybridization techniques failed to demonstrate TF in endothelial cells lining normal mammary artery and saphenous vein. Furthermore, no TF mRNA or protein is detected in either the endothelium lining the vascular surface or small vessels within human atherosclerotic plaque specimens, whereas high levels of TF are associated with Macs (Wilcox et al., 1989). Histologicalevidence of TF associated with fetal stem vessel endothelial cells in areas of placental chorionic villi associated with chronic inflammation has been presented by Faulk et af. (1990), although these lesions contain activated Macs which represent a potential source of TF. Endothelial cells in culture were first shown to produce TF by Maynard et a!. (1979, who concluded that it was functionally dormant in undisturbed TABLE II Inducersof Endothelial Cell TF

Agent Extrinsic LPS Phorbol esters Intrinsic Platelets Thrombin Interleukin I Tumor necrosis factor-a Allogenic lymphocytes Vascular permeability factor Diacylglycerol Normal plasmalserum "Procoagulant" albumin Antibody, immune complexes Antiphospholipid antibodies Minimally oxidized LDL Histamine EpineDhnne

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cells but that scraping, freeze-thawing, or tryptic digestion-procedures that perturb the cell surface-made TF available to initiate extrinsic coagulation. These findings confirmed earlier histological observations by Ashford and Freiman (1968) that the endothelium performed a protective function and had no coagulation activity in uiuo unless it was traumatized. The first clear demonstration that TF was an inducible protein in endothelial cells (reviewed by Galdal, 1984; Prydz and Pettersen, 1988) has led to many studies to define the mechanisms of TF induction by these cell types. The TF of unstimulated endothelial cells is intrinsically low but is enhanced to varying degrees by the agents listed in Table 11. Functional activity has usually been measured using lysates of human umbilical vein endothelial cells (HUVEC). It occurs optimally after 4-8 hr in culture and returns to basal levels after 24 hr. Decay of TF activity is accompanied by a reduction in TF antigen, suggesting that it is degraded in culture rather than inhibited (Andoh et al., 1990). Cell surface expression of T F induced by LPS comprises approximately 30% of the total TF activity (Bevilacqua et al., 1984). In one study using a monoclonal antibody to assess surface TF, maximum antigen expression in response to LPS preceded maximal functional activity and capacity to bind radiolabeled factor VIIa by 2-4 hr (Noguchi et al., 19891, and followed kinetics similar to those described for induction of mRNA synthesis (Crossman et al., 1990). Noguchi et al. (1989) propose that the TF apoprotein may require a post-translational conformational change at the cell surface as described for the insulin proreceptor (Olson and Lane, 19871, reorganization of phospholipid cofactors important for factor VII binding, and/or proteolytic processing to achieve optimal functional capacity. The functional properties of surface T F on HUVECs stimulated with thrombin has been compared with purified TF apoprotein in reconstituted mixed phospholipid vesicles. With both types of TF, the rate of factor VIIa/TF activation of factor X is severalfold faster than that of factor IX, indicating that the microenvironment of T F within the membrane does not alter the kinetic parameters of factor VIIalTFdependent activation in favor of factor IX as the substrate (Almus et al., 1989). The estimated affinity constant for TF-mediated binding of factor VII/ VIIa on LPS-stimulated HUVECs is 17.2 ?Z 5.2 nmol and approximates levels of FVII in plasma (10-20 nmol) with an estimated 342,000 +- lo00 binding sites/cell (Clozel et al., 1989). Effective triggering of the extrinsic coagulation pathway on the cell surface may depend on the number of molecules of FVIWIIa bound rather than on the affinity of FVII/VIIa for TF, as long as the affinity is sufficient to achieve receptor occupancy under physiological conditions. Furthermore, the recent demonstration

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of at least two independent binding sites for factor VIUVIIa on stimulated endothelial cells, of which about 10% are TF-specific and the rest are shared with equal affinity by other vitamin K-dependent proteases via the Gla-domain common to these proteins, suggests that the nonspecific sites may facilitate the assembly of components of the extrinsic coagulation pathway on the cell surface by lateral recruitment of these factors (Reuning et al., 1993). Treatment of cells with cycloheximide or actinomycin D during T F induction confirmed the response to be dependent on de nouo protein and RNA synthesis (Galdal et al., 1985; Lyberg et al., 1983a; Nawroth and Stem, 1986; Schorer et al., 1986). Northern blot analysis indicates that T F mRNA is rapidly induced in HUVECs by LPS, phorbol myristate acetate (PMA), TNF-a and ILI-@,with maximal peak activity variously reported to be between 1 and 3 hr, after which it rapidly declines (Busso et al., 1991; Crossman er al., 1990; Scarpati and Sadler, 1989). Recent studies confirm that PMA stimulates transcription of the TF gene (Scarpati and Sadler, 1989), causing a 10-fold increase compared with the twofold increase in transcription levels following LPS stimulation and that these values parallel levels of functional acti-.,i:y (Crossman et al., 1990). Crossman er al. (1990) suggest that LPS stimulation of T F mRNA in HUVEC is substantially controlled by message stability whereas PMA stimulates transcription. Functional studies by Andoh et al. (1990) indicated that cycloheximide increased the half-life of TF induced by PMA from about 8 to 30 hr, and other studies suggest that a labile protein may mediate T F transcription (Busso et al., 1991; Scarpati and Sadler, 1989). In addition to direct induction by a single agent, T F expression can be augmented either by coculture with a second reagent or by some other cell types. LPS has been almost universally used as a standard stimulant for T F induction in cultured endothelial cells. In contrast to the exquisite sensitivity of human Mos to LPS (in the picogram range; see earlier discussion), endothelial cells require approximately 103-106higher doses. These levels are higher than those frequently measured in plasma from humans suffering with sepsis or in patients with sustained organ failure (Wharram et al., 1991), suggesting that the potential contribution of endothelial cell T F to the pathological consequences of septicemia may be due to a combination of LPS and cytokines. Furthermore, Drake and Pang (1989) suggested that the fibrin formed in the vegetations of infective endocarditis was apparently not a consequence of endothelial cell T F produced as a result of infection. Using endothelial cells isolated from human cardiac valves, these authors demonstrated high levels of T F in response to LPS but not following exposure to viable enterococci, viridans streptococci, or an enterococcal cell wall preparation. In this situation, indirect activation by other mediators produced locally as a result of

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infection (e.g., thrombin and ILI and TNF-a produced by activated Macs) may contribute to TF-initiated thrombosis in endocarditis. Early studies demonstrated that TF induction on HUVECs cultured with phytohemagglutinin (PHA), PMA (Johnsen et al., 1983)or LPS (Brox et al., 1984) or heat-aggregated IgG and sera from two patients with lupus (Galdal et al., 1985) was augmented by platelets and platelet aggregates whereas platelet-derived mediators played only a minor role. Although the procoagulant activity had plasma coagulation factor dependencies consistent with TF, it is possible that platelets provide an increased phospholipid surface that serves to assemble the prothrombinase complex in close proximity to TF induced on cultured cells, rather than enhancing TF gene transcription/antigen per se. Studies by Stern and colleagues (1985a) support this view. Scanning electron microscopy (EM) showed that LPS-activated rabbit aorta endothelial cells generate fibrin in the presence of factors VIIa, IX, VIIJ, X, prothrombin, and fibrinogen. Platelets increased thrombin formation about S f o l d , and platelet-derived factor V was thought to contribute to the response. Platelet-associated ILI expressed following activation by thrombin, ADP, and collagen (Hawrylowicz, 1993) may contribute to this response. Cooperation between endothelial cells and leukocytes and mononuclear cell lines has also been described (Lyberg et al., 1983a). Lymphocytes and Mos directly stimulated TF synthesis, which was augmented by PMA or PHA. Both CD4+ and CD8+ allogenic lymphocytes induce approximately a 6-fold increase in HUVEC TF. In contrast to other agents, this response is biphasic, with peak activity evident following 16 and 72 hr of coculture and may be mediated, at least in part, by cytokines released into the medium during allogenic stimulation although these were not identified (Lyberg et af., 1983a). Normal neutrophil/Mo suspensions, either directly in contact with cultured endothelial cells or separated by a semipermeable membrane, also enhance procoagulant levels in the absence of an exogenous stimulus (Schaub et af., 1990). Approximately 45% of the activity was attributed to ILl-/3 and was possibly produced by any or all of the three cell types in the mixture. The contribution of ILI to endothelial cell TF induction has been confirmed in coculture experiments by Wharram e? al. (1991), who showed that human Mos slightly increased basal procoagulant levels whereas they act in a synergistic manner to increase the sensitivity of endothelial cells to LPS by a factor of lo4. The response was amplified some 40-fold by heat-aggregated IgC, which has been shown by others to provoke cytokine secretion by LPS-sensitized monocytes and/or endothelial cells (Arend et al., 1985). Stern and colleagues (1985a) suggested earlier that an integral part of the endothelial cell response to injurious stimuli was the regulatory effect

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of endothelial cell-derived ILI on both the inflammatory process and the vessel wall, and showed that thrombin and LPS-stimulated endothelial cell supernatants contained procoagulant-inducing activity attributed to IL1. Supernatants from the stimulated cocultures described above (Wharram et al., 1991) induced TF, 98% of which was inhibited by anti rILI-p but not by anti-r ILI-a or anti-rTNF-a, which suggests that IL1-p is the principal cytokine involved. Although human ILl-a and -p stimulate endothelial cell procoagulant with approximately equal efficiencies (Dejana er al., 1987), ILl-p is the major soluble product of activated Mo. On the other hand, TNF-a is also a major product of activated Mo and was detected in these supernatants at levels approximating 20 ng/ml. In comparison with ILI-p, TNF-a induces weak T F activity in endothelial cells (Yong et al., 1991); the activity of rILI-0 is about 200 times that of rTNFa and these two cytokines act in synergy over a narrow dose range to induce TF. Additional factors are apparently also involved because anti-r-lL 1-p and -aand anti-TNF inhibit stimulated cell coculture by only 58%. This proposal is supported by the fact that T F induced by rIL1-P alone peaks at 6 hr and rapidly declines whereas the procoagulant response is prolonged through at least 24 hr by Mos. Although the majority of studies describe T F induction 4-8 hr after stimulation with recombinant TNF-a or IL1, Schorer et al. (1986) describe persistent T F activity induced by native ILI on HUVECs over 24 hr. The differences may reflect variations in assay or culture conditions or ILI preparations. The contribution of ILI-p derived from other cell types is also worth considering. The rapid release of neutrophil-derived ILI may play a role in early inflammatory response (Goto et al., 1984), particularly in conditions such as vasculitis, in which these cells play a key role (Bacon, 1991). Furthermore, neutrophil ILl-/3 is upregulated by a number of stimuli, including LPS, GM-CSF, TNF, and IL1 (Goh et al., 1989; Lindemann et al., 1988; Marucha et al., 1990). The combination of IL1-p and TNF cooperatively induce on neutrophil IL1-p gene expression, which is rapid and which may augment inffammationbefore mononuclear cells are prominent within the exudate. IFN-y is an important T-lymphocyte-derived mediator involved in activation of a number of cell types and which can also influence endothelial cell function (Pober, 1988). As described earlier, IFN-y can profoundly augment the procoagulant responses of inflammatory Macs. rIFN-y weakly enhances T F induction by LPS, PHA (Almus et al., 19891, and IL1-p (Yong et al., 1991) on HUVECs although it may not act through priming since it increased T F when it was added after LPS or IL1-p, and prolonged responses to these agents over 16 hr. In contrast to its effect on TG-elicited Macs, the reported enhancement of the LPS response

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on HUVECS is comparatively weak. The increase in total cell activity (lysed cells) induced was only about 2-fold and much higher doses (102-104U/ml) were required for endothelial cells than for Macs (0.1lo2 U/ml). The significance of IFN-y-enhanced endothelial cell TF is unclear although its principal function may be to maintain the response induced by other stimulants. A recently described 44-kDa protein isolated from supernatants of murine fibrosarcoma cells and related to vascular permeability factor (Keck et al., 1989) amplified the TF mRNA expression induced in HUVECS by low concentrations of TNF-a (Clauss et al., 1990a,b).The factor had little effect alone but enhanced TF antigen expression approximately threefold over 4-6 hr when it was added in the picomolar dose range. Clauss and colleagues suggest that it may increase the affinity and number of endothelial binding sites for TNF-a to cause enhanced TF activity. This may result in the formation of occlusive thrombi within the vasculature and alterations in vascular function within the tumor microenvironment. In addition to the cytokines discussed above, endothelial cell procoagulants are induced by some modified proteins, including glycosylated albumin, which are produced to resemble advanced glycosylation end products (AGE) of proteins (Esposito et al., 1989), and a “procoagulant albumin” constituent of normal plasma, the precise modification of which is unknown (Faucette et al., 1992). Such modified proteins may accumulate in the vasculature of older subjects and patients with diabetes, and may be associated with vascular damage. The effect of AGE-bovine serum albumin (BSA) on endothelial procoagulant was slower in onset than that observed for “procoagulant albumin” and other cytokines and required at least 2-3 days for induction of maximal activity. Both forms of albumin induced relatively weak activity approximately equivalent to that produced by TNF-a whereas AGE-BSA-activated cells (2 days) expressed about eightfold more activity when subsequently stimulated for 6 hr with TNF-a. This suggests that such proteins may sensitize the vasculature to exhibit amplified procoagulant activity in response to other inflammatory stimulants. Because of the potential importance of cellular procoagulants in mediating tissue injury and clot formation in atherosclerosis, modified lipoproteins have been implicated in TF induction in endothelial cells. Native low-density lipoprotein (LDL) is ineffective whereas minimally oxidized LDL (mm-LDL), a form of LDL which alters several functions of endothelid cells that modulate interactions with blood cells, enhances TF mRNA levels approximately 30-fold within 2 hr, with peak activity in lysed cells evident after 4-6 hr (Drake et al., 1991). These studies differ from the

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majority of those described above in that cultured aortic and cardiac valve endothelial cells were used in the experimental system. The effective form of mm-LDL was recognized by the LDL receptor, indicating minimal modification of the apoprotein whereas both highly oxidized LDL and malondialydehyde-modified LDL, forms of LDL recognized by the scavenger receptor, failed to induce TF. In contrast to the levels of TF produced by many agonists, optimal doses of mm-LDL preparations (40 pg/ml) enhanced TF activity from 8- to 50-fold, which is equivalent to levels achieved for maximal stimulation with LPS. Low concentrations had an additive rather than synergistic effect when tested in combination with IL-I , whereas low levels of LPS caused no enhancement. In spite of the large number of studies of endothelial cell TF induction in uitro, the paucity of reports demonstrating TF antigen or mRNA in pathological specimens raises questions about its physiological relevance. Furthermore, discrepancies exist within the literature concerning the optimal time of induction of TF mRNA by a variety of stimulants and the duration of TF expression. These differences may be explained, in part, by the source of endothelial cells used (generally human umbilical vein, which may be inappropriate for studies aimed at questions concerning thrombosis or atherosclerosis), the growth conditions and factors used, the number of passages cells have undergone before testing, and measurement of total cell (lysed) versus viable cell TF activity. For example, secondary cultures (4-5 doublings)apparently respond better than primary cultures, but at higher doublings the response declines (Andoh et af., 1990; Busso et al., 1991; Prydz and Pettersen, 1988). Endothelial cells develop hyporesponsiveness to a stimulant followingprolonged incubation so that serum factors or LPS may alter the procoagulant potential of these cells even though they may respond well to a second agent (Busso et al., 1991; Moore et al., 1987). Sensitization is not limited to TF induction and may represent another level of modulation of endothelial cell function by inflammatory agents in uiuo. Growth factors may also influence the experimental outcome (see later discussion). A variety of model systems have been used in attempts to simulate conditions in uivo. Effects of human rIL-IP (1 pg/kg) on coagulation in rabbits 0.5 or 4 hr after systemic injection, after which stasis was induced in isolated segments of each jugular vein, were studied microscopically. Perturbed endothelium was indicated by increased surface microvilli, blebs, and gaps at cell junctions. Fibrin was seen as single strands in close association with the surface rather than as a clot, and single disk-shaped platelets (nonactivated) sometimes overlay the fibrin strands (Merton et al., 1991). Fibrin formation was consistent with studies by Nawroth et af. (1986) who infused ILl into rabbits and demonstrated fibrin deposition

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on intact arterial endothelium. Although activated platelets, leukocytes, and red cells were found within the fibrin network, Merton and colleagues suggest that the amplification of procoagulant activity by IL1 alone is insufficient for the formation of an acute occlusive venous thrombosis. Studies using perfusion chambers to simulate conditions of blood flow showed that HUVECs cultured with LPS or TNF-a induced fibrin deposition at relatively low shear rates (100-300 sec-I) (Clozel et al., 1989; Tijburg et al., 1991b). Under these conditions LPS exposed some of the extracellular matrix (ECM) between cells and this also supported platelet and fibrin deposition. Weiss et al. (1989) found that the critical pathway of fibrin deposition on subendothelium of segments of rabbit aorta after exposure to flowing blood was TF dependent. Procoagulant activity was located immediately beneath the endothelium in both human umbilical artery and rabbit aorta, confirming the immunohistochemical location of TF in the deep layers of the vessel wall rather than at the surface. Although TF may be derived from fibroblasts or smooth muscle cells (Maynard et al., 1975; Zacharski and McIntyre, 1973a), endothelial cell-derived TF has been shown within the ECM. Using small diameter capillaries lined with HUVECS, Lindhout et al. (1992) demonstrated a 20-fold greater activity in the ECM than at the surface following perfusion with TNF for 4-8 hr. Scanning electron microscopy indicated reduced integrity of the monolayer under these conditions, exposing the ECM. The cellular origin of TF protein trapped within the ECM of the necrotic core of atherosclerotic plaque (Wilcox et al., 1989) is, however, unknown. Initiation of coagulation by TF-factor VIIa on perturbed endothelium is some 10-fold greater in the presence of factors IX and VIII than when only factor X is present (Stern e f al., 1985b; Tijburg et al., 1991b). Highaffinity factor IXa binding sites are proposed to mediate this response (Dorian et al., 1989) although the relationship between this receptor and the vitamin K-dependent Gla-binding domain of TF described by Reuning et al. (1993) is unclear. Active site-blocked IXa (IXaJ blocked the TFfactor VIIa-initiated responses of TNF-activated endothelial cells in plasma whereas activity of the ECM (some 10- to 20-fold greater than on cells) was inhibited by IXq only when low concentrations of TNF were used to induce TF. By contrast, Xa(i) inhibited reactivity induced by all concentrations of TNF. These studies emphasize the link between TF-factor VIIa-mediated activation of factors IX and X, and again indicate the importance of the IX/IXa pathway in circumstances where low levels of TF are expressed. TNF-a-induced TF that is associated with membrane vesicles at the subendothelial matrix but not on the apical surface has recently been

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described (Ryan et al.. 1992).TF that is associated with membrane vesicles shed from a variety of TF-bearing cells has been reported by others (Bastida et al., 1984;Bona et al., 1987)and this may represent amechanism of sequestering TF in the subendothelium to promote the formation of thrombin at sites of vascular injury. The importance of the subendothelium in the rapid initiation of the hemostatic process after its exposure is well accepted. In addition to sequestering TF, thrombin interacts with cell surface heparan sulfate receptors (Shimada and Ozawa, 1985) and with dermatan sulfate in the ECM (Bar-Shavit et a!., 1989). Thrombin bound to the ECM can convert fibrinogen to fibrin and activate platelets. Sequestered thrombin may contribute to the enhanced platelet adhesion to the ECM observed followingperturbation of endothelial cells (de Groot et al., 1987). Furthermore, ECM-immobilized thrombin is protected from inactivation by antithrombin and heparin, which suggests an altered conformation of the bound enzyme (Bar-Shavit et al., 1989). The sequestration of procoagulants on the ECM may not only play an important role in acute thrombosis, but may have functional significance during inflammation. 4. Tissue Factor on Other Cell Types

Zacharski et al. originally described a coagulant expressed by fibroblasts in tissue culture which had properties associated with TF (Zacharski et al., 1973; Gordon and Lewis, 1978; Green et al., 1971). The procoagulant increased several hundredfold over 12-24 hr of incubation, was correlated with cell adhesion (Zacharski and McIntyre, 1971), and required new protein and RNA synthesis (Zacharski and McIntyre, 1973a,b). Subsequent studies indicated that TF associated with the surface of cultured smooth muscle cells and fibroblasts was in a dormant state until perturbed (Maynard et af., 1975). This lead to the proposal that TF expression by selected extravascular cells is fundamental to hemostasis and initiates clot formation as a consequence of injury. In spite of the fact that TF expression by these cells has been known for many years, and murine (Hartzell et al., 1989; Ranganathan et al., 1991)and human (Morrissey et af.,1987)fibroblasts were used as a source of cDNA for its cloning, relatively few studies have been performed using these cells. The sequential expression of specific genes designated immediate-early genes, which mediate the growth response of fibroblasts to growth factors, include those encoding TF (Hartzell et al., 1989). Platelets enhanced TF expression (Smariga and Maynard, 1982a), possibly via a soluble platelet protein (Smariga and Maynard, 1982b). TF mRNA is now known to be upregulated in mouse 3T3 fibroblasts within 20 min by

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platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) (Hartzell et al., 1989). The gene is transcriptionally activated by growth factor and superinduced by cycloheximide. In comparison with PDGF, epidermal growth factor, and insulin, which induce relatively low levels of T F activity on murine AKR-2B fibroblasts, TGF-/31 induces high amounts and this effect is apparently not coupled to mitogenesis (Ranganathan et d., 1991). These studies have resulted in speculation that T F may have functions in addition to those involved in hemostasis and that they may include embryonic development, cell growth, and wound healing. Calcium ionophore A23 187 rapidly and reversibly enhances the surface expression of TF on bovine fibroblasts to levels achieved by disruption. Bach and Rifkin (1990) suggest that surface expression of TF is mediated by Caf2-dependent changes in the asymmetric distribution of phosphatidylsenne in the plasma membrane. Redistribution of phosphatidylserine may be important in the association of TF with factor VIIa. Human fetal lung fibroblasts bind factor VIIa in a Ca2+-dependent manner to about 100,000 high-affinity binding sites per cell (Ploplis et al., 1987). Unlike Mos, specific high-affinity binding is abolished by high levels of Ca2+ whereas dissociation constants for factors VII or VIIa are similar on both cell types. In addition, fibroblasts bind factor X, with the estimated number of binding sites approximately 7% of those estimated on endothelial cells. Association of factor VIIa and X at levels less than normally present in plasma (10 and 100 nM, respectively) with the fibroblast surface may be important in inflammatory lesions, or in tissue injury, where plasma extravasation would provide sufficient protein to render these cells functionally active. In addition to the procoagulant activity associated with alveolar Macs in inflammatory lung diseases, and in support of the immunohistological data of Drake et al. (1989a), alveolar epithelial cells express some 10- to 20-fold greater TF activity than Macs. The activity is on the surface of unstimulated cells, and mRNA and activity levels increase about twofold following stimulation with PMA (Gross et al., 1992). The failure of LPS to enhance TF expression may be due to the desensitization of these cells in a manner similar to that described for endothelial cells and Macs, due to the likelihood of their constant exposure to inhaled LPS. Mesangial cells and epithelial cells within the glomerulus also normally express TF, which is increased in glomerulonephritis (Drake et al., 1989a; Neale et al., 1988)although the majority of the procoagulant activity may be derived from infiltrating Macs. Little more is known about the regulation of epithelial cell procoagulant activity, particularly its modulation by cytokines, and its physiological importance may be restricted to specific tissues.

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111. Procoagulants A. Regulation of Procoagulant Activity by Adhesion Receptors

It has been known for many years that adherence of Mos to plastic is sufficient to induce TF expression (van Ginkel et af., 1977). Many earlier studies of Mo/Mac TF induction used cells (blood Mos, splenic Macs) adhering to plastic or fibronectin-coated plates and activity of cell lysates was usually measured. By contrast, all the studies carried out in our laboratory have used nonadherent conditions to study procoagulant expression by viable cells. It is possible that adhesion may itself function as a priming step to amplify responses to other agents, but few careful comparative studies have been made. Miserez and Jungi (1992) recently showed that although IFN-y is a strong inducer of TF on human Moderived Macs grown in suspension, it fails to activate adherent Macs whereas LPS induces activity under both circumstances. The leukocyte-restricted differentiation antigen, CDll/CD18, plays a major role in inflammation. CDl lb/CD18 (Mac-l) is important in adhesion reactions of neutrophils and Mos. Furthermore, CDl lb/CD18 on Mos binds factor X with reasonable affinity (2 x M ) in a Ca’+-dependent manner (Altieri and Edgington, 1988a; Altieri et af., 1988b). Altieri and colleagues have shown that a number of agonists, including ADP, ionomycin, and the chemotactic peptide FMLP, induce functional epitopes within CD1 lb/CD18, absent under resting conditions, to an “activated” state capable of binding factor X (Altieri and Edgington, 1988b).Activation is rapid, with optimal binding after 20 min; factors 11, VII, IX, IXa, or Xa fail to compete for binding. A sustained increase in cytosolic free [Ca2+Ii,coupled with variations in transmembrane potential, is produced by ADP. An intracellular signaling pathway, possibly involving protein kinase C in a manner similar to that of other classical mobilizers of cytosolic Ca2+,such as FMLP, C5a, and IL8, is proposed to modulate the transient and functional upregulation of this receptor (Altieri et al., 1990). In the absence of demonstrable TF or TF-VIINIIa complex, ADPstimulated Mos and myeloid cells expressing Mac- 1 directly convert surface-bound factor X to the active form, Xa. Neutralizing antibodies to TF fail to block this response and an unknown enzyme of cellular origin is thought to process the inactive zymogen (Altieri et af.,1988a). Furthermore, engagement of the CDl lbKD18 complex either with antibodies or by binding to immobilized fibrinogen enhances the Mo TF response to LPS or MPIF some two- to eightfold (Fan and Edgington, 1991).

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These findings suggest that augmentation of the procoagulant response after ligation of CD1 lblCD18 may represent an important amplification mechanism in inflammatory lesions. It may be particularly important in the enhanced TF production which may occur under some circumstances of T cell-Mo cooperation (Farram et al., 1983) and in the interactions between endothelial cells and Mos or neutrophils described later. In addition, CD1 lb/CD18 binding of opsonized (C3bi) microorganisms may have a similar effect, thereby potentiating fibrin formation in infection. The levels of CDllb/CD18 on neutrophils are also modulated by a number of proinflammatory agents. Both blood neutrophils and a myeloid cell line HL60 (differentiated with dimethylsulfoxide to a cell with granulocyte properties) express functionally competent Mac- 1 receptor after ADP stimulation, with characteristics of factor X binding similar to those of circulating Mos or related cell lines (Altieri and Edgington, 1988a; Altieri et al., 1988b).This pathway of procoagulant induction has many physiological implications. Adenine nucleotides are continuously generated during normal hemostasis at sites of vessel injury and inflammation (Born and Kratzer, 1984),and platelets represent a major source. Inflammatory sites characteristically involve localized activation of neturophils and/or Mos, and migration of these cells from the blood in response to chemotactic stimulation upregulates CD11b/CDl8. Because neutrophils possess no

activates endothelial

Factor Xa on Mo/Mac endothelium

thrombin

FIG. 3 Neutrophils can mediate procoagulant responses (see text).

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other known activators of the extrinsic coagulation cascade, the CDl Ib/ CD 18-mediated pathway may induce rapid localized fibrin formation following transmigration of these cells. Furthermore, CDl 1b/CD18 rapidly binds fibrinogen although under comparable conditions factor X competes for fibrinogen binding (Altieri et al., 1988a). In addition, CDl lc/CD18 on TNF-a-stimulated neutrophils functions as a fibrinogen receptor that recognizes the sequence Gly-Pro-Arg in the N-terminal domain of the Aa chain of fibrinogen (Loike et al., 1991). Neutrophil binding to fibrin occurs via the 95-kDa P-chain common to the three members of the leukocyte integrins (Cooper et al., 1984). The properties of neutrophils which indicate their potential role in activation of coagulation are illustrated in Fig. 3. Neutrophils rapidly accumulate within fibrin thrombi (Cooper et al., 1984), and Macs at sites of antigen challenge are enmeshed in a fibrin network (Hopper et al., 1981). A number of years ago we suggested that fibrin formation may serve to localize phagocytic cells at inflammatory sites and that this process was the basis of migration inhibition by such cytokines as macrophage migration inhibition factor (MIF) (Geczy and Hopper, 1981; Hopper et al., 1981). The data reported here indicate the feasibility of this hypothesis although the ability of the recently cloned MIF (Weiser et al., 1989) or MIF-related proteins (Odink et al., 1987) to influence CDl 1/CD 18 expression and/or procoagulant induction has not been reported. B. Other Cellular Procoagulants

Activated Mo or Mac have been reported to assemble or express all of the proteins of the extrinsic coagulation pathway. Differences in experimental design and assay procedures, source of cells (differentiation and maturation state of the cells, for example, can affect the procoagulant response), and a variety of stimulants may contribute to the differences in results obtained in different laboratories. In addition, many studies using animal cells, particularly those from mice, have used human or bovine plasmas and/or coagulation factors to define a particular procoagulant response and the species specificities of these factors makes identification difficult (Bach, 1988; Janson et al., 1984). In the absence of specific reagents to identify the murine proteins, MoMac procoagulant characterization was mainly derived from functional studies. Shands (1984) summarized the early work describing procoagulants, in addition to TF, detected on murine peritoneal Macs (factors IX, X, VII, V, 11),murine TG-PEC (factor VII, factor X activator), and murine and human blood Mos (prothrombinase and factor VII). It is surprising

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that although cDNA probes and antibodies to human factors VII and X have been available for some time, little is known about the regulation of these factors in human Mo/Mac or other cell types. Only activities characterized recently are described here (see Table 111). A unique prothrombinase produced in response to specific lipoproteins and first reported by Schwartz, Levy and colleagues (Levy et al., 1981; Schwartz er al., 1981, 1982b) was not neutralized by antifactor X serum or inhibited by a panel of factor Xa inhibitors, indicating the activity was not factor Xa (Ottaway er al., 1984). In response to murine hepatitis virus type 3 (MHV-3) infection, Mo/Mac prothombinase increased to maximal levels within 4-6 hr and its expression correlated strongly with susceptibility or resistance to the disease (Dindzans et al., 1986).The Ca2+-dependent activity within the cell membrane is a serine esterase. Fung et al. (1991) recently produced a panel of monoclonal antibodies which failed to react with TF but some of which influenced the prothrombiTABLE 111 Some Cellular Activators of Coagulation

Activity

Cell type

Factor VII

Mo/Mac

Factor V/Va

Factor Va light chain-like protein (Effector cell protease receptor-], EPR-1) Cysteine protease

Platelets, Mo/Mac, lymphocytes, neutrophils, endothelial cells Mo, monocytoid/myeloid cell lines, some neutrophils, NK cells, and some lymphocytes Endothelial cells

Glycoprotein gC

Endothelial cells

CDIIb/CD18 (Mac- 1)

Mo/Mac, neutrophils

Unique prothrombinase (two-chain protein, 74 and 70 kDa)

Mo/Mac

Properties May be inadequately ycarboxylated. Antigensensitized lymphocytedependent induction Induced by thrombin, LPS, PMA on endothelial cells

Binds factor Xa, lymphocyte EPR-I enhanced by PHA, conA, allogenic cells Activates factor X induced by hypoxia Facilitates binding and activation of factor X; HSV infection; gC encoded by HSV genome Binds factor X-processed to factor Xa, binds fibrinogen; activated by ADP, FMLP, IL8 Activates prothrombin induced by viral infection (murine hepatitis virus)

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nase activity on MHV-Istimulated murine Macs. The protein is not detected on normal cells and requires new protein and RNA synthesis for expression. Numbers of positive Macs and staining intensity correlated with multiplicity of infection. Antibodies reacted with proteins of 140, 74, and 70 kDa (nonreduced) and 74 and 70 kDa (reduced). Interestingly, LPS-stimulated Macs (TF-positive)failed to bind the antibodies and the molecular weight of the prothrombinase is substantially greater than the reported mass of TF. Although the amino acid sequence of the prothrombinase has not been reported, this is apparently a novel procoagulant associated with viral infection. Sinclair et al. (1990) suggest that Mo/Mac procoagulants induced by bacteria and viruses may represent disparate routes of procoagulant induction. Our finding of an as-yet uncharacterized prothrombinase-like activity together with TF induced by LPS following IFN-y-priming (Moon and Geczy, 1988; Wheeler et al., 1991) suggests a unifying pathway. In addition to its antiviral role, IFN-y is thought to be a key mediator in the Schwartzman reaction (Heremans ef al., 1990) and in endotoxemia (Heinzel, 1990), and may therefore contribute to the coagulopathies associated with bacterial sepsis. Furthermore, the synergistic response of two procoagulants induced on the cell surface by different combinations of stimulants would represent a potent amplification of the procoagulant potential at inflammatory sites. The role of platelets in the assembly of the prothrombinase complex is well documented and occurs via Ca2+-dependent I :1 stoichiometric complex formation of factor Xa with the nonenzymatic cofactor Va. The cofactor contributes to the amplification of prothrombin activation by altering the kinetics of its cleavage and by stabilizingthe enzyme-substrate complex (Mann er al., 1990). A number of agents which activate platelets induce the release of microparticles which are enriched for binding sites (possibly phospholipid in composition) for factor Va, and their release parallels the expression of the catalytic surface for the prothrombinase complex. Factor Xa activates the factor V associated with microparticles 50-100 times more effectively than thrombin (Monkovicand Tracy, 1990). Thus factor Xa generated by cellular TF-factor VIIa may activate factor V bound as platelet microparticles or aggregates. In addition, membrane surfaces provided by Mo, and lymphocytes (Tracy ef af.,1983), neutrophils (Tracy et af., 1985), and intact (Rodgers and Shuman, 1983) and perturbed (Annamalai et af., 1986; Rodgers and Kane, 1986; Rodgers and Shuman, 1983) endothelial cells, but not nonvascular cells such as fibroblasts and visceral lung cells, express factor V. Rodgers and Shuman suggest heterogeneity within the endothelial cell population with aortic cell 2 umbilical cell > microvascular cell activity. Most of the factor V is present within the membrane of endothelial cells although a

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secreted, unactivated form is produced by cultured cells (Cerveny et al., 1984). Thrombin, LPS, and phorbol esters upregulate factor V production on endothelial cells but to date there is no evidence of cytokine involvement (Rodgers, 1988).In contrast to earlier studies suggesting that platelets were required for the formation of prothrombinase on the endothelial cell surface, Stem et al. (1985b) and Tijburg et al. (1991a) showed no requirement for exogenous factor V and propose that endogenous factor V(a) is involved in prothrombin activation on the cell surface and that activation of the zymogen by thrombin may regulate the rate of reaction. Relatively few studies have been performed to define the contribution of factor V to the procoagulant response of mononuclear cells. Tracy et al. (1983) showed that Mo prothrombinase activity was mediated by highaffinity factor Va binding sites, whereas factor V was intracellular rather than membrane-associated, More recent studies by this group (Worfolk et al., 1992) indicate that factor Xa interacts with the Mo through two sites distinguished by their requirements for factor Va and their expression of different functional activities. Thrombin is generated from prothrombin solely by a membrane complex of factors Va and Xa, whereas in the absence of added factor Va, Mo-bound factor Xa cleaves factor IX to the nonenzymatic intermediate IXa. This may be processed to the active form by TF-factor VIIa (Lawson and Mann, 1991) following Mo stimulation. Factor Xa binds to monocytoid/myeloid-cell lines THP, U937, and HL60 via a membrane protein of approximately 74 kDa which cross-reacts with a monoclonal antibody to factor Va light chain (Altieri and Edgington, 1989). The term “effector cell protease receptor 1” (EPR-1) was given to this protein, which it is suggested represents a cell-surface analog of plasma factor V/Va (Altieri and Edgington, 1990). The claim that EPR-1 provides a cofactor-like effect in factor Xa-catalyzed prothrombin activation is disputed by Worfolk et al. (1992) who found that added factor Va is essential for this activity on Mo and that factor IX is the preferred substrate in its absence. These studies are in keeping with the current knowledge of how factor Va functions in the prothrombinase complex. The light chain of factor Va forms part of the factor Xa binding site at the cell surface (Higgins and Mann, 1983;Tracy and Mann, 1983)whereas the heavy chain binds prothrombin (Guinto and Esmon, 1984), indicating a requirement for both subunits to assemble the enzyme and substrate of the prothrombinase complex. Apart from the reported ability of lymphocytes to assemble a functional prothrombinase complex (Tracy et al., 1983), normal lymphocytes were considered to be devoid of procoagulant activity. However, Altieri and Edgington (1990) recently reported EPR-1 expression on a subset of CD3+ cells coexpressing CD2, CD4 or CD8, CD57, CDl lb, and apT-cell receptor. Furthermore, peripheral blood lymphocytes stimulated with PHA,

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81 concanavalin A, or allogenic cells expressed 8- to 10-fold more EPR-1. A population of natural killer (NK) cells and a heterogeneous population of neutrophils also express EPR-1 although nothing is known concerning its regulation in these cells. These findings suggest a role for EPR-1 in immune effector function and possibly in fibrin formation in the early stages of DTH reactions. Together with the capacity of CDIIb/CD18 on infiltrating leukocytes to bind and activate factor Xa, these localized cellular reactions may result in sufficient thrombin formation to initiate an early response. In addition to its ability to alter the procoagulant potential of endothelial cells and platelets, thrombin is chemotactic for phagocytic cells (Bar-Shavit et al., 1983), processes IL8 (Herbert et al., 1990) and ILl (Jones and Geczy, 1990), and alters endothelial cell permeability, all of which would contribute to enhanced and/or sustained inflammation. Infection of endothelial cells with herpes simplex virus 1 (HSV-1) (Visser et al., 1988) or cytomegalovirus (CMV) (van Dam-Mieras et al., 1992) leads to membrane perturbations resulting in enhanced factor X binding and prothrombinase activity. Visser et al. (1988) suggest that infection causes a rearrangement of negatively charged phospholipids on the outer leaflet which facilitate factor Va and Xa assembly. The recent demonstration by Etingin et al. (1990), of an HSV-encoded endothelial cell surface glycoprotein (gC) which binds and promotes activation of factor X on infected, but not uninfected cells, indicates a novel pathway for procoagulant induction. Although gC encoded by the HSV genome can function as a complement (C3b) receptor, it has no primary structural homology to Mac- 1 but may bind factor X in a manner similar to that described above for this ligand. These findings have been implicated in the pathogenesis of atherosclerosis since both HSV (Hajjar et al., 1987) and CMV (Adam et al., 1987) have been located in human atherosclerotic plaque, and in the coagulopathies associated with HSV infection (Visser et al., 1988). There are several reports suggesting that factor VII is produced by activated Mo/Mac although further definition of this activity is required. Factor VII-like activity secreted by cultured murine LPS-stimulated Macs (Shands, 1983) may contribute to the factor X activator activity of these cells by binding to TF. In contrast, human Mos can be induced by LPSactivated CD4' T lymphocytes to express a factor VII/VIIa activity which is predominantly of intracellular origin since viable cells express only 5-20% of the total functional activity (Tsao et al., 1984). Similarly, factor VII expression on sensitized blood Mos stimulated with antigen (tuberculin; Godfrey et al., 1986) or allogeneic cells (Carlsen et al., 1989) indicates a requirement for T cells and suggests involvement of factor VII in fibrin formation in cell-mediated responses. In addition, factor VII synthesized and secreted by alveolar Mac may contribute to inflammatory diseases of the lung, including idiopathic pulmonary fibrosis and adult respiratory

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distress syndrome (Chapman et al., 1985). Interestingly, these authors found increased alveolar Mac-associated factor VII activity in sarcoid patients, and propose that the disproportionate increase in alveolar T cells which occurs in these individuals may contribute to its expression. Alveolar Macs synthesize factor VII in the 48-kDa single-chain zymogen form but not the two-chain activated derivative, factor VIIa. Chapman el al. (1985) and Carlsen et al. (1989) suggest that much of the synthesis may occur in the undercarboxylated precursor form. Although factor VII mRNA has been demonstrated in extracts of freshly isolated alveolar Macs, the lack of vitamin K-dependent y-carboxylase, which is required for processing the factor into the functional form, implies that activity may be limited (McGee et al., 1989). The effect of lymphocytes on ycarboxylase activity is worthy of study. Interestingly, we found that IFNy, the T-lymphocyte product, induced a factor VIIa-like activity in murine inflammatory Macs (Moon and Geczy, 1988). The regulation of functional factor VIIa, by virtue of cytokine induction of a y-carboxylase, may represent another level of control by these mediators. The relationship of a tumor-derived cysteine protease and the factor X activator recently described by Ogawa et al. (1990) is unclear. The latter enzyme is expressed maximally 72 hr after endothelial cell cultures are exposed to hypoxic conditions but is not observed with intact or disrupted endothelium grown in ambient conditions. The Ca2+-dependentenzyme directly activated factor X. It was not inhibited by neutralizing anti-TF antibody, but HgCI, blocked activity, suggesting a cysteine protease. The physiological relevance of this enzyme is still to be determined, particularly since the V,, is lower than that of the factor IXa-VIIIa complex although alterations in the endothelial milieu (reduced pH and blood flow) may favor the factor X activator during hypoxic episodes. These data suggest that this enzyme may be expressed in response to stress, and experiments investigating this possibility may be informative. C. Procoagulants and Malignancy

The association between recurrent thrombotic episodes and malignancy was described over a century ago by Trousseau (Rickles and Edwards, 1983). The prevalence of subclinical coagulation abnormalities in patients with cancer has been reported to be as high as 98% (Sun et al., 1979) although the differences in study design among various groups make comparisons difficult. Nanninga et al. (1990) recently concluded that approximately 22% of untreated primary cancer patients exhibited subclinical coagulopathy although the percentage may be higher in patients with more advanced disease (Nand and Messmore, 1990).

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Fibrin has been demonstrated histologically within the mass of many tumors, and anticoagulant drugs have antimetastatic effects. The role of coagulation in metastasis has been discussed for many years and is thought to contribute to tumor cell invasion and localization in normal tissue (Goldfarb and Liotta, 1986), and to development of blood supply (Laki, 1974). The formation of a fibrin barrier may prevent immune recognition and lytic interaction of tumor cells with cytotoxic lymphocytes (Donati et a f . , 1986) although activated macrophages associated with tumor cells within a fibrin meshwork have been associated with tumor rejection (Dvorak et al., 1978). Alternatively, fibrin may inhibit tumor cell detachment, thereby protecting the host from tumor cell invasion (Dvorak et af.,1979). It is now clear that fibrin formation may be a consequence of direct activation of the extrinsic coagulation cascade by tumor procoagulants or their shed membrane vesicles, or indirectly by stromal cells (e.g., Macs) stimulated to express procoagulant activity by soluble tumor-derived products or by cytokines produced as a consequence of an activated immune response to the tumor. A thrombosis-inducing activity in the plasma of lung cancer patients has been described (Maruyama et al., 1989). A summary of the characteristics of human tumor procoagulants has recently been published (Edwards et al., 1993). Immunohistochemical techniques indicate a heterogeneous pattern of procoagulant expression in which cells from some tumors are poorly reactive whereas in others coagulation factors are localized either to the tumor cells or to tumor-associated Macs (Zacharski et al., 1990). T F activity is found in cells from a number of solid tumors, including adenocarcinomas of the colon, ovary, gastrointestinal tract and pancreas; squamous cell carcinomas; melanoma; neuroblastoma; and bladder; lung, and teratanocarcinomas (Edwards et al., 1993). TF is the major procoagulant identified on cells and cell lines of the myeloid lineage, many of which have been used in the studies discussed earlier to investigate T F regulation. A "thromboplastic activity of leukemic white cells" was reported by Eiseman and Stefanini in 1954 although definitive proof of the involvement of TF has been relatively recent, Increased TF activity by leukemic cells from patients with acute nonlymphoid leukemia closely correlates with the occurrence of DIC (Andoh et al., 1987;Guarini et al., 1985). Inducible T F activity has been reported on monocytoid, early myeloid, monoblastic, and myelomonocytic cell lines (Edwards et al., 1993). In addition, and unlike normal or activated blood lymphocytes, TF has also been reported on a variety of T-lymphoblastoid lymphoma cell lines (Barrowcliffe et al., 1 989). Acute promyelocytic leukemia is associated with a high incidence of hemorrhage attributed to DIC and secondary fibrinolysis (Bauer and Rosenberg, 1984). HL60 cells are derived from human leukemic promyelo-

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cytes and have high basal levels of TF activity (Wijermans et af., 1989). Differentiation into Mac-like cells with phorbol esters (Rovera et af., 1979) induces high levels of TF expression before these cells acquire other Mo/ Mac characteristics. LPS and PHA, which stimulate TF expression on normal Mos, have no effect on these cells (Kornberg et af., 1982). If HL60 cells are differentiated into granulocytes by culturing with retinoic acid or dimethylsulfoxide, there is a concomitant decrease in procoagulant activity (Wijermans et af., 1989). Cells differentiated to either Mos or granulocytes also have decreased proteolytic activity (elastase, cathepsin G), which may account for the fibrinolytic activity of normal neutrophils. The combination of high TF and granulocyte protease levels may be unique to the promyelocyte and may explain the common bleeding complications in patients with acute promyelocytic leukemia (Wijermans et af., 1989). The recent finding that neutrophil elastase and cathepsin G both inactivate tissue factor pathway inhibitor by a cleavage which restores factor Xa activity after its initial inhibition by TFPI (Petersen et af., 1992) would strengthen this hypothesis. In addition to TF, hepatoma cells secrete single chain factor X and prothrombin (Fair and Bahnak, 1984),and some carcinoma cells and their shed membrane vesicles can support assembly of a functional prothrombinase complex. Factor Va bound to some tumor cell surfaces serves as a receptor for factor Xa, with the number of factor Va-Xa binding sites comparable to values reported for platelets (Van de Water et al., 1985). In addition to the expression of normal extrinsic pathway activators, a malignancy-associated procoagulant has been described. A factor Xactivating activity first isolated from extracts of colon and kidney carcinomas and rabbit V, carcinomas (Bauer and Rosenberg, 1984) acts independently of TF/VIIa. It is a unique 68-kDa acidic protein with cysteine protease activity (Falanga and Gordon, 1985; Gordon and Cross, 1981) which has only been described in neoplastic and fetal cells (Gordon et af., 1985). Varying levels of the enzyme were isolated from primary and metastatic human melanomas, but not benign nevi, with the highest activity in metastatic samples (Donati et al., 1986). Its involvement in metastasis has been strengthened by a close correlation between its expression and the metastatic potential of B16 melanoma cell variants (Gilbert and Gordon, 1983). This procoagulant has also been located in extracts of blast cells from patients with acute nonlymphoid leukemia, and Falanga et af. (1988) suggest varying proportions of factor X activator and TF in different cytological subtypes although the relative contributions of these two procoagulants in coagulopathies is still unclear. Furthermore, the recent demonstration in extracts of rat sarcomas of another factor X activator, a serine protease with properties distinct from TF-factor VIIa, which acti-

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vates factor X perfused within the tumor bed in uivo (Pangasnan et al., 19921, indicates that additional factors may also be involved. Increased incidences of thrombotic problems in nonlymphoid malignancies have been associated with the use of chemotherapeutic drugs (Doll et al., 1986; Goodnough et al., 1984; Levine et al., 1988; Sills et al., 1978; Weiss et al., 1981) and Fibach et al. (1985) suggested that these agents may contribute to the thrombotic complications of patients undergoing treatment for acute promyelocytic leukemia. Our earlier experiments indicated that doses of cycloheximide suboptimal for the inhibition of protein synthesis (0.5-1 pg/ml) superinduced procoagulant activity on murine TG-PEC in response to IFN-y or LPS (Moon and Geczy, 1988). Similar effects were noted with human blood Mos although we demonstrated a discordant relationship between the levels of TF antigen, which fell by about 70%, and functional activity, which rose by approximately 100% (Walsh and Geczy, 1991). The binding capacity of TF on LPS/cycloheximide-stimulatedMos is about the same as that of LPS-stimulated cells, suggesting that factor VII binding need not directly correlate with TF cofactor activity as suggested by Rodgers et al. (1984). We propose that the reduction in protein synthesis caused by cycloheximide may affect membrane fluidity and/or stabilization by altering protein and phospholipid turnover. Whereas exogenous phospholipid enhanced the TF activity of LPS-activated human Mos, it had no effect on LPS/cycloheximide-stimulatedactivity, which suggests that optimal levels of acidic phospholipids were available within the membrane to facilitate factor VII binding and the allosteric change in factor VIIa necessary for enhanced catalytic function (Walsh and Geczy, 1991). Bach and Rifkin (1990) suggest that modulation of the distribution of phosphatidyl serine in the plasma membrane of cells expressing TF may regulate its function. These studies prompted us to investigate whether other cytotoxic agents which intercalate with DNA or RNA, may modulate Mo/Mac procoagulant activity in a similar manner. In contrast to the antimetabolites methotrexate and 5-fluorouracil, we found that pharmacological levels of both cisplatin (which blocks DNA polymerase) and the anthracycline drugs doxorubicin and donarubicin (which inhibit DNA strand separation during replication) modulate TF expression on both murine TG-PEC and WEHI 265 monocytoid cells (Wheeler and Geczy, 1990) and on human Mos and RC2a cells (Walsh et al., 1992; Yen et al., 1993). Cisplatin and Adriamycin induce weak TF activity and synergize with low levels of LPS (10100 pg/ml) to enhance TF expression on human Mos. At concentrations which do not cause cell death, cisplatin (20 pg/ml) enhances TF induced by LPS some 6- to 20-fold whereas the quantity of TF antigen on viable cells indicates that the synergy observed with these agents is not due to

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enhanced TF antigen expression even though all the procoagulant activity is neutralized by an anti-TF monoclonal antibody. We propose that a mechanism similar to that observed with cycloheximide-treated cells is involved. D . Suppression of Procoagulant Expression

In contrast to the large number of reports describing the induction of TF by the cells described, there is relatively little information on factors regulating this response. The contribution of TFPI, which is produced by activated platelets, endothelial cells, and possibly by Mo/Mac (Werling et al., 1993), and which inhibits the TF-factor VIIa complex in a factor Xadependent reaction, is the subject of a number of recent reviews (Rapaport, 1991; Sandset and Abildgaard, 1991). Although it is an important regulator of TF-mediated reactions, it will not be described here. Apart from the inhibitory effects of TFPI, there are few reports of effective suppression of cellular TF once the thrombotic reaction is under way. Since the activity of TFPI is dependent on factor Xa and because the only requirement for activation of coagulation is the binding of factor VII/VIIa to TF, other regulators of this complex may be expected. Conkling et al. (1989) recently showed that sphingosine, the basic building block of the sphingolipids, profoundly inhibited TF on activated human Mos. Although it may function by inhibiting protein kinase C, the inability of a number of sphingosine analogs to alter activity suggests that the amino group of the polar head of sphingosine inhibits formation of the TF-factor VII complex. Alternatively, sphingosine may disturb the phospholipid cofactor activity within the cell membrane by becoming inserted into the bilayer. Although sphingosine and other glycosphingolipids may represent a new class of inhibitors of coagulation, their pharmacological use has not been explored. Several investigators have tested a number of agents known to affect the inflammatory response in an attempt to find clinically useful inhibitors of the development of TF (see Table IV). Dexamethasone inhibits TF generation in Mos stimulated with LPS in uitro (Lyberg, 1984a) although PEC from rabbits injected with LPS have higher procoagulant levels when injected simultaneously with cortisone acetate (Robinson et al., 1978). This observation is in accord with the finding that cortisone can substitute for the first of the two LPS injections which provoke the Schwartzman reaction (Thomas and Good, 1952). Our studies support this observation and indicate that in contrast to blood Mos, glucocorticoids enhance the procoagulant potential of inflammatory Macs, particularly in response to IFN-y ( J . D. Walsh, H. Wheeler, and C. L. Geczy, unpublished observa-

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TABLE IV

Some Modulators of TF ~

Modulating agents CD8 lymphocytes +

Interferon-y Interleukin 4

Heparin-binding growth factor1 plus heparin Agents elevating [cAMPIi (including pentoxyfylline, HSW 138) Prostacyclin, iloprost Sphingosine Retinoids

Effect Inhibit TF induction on endothelial cells Suppresses LPS-induced TF on Mos Variably suppresses LPS, ILl, and TNF-induced TF on endothelial cells and Mos Inhibit TF induction on endothelial cells Inhibit TF induction

Inhibit TF induction Inhibits preformed TF Inhibit TF induction

tions), a key mediator of the Schwartzman reaction. On the other hand, suppression of production and secretion of several cytokines, including ILl, TNF-a, IL2, and GM-CSF by steroids (Kelley, 1990)could indirectly downregulate procoagulant induction in a number of clinical situations. Retinoids modulate several functions of mononuclear phagocytes. Human blood Mos pulsed with therapeutic doses of retinoids for 10 min exhibit markedly depressed TF expression in response to LPS whereas there is little reduction of preformed TF (Conese et al., 1991). Inhibitor studies suggest that the effect may be mediated by a product of the lipoxygenase pathway although this is undefined. The ability of retinoids to downregulate procoagulant generation may contribute to the antiinflammatory effects of these compounds. Pharmacological mediators of intracellular metabolic events can also regulate TF expression. Elevation of intracellular levels of CAMP by dibutyryl CAMP inhibits induction of TF on Mos (Lyberg, 1984a),and phosphodiesterase inhibitors suppress TF expression on HUVECS (Galdal et al., 1984).Although the mechanisms of TF induction are still largely unknown, protein kinase C is thought to be involved in the processes leading to TF synthesis (Brozna, 1990; Pettersen et al., 1992) and inhibitors of protein kinase C, H7, H8, and sphingosine suppress the induction of TF on endothelial cells by allogenic lymphocytes and ILI-j3 (Pettersen e l al., 1992).

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The regulatory role of arachidonic acid metabolites on inflammatory reactions is generally well accepted. Although cyclooxygenase inhibitors have little effect on TF induction on Mos (Crutchley, 1984; Edgington and Pizzolato, 1983; Prydz and Lyberg, 1980), suggesting that the prostaglandins (PG) are not involved, accumulated PGE, and PGE, suppress Mo TF activity, possibly by increasing intracellular levels of CAMP. A recent study by Crutchley and Hirsh (1991) confirms the earlier observations and shows that the stable prostacyclin analog, iloprost, and to a somewhat lesser extent, prostacyclin, inhibit development of TF on stimulated blood Mos and THP-1 cells although the mechanism remains speculative. These observations add to the clinical potential of prostacyclin analogs which may inhibit both platelet aggregation and Mo procoagulant activity, in antithrombotic therapy. Furthermore, a recent report describes the protective effect of the xanthine derivative HSW 138 on the life-threatening coagulopathy induced in rats by LPS. Like pentoxifylline and theophylline, it acts by inhibiting phosphodiesterase and increasing intracellular levels of CAMP. Pentoxifylline suppresses TF induction on Mo and endothelial cells (Archipoff et al., 1989; de Prost et al., 1989) and TNF production (Bahrami et al.,1992), supporting a role for CAMPin the procoagulant response. PGE, suppresses the development of fulminant hepatitis in MHV-3-infected mice, and mononuclear cells recovered from infected mice treated with PGE, have no prothrombinase activity. Furthermore, PGE, prevents prothrombinase induction by MHV-3 in uitro, suggesting that the immunosuppressive effect of this agent may be linked with the procoagulant potential of the cells involved (Levy and Abecassis, 1989). However, an indirect effect of the prostaglandin on induction of a T-cell-dependent cytokine thought to mediate this response cannot be excluded. Induction of TF on endothelial cells induced by allogenic stimulation with CD4+-Tlymphocytes is suppressed by CD8+ lymphocytes (Pettersen et al., 1992). This may be mediated by IL4, a CD8+-Tlymphocyte product recently shown to downregulate a number of proinflammatory functions of blood Mos (Hart et al., 1989). IL4 suppresses the expression of TF on cultured bovine aortic endothelial cells by ILl-j3 and TNF-a (Herbert et al., 1992). Although IL4 suppressed induction of TF on HUVECs by LPS, activity expressed in response to ILl-p and TNF-a was not affected by IL4, confirming the results of Kapiotis et al. (1991). In addition, TF induction on adherent Mos by ILl-j3 was reduced by IL4 whereas the LPS response was not. Herbert et al. (1992) found no suppressive effect of IL4 on murine TG-elicited Macs stimulated with LPS. The apparent discrepancies in responsiveness of different cell types may reside in the differences in magnitude of procoagulant responses induced by different

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stimulants (e.g., ILl-/3 is a weaker stimulant of Mo T F than LPS) and in the origin of the cell used. Recent experiments by Hart and colleagues suggest that whereas blood Mos are sensitive to the suppressive effects of IL4, human Macs isolated from inflammatory sites are refractory (P. Hart, personal communication), again indicating the importance of the maturation and differentiation state of these cells in the outcome of the inflammatory response. Furthermore, the dichotomy of responsiveness of Mo/Mac to IFN-y (it suppresses LPSinduced blood Mo T F and inhances Mac T F expression) suggests additional levels of control. Suppression by varying levels of TFPI, as may occur on Macs in some tumors (Werling et al., 1993), and by other anticoagulants such as antithrombin (upregulated by IFN-y in inflammatory Macs; Kakakios and Geczy, 1994) or other cytokines which may affect T F gene transcription, offer a number of possibilities. Investigations with other cytokines, particularly ILlO and IL13, recently shown to downmodulate a number of Mac functions, should be pursued. The functional properties of TF-factor VIIa can be modified by heparin. Heparin increases the substrate specificity of TF-factor VIIa to enhance activation of factors X and IX by HUVEC in a pure system (Almus et al., 1989). It does not affect the cofactor activity of factor Xa for TFPI, indicating that it does not regulate the anticoagulant potential of TFPI in plasma. We also found that heparin weakly enhances the T F response of blood Mos (Kakakios and Geczy, 1994) measured in a plasma recalcification assay, suggesting a possible procoagulant role for heparin in clinical situations, such as DIC, in which anticoagulant proteins are depleted by excessive coagulation. Primary cultures of HUVEC grown with heparinbinding growth factor 1 (HBGF-1) and heparin have decreased levels of both surface and total T F in response to thrombin than when grown without heparin. Decreased T F mRNA coincided with decreased TF antigen (Almus et al., 1991) in a manner similar to suppression of PAI-1 expression (Konkle and Ginsburg, 1988), fibronectin synthesis (LyonsGiordano et al., 1990), and diminished prostacyclin production (Hasegawa et al., 1988),which suggests that heparin modulates a generalized response by endothelial cells to HBGF-1, which could alter the procoagulant potential of these cells. IV. Conclusion

The importance of cellular procoagulants in the pathology of sepsis, infection, thrombosis, and inflammation has become increasingly obvious over the past decade. The role of T F in these conditions is now relatively

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well defined although there are many questions still unanswered. It is interesting that the expression of TF in several cell types is induced in what seems to be a “cytokine-specific” manner. For example, IL1-p is a potent inducer of endothelial cell TF whereas its effect on MolMac is rather weak, and FGF is effective on fibroblasts but not Mo/Mac or endothelial cells. Because T F is apparently an early-response gene product of some cell types, studies investigating its role in cell growth and embryonic development and its transcriptional control in various cell types may yield interesting results. Furthermore, the relative contributions of TF, sequestered to the ECM of stimulated endothelial cells, and of Mo/Mac TF, which is expressed at much higher levels, to initiation of coagulation, should be objectively assessed. It is possible that the sequestration of coagulation factors to the ECM may have additional functions in inflammation. Under these circumstances, their effects on cell migration and activation may be of major significance. Early experiments using anticoagulants suggested that fibrin formation was an important event in inflammatory reactions. However, with the exception of thrombin, the possible contributions of products of activated coagulation to other components of the inflammatory response (e.g., cytokine processing, cell activation) warrant further investigation. An understanding of the control of procoagulants, in addition to TF, by cytokines and growth factors is essential for assessing their contribution to fibrin formation, particularly within the extravasculature. Downregulation of procoagulant expression may occur via inhibitors, cytokines, and other agents which regulate gene transcription and translation, by availability of phospholipids and other cofactors, and by the physiochemical presentation of these factors within the cell membrane. Further investigation in this complex area are likely to have important clinical relevance, particularly in the development of more specific and potent inhibitors, and should be extended to include the possible interactions of TF/factor VIIa/Mac-l/factor X on adhesive substrates and on the ECM. Studies of the biology of cellular procoagulants will continue to be important in linking our understanding of the basic mechanisms of the activation of blood coagulation and inflammation, and human diseases as diverse as atherosclerosis, thrombosis, multiple sclerosis, and cancer.

Acknowledgments The author acknowledges support from the National Health and Medical Research Council of Australia and the members of her laboratory who have contributed to the work. The excellent secretarial assistance of Tina Lum is gratefully acknowledged.

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bond of the second disulfide loop of tissue factor is required for binding of factor VII. J . Biol. Chem. 266, 10294-10299. Reuning, U., Preissner, K. T., and Muller-Berghaus, G. (1993). Two independent binding sites on monolayers of human endothelial cells are responsible for interaction with coagulation factor VI and factor VIIa. Thromb. Haemostasis 69, 197-204. Rickles, F. R., and Edwards, R. L. (1983). Activation of blood coagulation in cancer. Trousseau's syndrome revisited. Blood 62, 14-3 1. Robinson, A. J., Rapaport, S. I., and Brown, S. F. (1978). Procoagulant activity of peritoneal leukocytes: Effect of cortisone and endotoxin. Am. J. Physiol. 235, 333-337. Rodgers, G. M. (1988). Hemostatic properties of normal and perturbed vascular cells. FASEB J . 2, 116-123. Rodgers, G . M.. and Kane, W. H. (1986). Activation of endogenous factor V by a homocysteine-induced vascular endothelial cell activator. J . Clin. Invest. 77,1909-1916. Rodgers, G. M., and Shuman, M. A. (1983). Prothrombin is activated on vascular endothelial cells by factor Xa and calcium. Proc. Natl. Acad. Sci. U.S.A. 80, 7001-7005. Rodgers, G. M., Broze, G. J., Jr., and Shuman, M. A. (1984). The number of receptors for factor VII correlates with the ability of cultured cells to initiate coagulation. Blood 63, 434-438. Rothberger, H., Zirnmerman, T. S.,Spiegelberg, H. L., and Vaughan, J. H. (1977). Leukocyte procoagulant activity. Enhancement of production in vitro by IgG and antigenantibody complexes. J. Clin. Invest. 59, 549-557. Rothberger, H., Dove, F. B., Lee, T.-K., McGee, M. P., and Kardon, B. (1983). Procoagulant activity of lymphocyte-macrophagepopulations in rabbits: Selective increases in marrow, blood, and spleen cells during Shwartzman reactions. Blood 61, 712-717. Rovera, G., Santoli, D., and Damsky, C. (1979). Human prornyelocytic leukemic cells differentiate into macrophage-like cells when treated with phorbol diesters. Proc. Natl. Acad. Sci. U.S.A. 74, 2458-2462. Roy, S ., Paborsky, L. R., and Vehar, G. A. (1991). Self-association of tissue factor as revealed by chemical cross-linking. J. Biol. Chem. 266, 4665-4668. Ruf, W., Rehemtulla, A., and Edgington, T. S. (1991). Phospholipid-independent and dependent interactions required for tissue factor receptor and cofactor function. J . Biol. Chem. 266,2158-2166. Ryan, J., and Geczy, C. L. (1986). Characterisation and purification of mouse macrophage procoagulant-inducing factor. J. Immunol. 137, 2864-2870. Ryan, J., and Geczy, C. L. (1987).Coagulationand the expression of cell-mediatedimmunity. Immunol. Cell Biol. 65, 127-139. Ryan, J., and Geczy, C. L. (1988).Macrophage procoagulant-inducingfactor. In vivo properties and chemotactic activity for phagocytic cells. J. Immunol. 141, 21 10-2117. Ryan, J., Brett, J., Tijburg, P., Bach, R. R., Kisiel, W., and Stem, D. (1992).Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface. Blood 80, 966-974. Sakai, T., Lund-Hansen, T., Thim, L., and Kisiel, W. (1990). The y-carboxyglutamic acid domain of human factor VIIa is essential for its interaction with cell surface tissue factor. J. Biol. Chem. 265, 1890-1894. Sandset, P. M., and Abildgaard, U. (1991). Extrinsic pathway inhibitor-the key to feedback control of blood coagulation initiated by tissue thrornboplastin. Haemostasis 21,2 19-239. Scarpati, E. M., and Sadler, J. E. (1989). Regulation of endothelial cell coagulant properties. J . Biol. Chem. 264, 20705-20713. Scarpati, E. M., Wen, D., Broze, G. J., Jr., Miletich, J. P., Flandermeyer, R. R., Siegel, N. R., and Sadler, J. E. (1987). Human tissue factor: cDNA sequence and chromosome localization of the gene. Biochemistry 26, 5234-5238.

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van Deventer, S. J. H., Buller, H. R., ten Cate, J. W., Aarden, L. A., Hack, C. E., and Sturk, A. (1%). Experimental endotoxemia in humans: Analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood 76, 2520-2526. Van de Water, L., Tracy, P. B., Aronson, D., Mann, K. G., and Dvorak, H. F. (1985). Tumor cell generation of thrombin via functional prothrombinase assembly. Cancer Res. 45,5521-5525. van Ginkel, C. J. W., van Aken, W. G., Oh, J. I. H., and Vrecker, J. (1977). Stimulation of monocyte procoagulant activity by adherence to different surfaces. Br. J. Haemaiol. 37, 35-45. van Ginkel, C. J. W., Zeijlemaker, W. P., Wesbelh, A., Stricker, J., and van Aken, W. G. (1981). Enhancement of monocyte thromboplastic activity by antigenically stimulated lymphocytes: A link between immune reactivity and blood coagulation. Eur. J. Immunol. 11, 579-583. Vassalli, J.-D., Sappino, A.-P., and Belin, D. (1991). The plasminogen activator/plasmin system. J. Clin. Invest. 88, 1067-1072. Visser, M. R., Tracy, P. B.. Vercellotti, G . M., Goodman, J. L., White, J. G., and Jacob, H. S. (1988). Enhanced thrombin generation and platelet binding on herpes simplex virusinfected endothelium. Proc. Nail. Acad. Sci. U.S.A. 85, 8227-8230. Walsh, J. D., and Geczy, C. L. (1991). Discordant expression of tissue factor antigen and procoagulant activity on human monocytes activated with LPS and low dose cycloheximide. Thromb. Haemosiasis 66, 552-558. Walsh, J. D., Wheeler, H., and Geczy, C. L. (1992). Modulation of tissue factor on human rnonocytes by cisplatin and adriamycin. Br. J. Haemaiol. 81, 480-488. Warr, T. A., Rao, L. V. M., and Rapaport, S. I. (1990). Disseminated intravascular coagulation in rabbits induced by administration of endotoxin or tissue factor: Effect of antitissue factor antibodies and measurement of plasma extrinsic pathway inhibitor activity. Blood 75, 1481-1489. Weiser, W. Y., Temple, P. A., Witek-Giannotti, J. S., Remold, H. G., Clark, S. C., and David, J. R. (1989). Molecular cloning ofa cDNA encoding a human macrophage migration inhibiting factor. Proc. Natl. Acad. Sci. U.S.A. 86, 7522-7526. Weiss, H. J., Turitto, V. T., Baumgartner, H. R., Nemerson, Y., and Hoffman, T. (1989). Evidence for the presence of tissue factor activity on subendothelium. Blood 73,968-975. Weiss, R. B., Tormey, D. C., Holland, J. F., and Weinberg, V. E. (1981). Venous thrombosis during multimodal treatment of primary breast carcinoma. Cancer Treat. Rep. 65, 677. Werling, R. W., Zacharski, L. R., Kisiel, W., Bajaj, S. P., Memoli, V. A., and Rousseau, S. M. (1993). Distribution of tissue factor pathway inhibitor in normal and malignant human tissues. Thromb. Haemosiasis 69, 366-369. Wharram, B. L., Fitting, K., Kunkel, S. L., Remick, D. G., Memtt, S. E., and Wiggins, R. C. (1991). Tissue factor expression in endothelial cell/monocyte cocultures stimulated by lipopolysaccharide and/or aggregated IgG. J. Immunol. 146, 1437-1445. Wheeler, H. R., and Geczy, C. L. (1990). Induction of macrophage procoagulant expression by cisplatin, daunorubicin and doxorubicin. Ini. J . Cancer 46,626-632. Wheeler,H. R.,Rockett,E. J.,Clark,I.,andGeczy,C. L.(1991). ActinomycinDupregulates lipopolysaccharide induction of macrophage procoagulant expression and turnour necrosis factor-alpha production. Clin. Exp. Immunol. 86, 304-310. Wijermans, P. W., Rebel, V. I., Ossenkoppele, G. J., Huijgens, P. C., and Langenhuijsen, M. M. A. C. (1989). Combined procoagulant activity and proteolytic activity of acute promyelocytic leukemic cells: Reversal of the bleeding disorder by cell differentiation. Blood 73, 800-805. Wilcox, J. N., Smith, K. M., Schwartz, S. M., and Gordon, D. (1989). Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc. Natl. Acad. Sci. U.S.A. 86, 2839-2843.

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Function and Modulation of Expression of Auxin-Regulated Genes Yohsuke Takahashi, Sarahmi Ishida, and Toshiyuki Nagata Department of Biology, Faculty of Science, University of Tokyo, Tokyo 113, Japan

1. Introduction

Plant bodies arise as a result of the precisely controlled division of cells in specialized tissues, the meristems, followed by the continuous differentiation of cells throughout ontogeny. This morphogenetic pattern of plant development contrasts with the development of animals, which occurs only at distinct embryonic phases and involves complex cell movements and interactions. No complex rearrangements and interactions among different tissues, initiated by the movements of cells, are observed in plants because of the presence of cell walls. Vascular plants are sedentary, and they depend on a supply of nutrients from their immediate environment. These features make plants particularly susceptible to local environmental changes, to which they respond by adapting their developmental program. Plastic growth responses are essential in order to adapt to a fluctuating supply of light or other local resources, in competition with neighboringplants. Furthermore, individual plant cells retain a remarkable regenerative capacity, namely, totipotency, even after the initial program of differentiationis completed. In animal systems, this capacity is confined exclusively to early embryonic cells. In many instances, phytohormones are found to play a critical role in the plastic developmental responses of plants. The phytohormones are represented by five major types: auxins, cytokinins, ethylene, gibberellins, and abscisic acid. Because of the limited structural diversity of plant hormones and the wide variety of physiological responses that each can evoke, it has been argued that the mode of action of plant hormones is different from that of animal hormones. Among the classical plant hormones, auxin, which was first discovered as a plant hormone more than 60 years ago, is of foremost importance, since it has a dramatic effect on numerous aspects of plant growth and development. These include cell expansion, cell division, induction of lnrernarional Reuiew of Cyrology. Vol.

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YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

adventitious roots, control of apical dominance, and plastic responses to fluctuations in environmental factors that include light and gravity (Hoad et al., 1987). Therefore, it is likely that the expression of some regulatory genes involved in development and cell division is controlled by auxin. Despite a mass of literature on physiological studies of auxin, very little information is available on its mechanism of action in molecular terms. With the advent of techniques based on the polymerase chain reaction (PCR) (Saiki et al., 1988), it has become simple to isolate and identify plant regulatory genes whose products are related to signal transduction pathways, such as guanosine triphosphate (GTP) binding proteins (Ma et al., 1990) and protein kinases (Walker and Zhang, 1990), by analogy to mammalian cells and yeast. Although this strategy has provided a rich supply of information on the primary structures and has shown that many elements of signal perception pathways are structurally conserved in plants and other eukaryotes (Palme, 1993), the functional significance of these genes in plant cells and their relationship to the action of plant hormones have not been clarified. Three strategies can be used to gain insight into the mode of action of auxin at the molecular level. The first is a genetic approach; for example, several auxin-resistant mutants of Arabidopsis thaliana were isolated (Estelle and Sommerville, 1987; Timpte et al., 1992) with the use of toxic concentrations of auxin. In one of the mutant lines, resistance to auxin is ascribed to a dominant mutation, axr2. The axr2 mutation also confers resistance to two other plant hormones, namely, ethylene and abscisic acid. In addition, the mutant plants have a pronounced dwarf phenotype and display defects in both shoot and root gravitropism. Microscopic analysis showed that the morphological abnormalities are due to a reduction in both cell size and number (Timpte et al., 1992). The locus of axr2 has been mapped to chromosome 3 (Wilson et al., 1990), and chromosome walking for isolation of the axr2 gene has been initiated. The second strategy is a biochemical approach, which is especially suitable for auxin-binding proteins. Structural and functional analysis of natural and synthetic auxins has suggested that auxin activity depends upon a fractional positive charge located at a favorable distance from the carboxyl group of the acetic acid moiety of the hormone. The structural requirements for active auxins include a carboxylic acid moiety attached to an aromatic ring that may be connected in the ortho position to another aromatic ring or another planar arrangement of atoms (Palme et al., 1993). The structure-function relationships identified for auxins indicate that the primary step in response to this class of hormones must be an interaction of the plant hormone with a protein molecule that is subsequently mediated by second messengers to signal transduction chains. Many auxin-binding proteins have been identified over the past several decades, but biochemical progress in the characterization of these proteins

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has been retarded because of the many difficulties associated with classical ligand binding techniques. The problem has been overcome with the introduction of photoaffinity labeling techniques (Chowdhry and Westheimer, 1979), as well as improvements in other biochemical and immunological methods available for structural and functional studies. Hence, the list of diverse auxin binding proteins has been growing longer (Campos et al., 1 992). An endoplasmic reticulum (ERbassociated auxin binding protein from maize coleoptiles has been one of the most thoroughly studied (Inohara et al., 1989; Hesse et al., 1989). For example, an antibody against this auxin-binding protein inhibited the auxin-induced variation in the transmembrane potential of tobacco mesophyll protoplasts (Barbier-Brygoo et al., 1989). The third approach, which we are exploiting for the elucidation of the mechanism by which auxin acts, is to clone auxin-regulated genes and identify the functions of their products. A study of the hormonal activation of transcription of these genes should resolve the details of the signal transduction systems involved in this process. Current studies on mammalian systems suggest that a variety of receptors present at the cell membrane activate either preexisting transcription factors or their modulators via second messengers. As a consequence, transcription of a primary response gene is activated. Proteins newly synthesized from the primary response genes may mediate the expression of specific secondary response genes to produce ligand-specific and/or cell type-specific responses. Alternatively, they may have diverse and direct effects on metabolic processes. By unraveling the signal transduction pathway for auxin, we may be able to characterize the cellular signaling mechanisms that are common to animal and plant cells, as well as those that are specific to plants. In this chapter we provide an overview of recent developments in the analysis of auxin-regulated genes, with emphasis on our recent work with tobacco mesophyll protoplasts and tobacco BY-2 cells. We concentrate our discussion on transcriptional regulations and the functions of their gene products, since recent developments are mostly confined to these subjects. II. Cells Affected by Auxin A. Elongating Tissues

In the search for the primary events that are initiated by auxin, both shortand long-term effects on plant growth have been studied. A major problem has been to distinguish the primary hormonal responses in growth regula-

TABLE I Auxin-Regulated Genes

A

4

Genelclone

Material

Time of earliest detectable change in mRNA level

Function/ homology

Reference

ru

Upregulated genes aux22 aux28 GH2-4 (Gmhsp26A) SAURs GH3 arg 1 arg;!

IAA4/5 IAA6 parA parB pafl arcA

Soybean hypocotyls Soybean hypocotyls Soybean hypocotyls

15 min 30 min 30 min

Soybean hypocotyls Soybean hypocotyls Mung bean hypocotyls Mung bean hypocotyls Pea epicotyls Pea epicotyls Tobacco mesophyll protoplast Tobacco mesophyll protoplast Tobacco mesophyll protoplast Tobacco cell suspension culture (BY-2)

2-5 min 30 min lhr 1 hr 10-15 min 10-15 min 10-20 min 10-20 min 10-20 min (1 hr

P24

Fatty acid desaturase

Nuclear protein (p24) GST P24 WD40 repeat (G ,family)

Ainley et al. (1988) Ainley er al. (1988) Hagen et al. (1988); Czarnecka et al. (1988) McClure ef al. (1989) Hagen et al. (1991) Yamamoto et al. (1992) Yamamoto et al. (1992) Theologis et al. (1985) Theologis et al. (1985) Takahashi er al. (1989) Takahashi and Nagata (1992a) Takahashi and Nagata (1992b) Ishida et al. (1993)

103 107

pLS216

dbP An ACC synthase SARI SAR2 mas rolB nos

-

4

0

gene 5 Downregulated genes SAR5 ADR6 ADRll ADRl2

Tobacco cell suspension culture Tobacco cell suspension culture Tobacco cell suspension culture Arabidopsis seedlings Zucchini, winter squash

15-30 rnin

GST (p24)

van der Zaal et al. (1991)

15-30 rnin

P24

van der Zaal et al. (1991)

P24

Dorninov et al. (1992)

Basic protein ACC synthase

Alliotte et a / . (1989) Huang et al. (1991); Nakagawa et al. (1991) Reddy et al. (1990) Reddy et al. (1990) Langridge et al. (1989) Maurel et al. (1990) An et al. (1990) Korber et al. (1991)

15 rnin

4 hr 2 hr

Strawberry receptacles Strawberry receptacles Ti plasmid Ri plasmid Ti plasmid Ti plasmid

12 hr 12 hr 10 hr 10 hr 24 hr 12 hr

Strawberry receptacle Soybean hypocotyl Soybean hypocotyl Soybean hypocotyl

2 hr 4 hr 4 hr 4 hr

Mannopine synthase Indole-8-glucosidase Nopaline synthase Indole-3-lactate synthase

Seed protein

Reddy et al. (1990) Datta et al. (1993) Datta et a / . (1993) Datta et al. (1993)

114

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

tion from the responses that are merely consequences of primary reactions. Cell elongation is one of the fastest responses, with a latency of a mere 10-25 min after the start of auxin treatment (Vanderhoef et al., 1976). Two major hypotheses have been proposed to explain the auxininduced elongation of cells. One is the gene activation hypothesis, which proposes that auxin regulates the activation of specific genes that are necessary for the growth process, such as enzymes important for maintenance of cell walls during expansion (Key, 1969). However, an effect of auxin on induction of specific mRNAs, that was rapid enough to cause cell elongation was not detected until the 1970s. Consequently, the other hypothesis, the acid growth hypothesis, which suggested that cell enlargement is initiated by the auxin-induced secretion of proton received more attention (Rayle and Cleland, 1970; Hager et al., 1971). Several lines of evidence point to acidification of the cell wall as part of the mechanism by which auxin changes cell wall plasticity. The addition of acidic buffers to cells initiates a transient growth response, and acid increases the plastic deformability of isolated cell walls (Luthen et al., 1990).It is not clear how acidification affects cell wall plasticity, but several hypotheses have been proposed. For example, it has been suggested that protons may break calcium cross-bridges between acidic side chains of polymers of dicot cell walls, or that they activate pH-sensitive hydrolases or modulate the supply of new wall material (Cleland, 1987). Vanderhoef and Dute (1981) combined the two hypotheses, suggesting that the auxininduced elongation of cells involves two phases: an early growth response, resulting simply from auxin-induced proton secretion and a later phase of cell wall synthesis involving auxin-induced gene expression. This compromise viewpoint was popular in 1980, but recent advances in molecular biology have changed the situation and have made it possible to isolate cDNA clones for auxin-induced genes that are expressed a few minutes after auxin treatment. Up to now, as summarized in Table I, auxin-induced genes, Aux22, Aux28 (Ainley et al., 1988), pGH2-4 (Gmhsp26-A);(Czarnecka er al., 1988;Hagen et al., 1988),pGH3 (Hagen et al., 1991),and SAURs (McClure ef al., 1989) have been cloned by differential screening from soybean hypocotyls and sequenced. Nuclear run-on experiments have shown that the accumulation of mRNAs transcribed from these genes in response to auxin is, at least in part, due to the activation of transcription. The proteinbinding sites within the promoter region of the Am28 gene have been investigated by DNase I footprinting and a gel mobility shift assay with nuclear extracts from soybean hypocotyls (Nagao et al., 1993), but the regulatory sequences involved in the hormonal activation of transcription have not been determined in the soybean system and the function of the

EXPRESSION OF AUXIN-REGULATED GENES

115

gene product has yet to be identified. At one time, the rapid initiation of cell elongation was a most attractive feature of this system for investigations on the primary mechanism of action of auxin. However, today this experimental system is not necessarily suitable for molecular biological and biochemical analysis. The lack of an efficient DNA delivery system in the soybean hampers present studies. Indeed, the GH3 promoter/GUS and SAURs promoter/GUS chimeric genes had to be introduced into a heterogeneous system, namely, tobacco (Li et al., 1991; Hagen et al., 1991), and the homologs of Aux22 and Aux28 were isolated from A . thaliana (Conner et al., 1990). Among the genes mentioned above, SAURs (small auxin up RNAs) are the best characterized. Accumulation of SAUR mRNA is induced at the level of transcription within 2.5 min after the start of auxin treatment (McClure and Guilfoyle, 1987). SAURs are most strongly expressed in elongating regions of hypocotyls and epicotyls in soybeans. The mRNAs are distributed symmetrically in epidermal and cortical cells of hypocotyls when seedlings are grown in the normal vertical orientation, but become asymmetrically distributed in the lower and upper halves of the hypocotyl within 20 min after seedlings are reoriented horizontally. An 832 bp fragment of the SAUR 10A promoter can elicit the hormonal activation of transcription and drive the expression in an asymmetric manner during gravitropism and phototropism in transgenic tobacco. Furthermore, auxin transport inhibitors, 2,3,5-triiodobenzoic acid (TIBA) and N-( 1-naphthyl)phthalamic acid (NPA), suppress both asymmetric growth and asymmetric gene expression of SAURs (Li et al., 1991). These observations suggest that an active pool of auxin becomes asymmetrically distributed during growth in both orientations and they are consistent with the hypothesis that an asymmetric distribution of auxin results in asymmetric growth or curvature of an organ (Evans, 1991). Hagen et a f . (1991) analyzed the tissue-specific expression of auxininduced GUS activity in transgenic tobacco plants that contained a 592 bp promoter region from the GH3 gene fused to a GUS reporter gene. In trangenic tobacco plants that had not been exposed to exogenous auxin, the expression of GUS activity was largely restricted to roots of young green plants and developing floral organs, including ovules, developing seeds, and the pollen of mature plants. In situ hybridization analysis of expression of the GH3 gene in the soybean revealed a similar pattern, with the exception that the expression in pollen was seen only in transgenic tobacco. In situ hybridization also demonstrated that GH3 and SAUR transcripts show different patterns of expression among various organs and that both transcripts were differentially induced in specific organs by treatment with auxin (Gee et al., 1991). GH3 transcripts became more

116

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

abundant in the vascular regions of all organs, in developing palisade mesophyll cells of leaves, cotyledons, and flowers of plants treated with 2,4-dichlorophenoxyacetic acid (2,4-D). By contrast, SAUR mRNA became more abundant in the epidermis, cortex, starch sheath, and pith tissues of epicotyls and hypocotyls after treatment with 2,4-D. Although such detailed analysis of the distribution of the transcripts of SAUR and GH3 did not directly reveal the functions of the gene products, it can be inferred that different tissues respond to exogenous auxins by expressing different auxin-regulated genes. It is possible that multiple types of signal transduction pathways that include auxin receptors operate in plants and that these signal transduction pathways occasionally coexist in the same cells and tissues, whereas in other cells only one or other pathway may be operative. Alternatively, cell typespecific auxin-inducible cis elements and trans factors could participate in selective auxin-induced expression of genes in different cells and tissues. In this context, comparison of the 5’ flanking region of GH3 with that of SAURs does not reveal any strikingly similar sequence motifs, suggesting that the presence of multiple types of auxin-inducible elements that bind to different types of auxin-responsive transcription factors or auxin-inducible elements may have some ambiguity. SAUR mRNAs accumulate in the absence of auxin when protein synthesis is inhibited. The accumulation of mRNAs upon treatment with cycloheximide has been reported for some other auxin-regulated genes, such as IAA4l5, IAA6, parA, 103, 107,parB, parC, and an ACC synthase gene (Theologis et al., 1985: van der Zaal et al., 1987; Takahashi et al., 1991; Huang et al., 1991; Takahashi and Nagata, 1992b). However, treatment of soybean hypocotyls with cycloheximide does not induce the accumulation of the GH2-4 (Grnhsp26-A) and GH3 (Hagen and Guilfoyle, 1985) mRNAs. The drug inhibits the 2,4-D-mediated accumulation of pGH2-4 (Grnhsp26-A)mRNA by 80% but does not affect the 2,4-D-mediated induction of GH3 mRNA. Several possible mechanisms by which inhibition of protein synthesis could cause accumulation of mRNA have been proposed to operate in animal cells. A gene might be negatively regulated by labile repressor proteins (Subramanian et al., 1989) or, alternatively, the level of mRNA may be regulated posttranscriptionally by short-lived ribonucleases. Another possibility is that a certain mRNA must be translated to be degraded (Gay et al., 1989). Transcription run-on experiments with isolated nuclei showed that, unlike 2,4-D, the inhibitors of protein synthesis did not activate transcription. Instead, it appeared that the inhibition of protein synthesis resulted in stabilization of SAURs mRNAs (Franco et al., 1990). Several studies in mammalian cells have revealed that the 3’-untranslated

EXPRESSION OF AUXIN-REGULATED GENES

117

regions of unstable mRNAs contain AU-rich sequences that confer instability (Shaw and Kamen, 1986).A sequence element in the 3’-untranslated regions of SAURs may be related to the decay of mRNAs and the accumulation of mRNAs in response to inhibitors of protein synthesis. Yamamoto et al. (1992) isolated two cDNA clones, ARGl and ARG2, for new auxin-regulated genes from sections of elongating hypocotyls of mung bean. Recently, it has been revealed (K. T. Yamamoto, personal communication) that ARGl encodes a protein similar to a fatty acid desaturase of Arabidopsis, designated fad3 (Arondel et al., 1992). Although the functional significance of this finding is not clear at present, lipid metabolism may be related to the auxin-dependent elongation of hypocotyls.

6. Tobacco Mesophyll Protoplasts It has been well established from earlier studies of plant tissues in culture that auxin is necessary for the initiation and maintenance of cell division (Skoog and Miller, 1957). Induction of the cell division cycle is a consequence of the activation of a multicomponent cascade system that includes binding of the hormone to receptor molecules, triggering of the signaling pathway, reprogramming of gene expression, replication of DNA, and structural reorganization of the cellular architecture. Methods for the culture of tobacco mesophyll protoplasts were established in 1970 (Nagata and Takebe, 1970). These protoplasts, which form a highly homogeneous population and are in the Go phase, are able to initiate DNA synthesis, divide, and form colonies rapidly when they are cultured under appropriate conditions. A method for regenerating whole plants has also been established. Thus, this material is suitable for molecular analysis because of the homogeneity of the cells and the feasibility of delivering genes. The addition of two plant hormones, auxin and cytokinin, is an absolute requirement for the induction of meristematic activity (Fig. 1). Two-dimensional gel electrophoretic analysis by Meyer et al. (1984) showed that auxin induces the appearance of two proteins that are not observed in freshly prepared tobacco mesophyll protoplasts and decreases in the levels of nine proteins. No significant effect of cytokinin is observed on the changes of protein. Time-course experiments showed that the auxin-inducible proteins can be detected within 30 min of application of the hormones and reach a constant level after 2 to 4 hr. It has been postulated that the proteins, the level of which is reduced by auxin, are involved in formation of cell walls, given that 2,6-dichlorobenzonitrile prevents formation of cell walls and a reduction in levels of specific pro-

118

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

00

/%l

50

FIG. 1 The time course of DNA synthesis and cell division of tobacco mesophyll protoplasts under various hormone conditions. Open triangles represent mitotic index (MI) in the protoplasts cultured in a medium containing both auxin and cytokinin.

teins by auxin. Meyer et al. (1984) suggested that the auxin-induced proteins are involved in the initiation of mitosis. However, this possibility has not been substantiated experimentally. Using cultured tobacco mesophyll protoplasts at an early stage of culture, we isolated three auxin-regulated genes, parA (Takahashi et al., 1989), parB (Takahashi and Nagata, 1992a), and parC (Takahashi and Nagata, 1992b).These three genes are expressed in response to exogenous synthetic and natural auxins (Fig. 2) and are not induced by other plant hormones. The accumulation of parA, B, and C mRNAs was detected as early as 20 min and reached a maximum 4 hr after the addition of 2,4-D (Fig. 3). Since active synthesis of DNA has been observed from 24 to 48 hr in cultured tobacco mesophyll protoplasts by monitoring the incorporation of [3H]thymidine (Fig. I), the time course of the accumulation of par mRNAs suggests that active expression of par genes may be specified during the transition from the Go to the S phase. After 48 hr, when active cell division was observed, the accumulation of par mRNAs was almost fully suppressed (Takahashi el al., 1991; Takahashi and Nagata, 1992a,b). parA, parB, and parC responded to treatment with 2,4-D in the range of concentrations between 4.5 x lo-’ M and 2.2 x M and these genes were expressed among the various plant organs of tobacco, primarily in roots. Thus, the modes of expression of parA, parB, and parC were essentially similar, which suggests that the expression of these three genes may be regulated by a single mechanism. DNA sequence analysis revealed significant homology between parA and parC. Therefore, we looked for other parA-related genes in the same cDNA library by cross-hybridization and, as a result, a new cDNA clone,

EXPRESSION OF AUXIN-REGULATED GENES

119

FIG. 2 Effect of several auxins and cytokinin on accumulation of parA mRNA. (A) RNA was extracted after tobacco mesophyll protoplasts were cultured for 24 hr in Nagata and Takebe (1970) medium (NVO medium) without auxins, with 2,4-D (4.5 x 10-6M), with IAA (5.7 x 10-6M), or with NAA (5.4 x 10-6M). Each lane received 20 pg of total RNA. (B) IAA (5.7 x 10-6M) was added to the protoplasts which had been precultured in the NT7O medium without 2,4-D for 24 hr. RNA was extracted after 1 or 24 hr of incubation with IAA. Each lane received 20 pg of total RNA. (C) Effect of 6-benzylaminopurine (BAR on the accumulation of parA mRNA. RNA was extracted after culture for 24 hr in the absence of BAP. (-BAP), in complete NT70 medium containing BAP and 2,4-D (NT70). and in the absence of 2,4-D (-2,4-D). Each lane received 10 pg of total RNA. Blots were also produced with A A-I, a clone isolated in the differential screening, whose expression is not affected by 2.4-D.

FIG. 3 Induction kinetics of parB mRNA by 2,4-D. 2,4-D (4.5 x 10-'M) was added to the protoplasts, which had been precultured in the NT70 medium (Nagata and Takebe, 1970) without 2,4-D for 24 hr. RNA was extracted at the indicated times after addition of 2,4-D. Each lane received 20 pg of total RNA.

120

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHIYUKI NAGATA

designated C-7, was isolated (Takahashi and Nagata, 1992b). Although C-7 exhibited homology to parA and parC, it did not respond to exogenous auxin. The expression of C-7was predominantly detected in mature leaves, while the expression of parA and parC was observed primarily in root tips (Fig. 4). These results provide us with an interesting example of the differential expression of closely related genes. Thus far, several genes have been found to exhibit homology to parA (Table 11). Gmhsp26-A (pGH2-4) from soybean responds to auxin as well as to various stresses that include heat shock (Czamecka et al., 1988; Hagen et al., 1988).The prpl from the potato responds to a fungal elicitor (Taylor et al., 1990). cDNA clones 103 and 107, isolated after the addition of auxin to auxin-starved cultured tobacco cells, respond to auxin (van

FIG. 4 Expression of parC, C-7, and parA in different organs of tobacco plants and in tobacco BY-2 cells. RNA was extracted from shoot tips, flowers, stems, roots, young leaves, and fully expanded leaves of plants that had been grown in a greenhouse and from the whole plant body of I-week-old seedlings. Protoplasts were cultured in N V O medium with or without 2,4-D for 24 hr. BY-2 cells were harvested 2 days and 5 days after transfer to fresh medium (Yasuda et al., 1988). Each lane was loaded with 1 pg of poly(A)+RNA.

TABLE II Characterizationof parA-Related Genes parA

PaK

c-7

103

I07

Prp 1

pLS216

Source

Tobacco rnesophyll protoplasts

Tobacco mesoph yll protoplasts

Tobacco rnesophyll protoplasts

Tobacco cell suspension

Tobacco cell suspension

Potato leaves

Tobacco cell suspension

Soybean seedlings

Maize

Inducer

Auxin

Auxin

Auxin

Auxin

Pat hogen

Auxin, c y tok i n i n

Stresses

ND

Tissue-specific expression (highest level)

Roots

All organs (roots)

ND"

ND

ND

Tassels

ATITA sequence Homology to parA Reference

All organs (roots)

Root tips

-

Gmhsp26-A

Bz-2

3

1

0

1

0

0

2

1

1

100%

68%

65%

42%

67%

51%

92%

37%

31%

Takahashi et al. (1989)

Takahashi and Nagata (1992bj

Takahashi and Nagata (1992b)

van der Zaal et al. (1991)

van der Zaal et a / . (1991)

Taylor et a / . (1990)

Dorninov el a / . (1992)

Czarnecka et a / . ( 1988)

Nash ct a / . (1990)

ND, not determined.

122

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

der Zaal et al., 1991). Expression of pLS216, which is regulated by auxins and cytokinins (Dominov et al., 1992), was found in a suspension culture of Nicotiana plumbaginifolia cells. Bronze-2 (Bz-2) from maize, whose product is supposed to be involved late in the anthocyanin biosynthetic pathway (Nash et al., 1990; Schmitz and Theres, 1992) is regulated by unidentified factors. Since each of these parA-related genes can be induced by various compounds, the effect of different stresses on the expression of these genes was examined. A heat-shock response was observed with Gmhsp26-A, while parA, parC, C-7, and 103 failed to respond to heat shock. CdC1, induced parA and Gmhsp26-A. However, inhibition of splicing was observed with Gmhsp26-A mRNA (Czarnecka et al., 1988) but not with parA mRNA (Takahashi et al., 1991). Treatment of CdC1, had no effect on the accumulation of parC, C-7, and 103 mRNAs. Thus, responses of these genes to various stimuli are very different, and, while they may have a common ancestor, they must have evolved in different ways. In this context it is worth noting that no large-scale similarities have been found in the 5' flanking regions of Gmhsp26-A, parA, pa&, C-7, 103-like genes, prpl, and Bz-2. An ATTTA sequence is found within the 3' untranslated region of parA (in triplicate), parC, 103, Gmhsp26-A, pLS216 (in duplicate), and p r p l , and it may have some significant correlation with the short-lived responses of these genes to external stimuli. This sequence was originally found in mRNAs of genes that respond to mammalian growth factors and is considered to confer instability on mRNAs (Shaw and Kamen, 1986). Proof of this hypothesis in plants awaits the results of further investigations. The available data suggest some common function for the gene products, since the proteins related to the product of parA exhibit homology similar to a 24-kDa protein of Escherichia coli (Fig. 5 ) (Serizawa and Fukuda, 1987), which forms an equimolar complex with the holoenzyme of RNA polymerase (Ishihama and Saitoh, 1979). This 24-kDa protein was initially thought to be identical to the stringent starvation protein (ssp), a major protein synthesized in stringent strains of E. coli under extreme nutrient starvation. However, it now turns out that the two proteins are completely different (A. Ishihama, personal communication). Thus, it is possible that the products of this family may play a role similar to that of the 24-kDa protein of E. coli, since it has been reported that eukaryotic RNA polymerases exhibit partial homology to RNA polymerase of E. coli (Alisson et al., 1985; Biggs et al., 1985; Sweetser et al., 1987). To study the localization of the parA protein, we have generated polyclonal antibodies against parA protein that was overproduced in E. coli. In tobacco mesophyll protoplasts that were cultured for 6 hr with auxin, the location of antibodies to parA protein indicated a nuclear localization

123

EXPRESSION OF AUXIN-REGULATED GENES 1

24-kDa protein

212

FIG. 5 Dot matrix plot of parA and a 24-kDa protein of E. coli. Homology was scored by

a dot when 15 out of 30 amino acids were equivalent.

for the protein (Fig. 6). These results suggest that the parA product may be involved in transcription. Auxin induces parA and then the parA product may regulate the expression of other genes that are involved in physiological responses. The accumulation of parB can be induced by synthetic and natural auxins, but not by other plant hormones or stresses, such as heat shock and CdCI,. The deduced gene product of purl? exhibits homology to glutathione S-transferase (GST; RX: glutathione R-transferase, E.C. 2.5.1.18). GSTs form a family of enzymes that catalyze the conjugation of a variety of electrophilic xenobiotics with glutathione (Chasseud, 1979). This family of enzymes is widely distributed in animals, insects, and higher plants. In humans, at least seven different GSTs have been identified (Rushmore et al., 1990). Most of the enzymes have distinct but overlapping specificities for substrates. Among the known GSTs, the product of parB exhibited greatest homology to maize GSTIII (Grove er al., 1988) and the extent of homology between these two proteins was 46% (Fig. 7). The homology to mammalian GSTs (Suguoka et al., 1985; Board and Webb, 1987) was limited to the region of amino acid sequence between residues 52 and 75, which suggests that this region could be the functional domain for the enzymatic activity of GST. It was found that this region has a homology to rat lysophospholipase (Han et al., 1987).

124

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

FIG. 6 Nuclear localization of parA protein. (A, C) Fluorescence micrographs of tobacco mesophyll protoplasts staining with antibodies against parA protein. (B, D) Fluorescence micrographs of the same cells stained with 4’,6-diamidino-2-phenylindole(DAPI). (A, B) Freshly prepared tobacco mesophyll protoplasts. (C, D) Protoplasts cultured in NT70 medium with 2,4-D for 6 hr.

We have demonstrated that a gene product of parB produced in E. coli showed GST activity by an assay with I-chloro-2,4-dinitrobenzene (CDNB) as a substrate (Takahashi and Nagata, 1992a) (Fig. 8). This is the first instance in which the gene product of a differentially cloned auxinregulated gene has been ascribed a specific enzymatic activity. At this time there are two possibilities for the function of the gene product of parB. One is that exogenous auxins, as xenobiotics, could be detoxified by GST, although thus far detoxification of auxins by GSTs has not been known. The observation that the GST from a membrane fraction of A. thafiana binds indole-3-acetic acid (IAA) might have some relevance to its role (Zettl et al., in press). The second possibility is that the expression of parB could be related to the proliferative activity of tobacco mesophyll protoplasts. It has been suggested that specific expression of placental GSTs would be closely related to the process of neoplastic transformation. A placental GST has

A ParB MGSTIII

1

50

I-KVHGSP PLDLYBlM

VPAFEDG IMLVga

100

DLKLFESRAITQY IAHWADNGYQLI LQDPKKMPSMS- - -VWnEVECQKFEPPATKLTWELC lEvllllllllllDt-a-6-I~ATA-$AAKLF##U#BSWH~!NDSIWF@L 150

IKPI IGHTTDD MIBLu1IApI-

-=

FTLVDLHHI --PNIYYLMS FAlw(IvLLLPA- -

200

SKVKE---VPDSRPRVSAWC-ADI LARPAWVKCLEK--LQK A R P P R P C C @ A - ~ C # I ' V A A WIP P S S S A

B

C ParB(53-76) RatLPL(404-427)

VPAFEDCD-LKLP--ESFIT YIAH m-#ImIm-&

FIG. 7 Amino acid sequence of parB cDNA is compared with those of (A) maize GSTIII (MGSTIII), (B) rat placental GST-P (rat GST), human GST 2 (human GST), and (C) rat lysophospholypase (Rat LPL). Identical amino acids are shaded. Positions of homologous regions of parB, rat GST,human GST, and rat LPL are indicated in parentheses with amino acid residue numbers.

A

Br.9

- - - - - - - - - A A ~ C A ~ ~ ~ ~ C A ATG ~ C MT C TCC CTA TAT CCA GM A l l ' A E CCG let Asn Ser Val Tyr Pro Glu Ile net Ala

B

rn L.

L . m

o

a

c

u

>

a

o r x c u e .

c 9lK

C

c 68K

c 43K

pTRCp arB Vector

8. 8 8 + 0 , 2 1

I


01

FIG. 8 Expression of parB cDNA in E. coli. (A) Structure of pTRCparB overproducing parB protein. (B) SDS/PAGE of the parB proteins overproduced in E. coli. Numbers on right are kilodaltons. (C) GST assay using CDNB as the substrate. SDs are calculated from five experiments.

126

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

been shown to be induced specifically at an early stage of chemical hepatocarcinogenesis in rats and the causal relationship is almost 100% (Satoh et al., 1985). Moreover, it has been reported that experimentally induced expression of the activated ras gene results in concomitant expression of a placental GST in cultured rat liver cells in association with carcinogenesis (Li et al., 1988). The involvement of glutathione, a substrate for GST, has been implicated in cell proliferation since Suthanthiran et al. (1990) showed that glutathione regulates the activation-dependent DNA synthesis in the T lymphocytes stimulated with antigens. In either case, it should be essential to search for substrates for the GST as a gene product of parB in tobacco mesophyll protoplasts. The 5' flanking region of the parA gene includes a lll-bp direct repeat and ten ATATAG repeats (Takahashi et al., 1990). When the auxinresponsive activity of the 5' flanking sequence of the parA gene was examined by a transient expression assay with GUS as the reporter gene, it was found that deletion of a 11l-bp segment abolished the auxin-induced GUS activity (Fig. 9). We have identified a nuclear protein that binds specifically to a 17-bp sequence in the 11 1-bp direct repeat. However, analysis of transgenic tobacco that carried the parA promoter fused to a GUS gene showed that exogenous auxin could induce the GUS activity without the 11 1-bp direct repeat. These data may suggest that the parA promoter has complex auxin-responsive elements. Recently, we determined the nucleotide sequences of the 5' flanking regions of the parB and parC genes, but no 111-bp direct repeat was be found in these sequences. Detailed analysis, such as a study of responsiveness to auxin after the sequences of promoter regions of parA, parB, and parC are transferred to a minimal promoter, might reveal the consensus cis-elements of auxinresponsive genes in tobacco mesophyll protoplasts.

C. Cell-Suspension Culture Cultures of plant cells in uitro have several unique advantages as experimental systems for molecular and cellular biology. First, they provide an

FIG. 9 GUS transient assay in electroporated tobacco mesophyll protoplasts. A M, A 41, A 45, A 106, A 108, A 2, and A X were introduced to protoplasts and followed by a 40-hr incubation. Activity is expressed in picomoles of 4-methylumbelliferone (4-MU) produced per milligram of protein in the protoplast extract per minute. Filled bars represent GUS activity from cell extracts of protoplasts cultured with 2,4-D; open bars represent protoplasts cultured without 2,4-D.

1 1 1-bp repeat I 1 1 1-bp repeat II

I

rK--xmlrnn

TATA Cap ATGl

GUS

6-bp repeats AM A4 1 A4 5 A106 A108 A2

AX

pmol4-MUlrninlrng protein 5w I

400 I

300 I

200

100

0

I

AM

A4 1

A4 5

A106

A108

A2

AX

128

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

apparently infinite supply of homogeneous material. Second, the cellular environment can be strictly controlled by use of a chemically defined medium. In studies with other plant systems, the events that follow wounding, exposure to light, and osmotic stresses may affect the experiments. Animal cell lines, such as NIH3T3 and HeLa cells, have played significant roles in the isolation of oncogenes and in the elucidation of the mechanisms of signal transduction pathways. Clearly, it is also important to establish standard plant cell lines for detailed molecular and cellular biological studies. Cultured plant cells generally require an exogenous supply of auxin for cell division. Removal of auxin from the medium can lead to cessation of cell division, which can be reinitiated by a resupply of auxin. Several auxin-induced genes have been identified from a few cell lines, for example, clones 103 and 107 from cultures of Nicofiana fabacum L. cv. White Burley (van der Zaal et al., 1991) and pLS 216 from cells of Nicotiana plumbaginifolia (Dominov et al., 1992). However, the genes were found to be similar to parA (see earlier discussion). The mode of expression of 103 was similar to that of parA. The 1.8-kb portion of the 5' flanking region of a 103-like gene, fused to a GUS gene, led to expression of GUS only in root tips of transgenic plants (van der Zaal et al., 1991). It may be worth noting here that the auxin-induced accumulation of pLS216 mRNA was blocked by a protein kinase inhibitor, staurosporin. Because the phosphorlylation of their specific amino acid residues, namely serine, threonine, and tyrosine, has been shown to serve general regulatory functions in both prokaryotic and eukaryotic cells, the signaling pathway for hormonal activation of transcription of this gene clearly involves phosphorlylation-mediated regulation, Recently, Droog et al. (1993) reported that the gene product of 103, produced in E. coli, had GST-like activity with CDNB as a substrate. One puzzling aspect of this observation is the limited similarity of the protein encoded by 103 to other plant GSTs (Grove et al., 1988; Dudler et al., 1991; Meyer et al,, 1991; Takahashi and Nagata, 1992a). Moreover, as noted above, 103 belongs to a gene family that includes parA, whose product was demonstrated to be localized in the nucleus. One possible explanation is that some of the plant GSTs, including 103, are involved in unidentified nuclear events. The other possibility is that parA-related genes might share an evolutionary common ancestor with genes for GSTs and may have acquired distinct functions that preserve a few common ancestral features. For example, a squid lens protein has limited similarity to GST (Tomarev and Zinovieva, 1988), which was interpreted to indicate that preexisting proteins were recruited as structural proteins of the eye lens during evolution. Proof of such hypotheses awaits the results of further investigations. We have began to analyze auxin action using tobacco BY-2 cells in

129

EXPRESSION OF AUXIN-REGULATED GENES

suspension culture. These cells are derived from Nicotiana tabacum L. cv. Bright Yellow 2 (see review by Nagata et al., 1992). The BY-2 cells multiply 80- to 100-fold in 1 week in culture and can be synchronized to 70-80% in terms of mitotic index by use of aphidicolin. The unique characteristics of BY-2 cells, namely their exceptionally high growth rates and a high degree of homogeneity, make them a suitable experimental system for studying molecular and cellular biology. After 2,4-D is applied to auxin-starved BY-2 cells, the frequency of cell division begins to increase at 10 hr and reaches a maximum at 12 hr. Without the application of 2,4-D, no such increase in cell division activity is observed (Fig. 10). Recently, we isolated a cDNA clone for the new auxin-induced gene, arcA, from this system (Ishida et al., 1993). Sequence analysis revealed that arcA belongs to an expanding gene family (Fig. 1 l), including the psubunits of G-proteins, which have a series of internal repeats of 40 amino acids called the WD-40 repeat (Simon et al., 1991) (Fig. 11). Members of the WD-40 repeat family are involved in various cellular functions, such as signal transduction (Sugimoto et al., 1985), RNA splicing (PRP4; Dalrymple et al., 1989), cell cycle regulation (CDC20; Goebl and Yanagida, 1991), transcriptional regulation (TUP1; Mukai et al., 1991), neurogenesis of Drosophila (enhancer ofsplit; Hartly et al., 1988)and photomorphogen-

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FIG. 11 Repeated segmental structures of the WD-40 repeat motif. (A) An alignment of the arcA product with mammalian G p subtypes, which contain a typical WD-40 repeat, was optimized and an appropriate gap was introduced by a computer program. The consensus amino acids in the repeated motif, in which aliphatic amino acid residues are represented by the 0, are shown at the bottom of the panel. Dots represent amino acid residues identical to those in G p 1. Identical and equivalent amino acid residues were boxed at the position corresponding to the consensus of WD-40 repeat and only identical residues were at other sites. (B) Seven repeat segments in the arcA protein are optimally aligned. Conserved (identical and equivalent) residues are boxed. Amino acid residues observed in the consensus sequence are shown at the bottom of the panel. (C) Homologous amino acid sequence within arcA protein and between arcA protein and mammalian G p protein. The dot matrix plot comparing the arcA protein with itself shows several repeats in addition to the identical diagonal (left). A comparison of arcA protein and mammalian G p protein sequence demonstrates the presence of conserved repeats (right).

132

YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

esis of Arabidopsis thaliana (COP1; Deng et al., 1992). Although the implications of the common function ascribed to this internal repeat have yet to be characterized, their widespread occurrence from yeast to humans suggests their functional importance. It is intriguing that many of the genes encoding WD-40 repeat proteins have been found to have functional interactions with members of another family of proteins, characterized by the presence of another internally repetitive domain, which consists of repeats of a 34-amino acid unit, tetratricopeptide repeat (TPR) Keleher et al., 1992). TPR proteins are involved in activities as diverse as RNA splicing (PRP6; Legrain and Choulika, 1990), mitosis (CDC23; Sikorski et al., 1990), and transcriptional repression (SSN6; Schultz and Carlson, 1987). Based on several lines of evidence, the attractive possibility has been suggested that each protein containing a WD-40 repeat can interact with a specific TPR protein. Examples of such proposed pairings include PRP4-PRP6, CDC20-CDC23, and TUP1-SSN6 (Goebl and Yanagida, 1991). The strongest evidence in support of this hypothesis is the observation that TUPl and SSN6 can actually associate to form a protein complex (Williams ef al., 1991). Recently, these two proteins were shown to function as a transcriptional repressor when paired together (Keleher et al., 1992). Analysis of the function of the arcA product in plant cells is of great interest. Both the families of the WD-40 repeat and TPR include genes that regulate mitosis. Therefore, it is possible that the gene product of arcA, with its WD-40 motifs, might form a complex with a protein having a TPR motif that could be related to cell division activity, since induction of cell division is the only phenomenon detectable after the application of 2,4-D to 2,4-D-starved BY-2 cells, and this induction is preceded by the expression of arcA. D. Other Auxin-Regulated Genes

Among products of other auxin-regulated genes, ACC synthase (S-adenosyl-L-methionine methylthioadenosine lyase, E.C. 4.4.1.14), which is the rate-limiting enzyme in the formation of ethylene, is of foremost importance. Ethylene plays a pivotal role in the cell separation processes that are involved in the abscission of leaves, softening of fruit, and, probably, in the formation of large intercellular air spaces (Abeles, 1973). Expression of ACC synthase is induced by auxin as fruit ripens and after tissue is wounded. This enzyme is encoded by a multigene family, the members of which are differently regulated. Nakagawa et al. (1991) reported that a gene for an auxin-induced enzyme (accA) is different from that for a wound-induced enzyme (accW). When sections of the hypocotyl of etiolated winter squash seedlings were treated with IAA, the expression of

EXPRESSION OF AUXIN-REGULATED GENES

133

accA was observed after 20 min while only a small amount of the transcript of accW appeared. However, a gene for ACC synthase in zucchini that is very similar (96%) to the wound-induced gene of winter squash was induced at high levels by IAA in wounded fruits (Huang et al., 1991). These results suggest that certain members of the family of ACC synthases are differentially induced by auxin in a tissue-specific manner. Alliotte et al. (1989) isolated an auxin-regulated gene, dbp, from Arabidopsis thuliana. The dbp gene is constitutively expressed in all parts of mature Arubidopsis plants and is expressed on an enhanced level in the root and shoot apex. In roots, the region of enhanced expression was localized by in situ hybridization and was shown to correspond to the zone of cell division in the apex, The accumulation of dbp mRNA in isolated peduncle segments could be enhanced by a 4-hr exposure to exogenous auxin. The product of dbp is lysine-rich and binds to doublestrand DNA nonspecifically. Since this protein shares several biochemical properties with histone H1, it will be of interest to determine whether the dbp protein is associated with chromatin. In strawberry fruits, achenes produce auxin that controls receptacle growth, and this production is dependent on pollination. Hence, receptacle growth can be stopped by preventing pollination, or by removing achenes from the pollinated fruit (Archbold and Dennis, 1985). Auxin applied exogenously to unpollinated fruits or to pollinated and then de-achened fruits can induce apparently normal development. Reddy et al. (1990) isolated two auxin-inducible cDNA clones (A SARl and A SAR2) from auxin-treated strawberry receptacles. Auxin induced the accumulation of mRNAs corresponding to A SARl and A SAR2, while no effect of ethylene was observed. Northern blot analysis suggested a positive correlation between growth of strawberry fruits and the accumulation of A SARl and A SAR2 mRNAs. Structural analysis is required to clarify the function of these gene products and their relationship to other auxin-regulated genes.

111. Prokaryotic Genes

T-DNA genes of Agrobacterium plasmids are actively transcribed in transformed plant cells. Observations that the T-DNA transcripts are polyadenylated and that the transcription is inhibited by a-amanitin in plant cells (Willmitzer et al., 1981) together indicate that the regulatory regions of the T-DNA genes interact with host regulatory systems to control expression of certain genes in plant cells. It has been shown that rolB of A . rhizogenes (Maurel et al., 1990), as well as mas (Langridge et al., 1989), gene 5 (Korber et al., 1991), and nos of A . tumefaciens (An et al., 1990)

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YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

are regulated by auxin. The product of rolB has P-glucosidase activity and is able to hydrolyze indole P-glucoside conjugates (Estruch et al., 1991). The gene 5 product increases the rate of conversion of tryptophan to indole-3-lactate, an auxin antagonist (Korber et al., 1991). Thus, rolB and gene 5 are induced by auxin and their products are involved in auxin metabolic pathways. Analysis of transgenic tobacco carrying the rolB promoter fused to a GUS gene showed that exogenous auxin could induce the GUS activity. Although rolB and rolC share common upstream sequences, the much lower efficiency of auxin-dependent activation of rolC, compared with rolB, may be attributable to orientation effects on these sequences (Maurel et al., 1990). Promoter analysis of gene 5 in transgenic plants revealed that sequences required for regulation by auxin and phloem-specific expression were mapped -202 to -292 and carried DNA sequence motifs common to SAURs and Am28 from soybean hypocotyls. However, it took 9-12 hr after initiation of treatment with auxin for activity of the reporter enzyme to be detected (Korber et al., 1991). This time lag is rather long when compared with that reported for other auxin-regulated genes. A similarly slow response to auxin was also observed with the rolB promoter. Thus, the induction by auxin of these two T-DNA derived genes may be a secondary response. In addition, it has been shown that the promoter activity of gene 5 is only induced by the presence of both auxin and cytokinin. Exploiting this observation, Boerjan et al. (1992) developed a bioassay for auxin and cytokinin using transgenic tobacco with a gene 5 promoter-GUS fusion gene. An et al. (1990) reported that the response by wounding of the nos promoter, derived from A . tumefaciens, is further enhanced by auxin. The enhancement was concentration dependent. Cytokinins, abscisic acid, and GA3 did not influence promoter activity. Deletion analysis of the nos promoter indicated that a 10-bp sequence, GCACATACGT, a potential zig-zag DNA (Z-DNA)-forming element located between - 123 and - 1 14 or an element that overlapped this sequence, is essential for the response to wounding and auxin. However, it has not yet been determined whether auxin can induce the nos promoter without wounding. The fact that TDNA promoters are active in tumor cells, in which auxin and cytokinin are abundant, may provide an explanation for the physiological basis of the induction of these prokaryotic promoters by auxin. Recently it has been reported that the as-1 sequence of the cauliflower mosaic virus (CaMV) 35s promoter, which is involved in root-specific expression, may be an auxin-responsive sequence (Third International Congress of Plant Molecular Biology, Tucson, 1991). However, the 35s promoter itself does not respond to auxin (An et al., 1990; Maurel et al., 1990). A detailed report on these results is eagerly awaited.

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IV. Genes Downregulated by Auxin

Previous studies have shown that auxin not only induces the expression of certain genes but also downregulates the expression of some genes (Zurtluh and Guilfoyle, 1980; Meyer et a!., 1984). As summarized in the preceding paragraphs, a number of genes that are upregulated by auxin have been characterized. However, there are only a few reports of the genes that are downregulated by auxin. Reddy et al. (1990) isolated a cDNA clone, A SARS, for an auxin-repressed gene from strawberry fruits, but analysis of the protein encoded by A SARS failed to reveal any homology to known proteins. In a study of soybean hypocotyls, three cDNA clones-ADR6, ADRl1, and ADR12-each representing a gene that is downregulated by auxin, were isolated and characterized (Datta et ul., 1993).The levels of mRNAs corresponding to these genes decreased 10- to 100-fold in response to applied auxin (Baulcombe and Key, 1980). Nuclear run-off transcription showed that the transcription rate for the genes that corresponded to ADR6, ADR11, and ADR12 was reduced in the presence of auxin, but the decrease was too small to account for the decrease in the amounts of these mRNAs after treatment with auxin. This result suggests that auxin regulates the amounts of these mRNAs through both transcriptional and post-transcriptional mechanisms. Furthermore, the expression of these downregulated genes is also controlled by light: expression of ADR6 and ADR12 appears to be upregulated by light, whereas that of ADRl 1 appears to be downregulated. Among these genes, ADRl 1 shows 46% homology to a seed protein from Viciafuba (Bassuner et al., 1988), although the functional significance of this similarity is not clear.

V. Future Prospects

One of the goals of plant science today may be to determine how plant hormones regulate the growth and differentiation that occur through the life of a plant and the adaptation to temporal changes in environmental conditions. The limited variety of chemical signals that correspond to the five classical types of plant hormones stands in marked contrast to the situation in animal systems. This difference raises the question of how such a limited number of chemical messages can control the myriad events that occur during plant development. A substantial diversity of signals may be achieved, in part, by the antagonistic or cooperative actions of a group of plant hormones. The balance between two or even more regula-

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YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

tory substances can theoretically create a vast potential for diversity and plant physiologists are inclined to look in this direction for a way to explain the diversity of processes that are regulated by plant hormones. Another possible explanation is that the various cellular responses to plant hormones might be due to the differential sensitivity of various plant cells to the hormones. Recent studies have raised further questions in molecular terms. Are multiple types of signal transduction pathways, including receptors for a certain plant hormone, operative in a single plant and, if so, are they distributed in a tissue-specific manner? Do the perception cascades for each plant hormone interact to produce a new response? What kinds of genes are activated in specific tissues as a consequence? As illustrated here, various experimental systems have yielded a number of auxin-regulated genes as a result of differential screening, and the primary structures and patterns of expression of these genes have been determined. The induction of these genes by auxin is mainly due to activation of transcription. However, our understanding of the precise functions of auxin-regulated genes and the mechanism of hormonal activation of transcription at the molecular level is just beginning to develop. With respect to identification of the functions of auxin-regulated genes, it is very difficult to infer any function if the sequences do not correspond to those of known genes in present databases. In this situation, accumulation of basic data, such as the determination of subcellular localization of the gene products, is important even though such data cannot directly reveal any function. Constitutive overexpression of auxin-regulated genes, as well as the expression of antisense mRNAs and ribozymes to decrease the level of expression of these genes in transgenic plants may provide clues to their function. Gene targeting (Thomas and Capecchi, 1987) may also be an effective strategy for elucidating the function of differentially cloned, auxin-regulated genes. To generate loss-of-function mutants in mice, the single gene of interest must be inactivated by homologous recombination in embryonic stem cells and the mutation must be introduced into the mouse germline, a procedure that requires great skill and experience. By contrast, regeneration of whole plants from plant somatic cells is now possible in various plant species. However, there have been no reports of successful gene targeting in plants to date. Much effort should be invested in this promising strategy. Recently, several genes have been isolated by tagging with T-DNA (Yanofsky et al., 1990) or transposons (Schmidt et al., 1987). Apart from conventional insertion mutagenesis, introduction of transcriptional enhancers into plant genomes could be expected to cause the overexpression of certain genes around the exogenous enhancer. Exploiting this strategy, Hayashi et al. (1992) isolated tobacco cells with a dominant mutation that

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were characterized by their ability to grow in the absence of exogenously applied auxin in the culture media. One of the plant genes rescued from these transformed cells retained the ability to confer auxin-independent growth when retransfected into tobacco protoplasts. It is important to note that this gene was directly or indirectly induced by auxin (H. Hayashi, personal communication). Although no significant homology between this gene and known genes, including auxin-regulated genes, has been found to date, unification of this new genetic approach and the analysis of auxinregulated genes can be expected in the near future. Attempts to characterize cis-acting regulatory elements required for activation by auxin has revealed several conserved motifs within hormoneresponsive promoters. Although certain sequences, for example, GCACATACGT from nos (An et al., 1990) and TGTCGGC from gene 5 (Korber et al., 1991), were proposed as consensus cis-elements, these sequences were merely deduced from available sequences of auxin-regulated promoters from heterogeneous systems. For further detailed analysis, it is necessary to study auxin responsiveness after these sequences are transferred to minimal promoters. Moreover, we must also consider the tissue specificity of the gene expression since, for example, transcripts of GH3 and SAUR, which were cloned from soybean hypocotyls, were induced in different tissues by treatment with auxin (Gee et al., 1991). The tissue specificity of the expression of auxin-regulated genes was examined by Northern blot analysis in transgenic plants carrying promoter/GUS chimeric genes (Alliotte er al., 1989; Langridge et al., 1989; Maurel et al., 1990; Reddy et al., 1990; Takahashi et al., 1991; van der Zaal et al., 1991). It is worth noting here that most of the auxin-regulated genes are strongly expressed in roots rather than in shoot tips. These results contrast with physiological data that indicate auxin is distributed at higher concentrations in shoot tips. Resolution of this puzzling situation awaits further investigation. One of the reasons for our present limited understanding of the molecular mechanisms of action of auxin and its consequences with respect to gene expression is the difficulty encountered in selecting mutations involved in the signal transduction process after stimulation by auxin. Little information is available on phenotypes that reflect deficiencies in the responses to auxin. Thus far, most relevant mutants have been isolated from searches for plants that are resistant to toxic concentrations of auxin. Now it is possible to screen for auxin-unresponsive mutants by monitoring the expression of fusion genes composed of an auxin-regulated promoter and the gene for GUS as a selectable marker in transgenic plants. The mutations are expected to be ascribed with members of signal transduction chains, from the perception of auxin to the activation of transcription.

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YOHSUKE TAKAHASHI, SARAHMI ISHIDA, AND TOSHlYUKl NAGATA

Barbier-Brygoo et al. (1989) demonstrated the presence and function of a plasma membrane receptor for auxin in tobacco mesophyll protoplasts. The relationship between the auxin receptor and the control of gene expression is intriguing. The elucidation of the pathways along which the auxin signal is transmitted, from the membrane to the nucleus, will require the characterization of receptors, the identification of signal amplification elements that produce second messengers, and the analysis of nuclear components, which include cis-elements and transacting factors. For detailed biochemical studies, it is important to establish a versatile experimental system of cultured cells that are both homogeneous and suitable for preparing large amounts of material. The mechanism by which auxin controls the growth and development of plants will be elucidated when results of the analysis of auxin-regulated genes, genetic approaches, and the biochemical analysis of the perception of auxin can be combined. Acknowledgment We thank Dr. Seiichiro Hasezawa for his help with the analysis by microscopy. Thanks are also due to Dr. Makoto Kusaba for his discussion. This study was supported by grants from the Ministry of Education, Culture and Science of Japan to (Y. Takahashi) and to (T. Nagata) from the Ministry of Agriculture, Forestry and Fishers of Japan to (T. Nagata), and from the Science and Technology Agency of Japan to (Y. Takahashi).

Note Added in Proof Recently, the cis-regions required for activation by auxin were defined between -318 and -154 in the promoter of PS-IAA45 (Ballus et al., 1993) and between -211 and -162 in the promoter of parB (Takahashi et al., in preparation).

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Regulation of Mitochondria1 Gene Expression in Saccharomyces cerevisiae Carol L. Dieckmann and Robin R. Staples Department of Biochemistry, University of Arizona, Tucson, Arizona 8572 1

I. Introduction

Although the title of this chapter suggests that it will focus on the regulation of mitochondrial gene expression in yeast, little is known about how this expression is regulated. What is known is that many nuclear gene products are needed for the expression of mitochondrial genes at the level of transcription, RNA processing, translation, post-translational modification, and complex assembly. Mutations in these nuclear genes often lead to respiratory deficiency, that is, the pet phenotype (Tzagoloff and Dieckmann, 1990). Whether one or more of these nuclearly encoded proteins controls the increase in mitochondrial gene expression on nonfermentable carbon sources has yet to be demonstrated. The bulk of this chapter summarizes current knowledge of the function of the various PET gene products needed for mitochondrial gene expression. Several very good reviews have been published in the past several years on this topic (Bolotin-Fukuhara and Grivell, 1992; Costanzo and Fox, 1990; Grivell, 1989; Tzagoloff and Myers, 1986); thus we have tried to focus on more recent developments. Finally, the last section reviews what is known about the direct and indirect modes of changing mitochondrial gene expression when yeast cells are switched from fermentation to respiration. Not covered is the import of proteins into mitochondria, a topic well reviewed recently (Glick et al., 1992; Schatz, 1993; Pfanner et al., 1992).

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Copyright 0 1994 by Academic Press. Inc. All rights of reproduction in any form reserved.

4

+o--

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t 146

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COX3

ORFZ

tRNAF-tRNAv-COX3

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tRNA

tRNAfM-RPM1-tRNAp

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ATPB

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COX1-ATPB-A TP6* *I Ikb

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CAROL L. DIECKMANN AND ROBIN R. STAPLES

II. Transcription A. Promoters

While mammalian mitochondrial genomes are transcribed from a pair of promoters, one on each strand, the yeast mitochondrial genome is approximately four times larger, and the genes are transcribed from several different promoters. Two current reviews lend greater depth to our understanding of yeast mitochondrial transcription (Schadel and Clayton, 1993; Jang and Jaehning, 1993). The 12 identified transcription units are shown in Fig. 1. All but tRNA,’hr are transcribed from the same strand of DNA. In addition, not shown in the figure are promoters at the four active origins of replication, which prime yeast mitochondria1DNA replication (Baldacci et al., 1984). The promoters have the consensus sequence S’ATATAAGTA 3‘, where the last A is the + 1 position of the transcript. Extensive mutagenesis of this sequence and analysis in an in vitro transcription system has shown that the -2, -4, -6, and -7 positions cannot be changed without destroying recognition by RNA polymerase. Conversely, at positions -8 and + 1, any nucleotide is tolerated (Biswas et al., 1987; Schinkel et al., 1987a; Marczynski et al., 1989). Some of the yeast mitochondrial promoters are “weak” due to a pyrimidine at the + 2 position, whereas the “strong” promoters have a purine at this position. Supplying dinucleotides in the transcription mix turns a weak promoter into a strong promoter, suggesting that polymerization of the first two nucleotides in the RNA is the rate-limiting step in transcription (Biswas, 1990). Analysis of the time required for promoter recognition, initiation, and subsequent elongation has shown that promoter recognition and first bond formation takes at least 10 times longer than elongation of the next 10 nucleotides (Biswas, 1992). In summary, the yeast mitochondrial FIG. 1 Transcription units in yeast mitochondria. The transcription units shown here are based upon the sequence published by de Zamaroczy and Bernardi (1986). tRNAs are depicted as lollipops @) with the one-letter amino acid designation. Open reading frames encoding exons or entire genes are shown as open boxes. Three open reading frames encoding potential proteins of unknown function are designated as ORFs. Hatched boxes denote maturase reading frames. Checkered boxes denote reading frames encoding homing endonucleases. The wavy box denotes an intronic open reading frame, the function of which is unknown. Arrows ( t ) indicate 5 ’ mRNA processing sites. Arrows ( t ) with an asterisk below indicate 3’ mRNA processing sites. Processing sites of RPMf , tRNAs, and rRNAs are not shown. The 5’ processing sites of COB, VARI, and ATP8IATP6 are from Bonitz er at. (1982), Zassenhaus et al. (1984). Cobon et al. (1982), and Simon and Faye (1984), respectively. The transcription units are ordered with respect to the clockwise circular map, the exception being the promoter of rRNAT, which lies between rRNAFand rRNAV on the opposite strand.

GENE EXPRESSION IN SACCHAROMYCES CEREVlSlAE

149 promoters are short sequences immediately adjacent to the site of transcription initiation. This arrangement is quite different from the - 10 TATAAT motif in Escherichia coli or the variable-distance TATA motif of nuclear yeast genes; it is more similar to the T-odd phage promoters. However, similarity to phage transcription does not stop at the promoter but extends to the polymerase.

6. RNA Polymerase Mitochondria1 RNA polymerase is composed of two subunits (Winkley et al., 1985; Schinkel et al., 1986)-a catalytic core subunit of 145 kDa (Kelly and Lehman, 1986;Ticho and Getz, 1988), and a promoterspecificity factor of 43 kDa (Schinkel et al., 1987b, 1988; Jang and Jaehning, 1991; Xu and Clayton, 1992; Riemen and Michaelis, 1993). It has been shown in in vitro reconstitution experiments that the specificity factor does not bind to promoter sequences independently but is required for the recognition of the promoter by the holoenzyme, much like the function of E. coli a factor. The genes for both the catalytic core subunit, RP042, and the specificity factor, M T F l , have been cloned and sequenced (Greenleaf et a f . , 1986; Kelly et al., 1986; Masters e f al., 1987; Lisowsky and Michaelis, 1988). Sequence comparisons have shown that the core polymerase has considerable similarity to the T-odd phage RNA polymerases (Masters et al., 1987), whereas the specificity factor has some limited homology to bacterial sigma factors (Jang and Jaehning, 1991).

C. Transcription Factor?

Another yeast mitochondrial protein, ABF2 (also named HM and scmtTFA), has been purified (Caron et al., 1979; Diffley and Stillman, 1988) and shown to stimulate yeast mitochondrial gene transcription three- to fourfold in vitro (Parisi et al., 1993). ABF2, first characterized as a “mitochondria] chromatin” protein similar to E. coli HU (Caron et al., 1979), is abundant enough to cover the entire mitochondrial genome at 15-bp intervals (Diffley and Stillman, 1991); however, it has a footprint of 2530 bp (Diffley and Stillman, 1992). The protein binds to DNA nonspecifically at most sequences but binds with higher affinity to a sequence adjacent to the promoter within an active origin of replication. The ability of ABF2 to wrap and bend DNA and unwind duplexes in the presence of topoisomerase suggests a role in facilitating transcription initiation (Diffley and Stillman, 1991, 1992; Fisher et af., 1992). The gene for this protein

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CAROL L. DIECKMANN AND ROBIN R. STAPLES

was cloned, and sequence analysis identified two HMG boxes, a motif found in a variety of nucleic acid binding proteins (Diffley and Stillman, 1991).

111. RNA Processing and Turnover A. tRNA and rRNA Processing and Modification

The yeast mitochondrial genome encodes 24 tRNA genes. Most of the genes are initially transcribed as multigenic RNAs containing various combinations of tRNAs, rRNAs, mRNAs, and the RNA component of RNAse P (see Fig. 1). A comprehensive review of the processing and modification of mitochondrial tRNAs has been published recently (Hopper and Martin, 1992). Regardless of the organization of the initial transcript, all precursor tRNAs have 5' and 3' extensions. The tRNA precursors are processed at the 5' end by mitochondrial RNase P (Martin et af., 1985) and at the 3' end by a specific endonuclease (Chen and Martin, 1988). RNAse P is made up of a mitochondrially encoded RNA of 490 nucleotides (Miller and Martin, 1983) and a nuclearly encoded 105-kDa protein (Morales et al., 1992). A disruption of the gene encoding the protein subunit, RPA42, results in the accumulation of mitochondrial tRNAs with unprocessed 5' extensions (Morales et al., 1992; Dang and Martin, 1993). The gene encoding the RNA has been studied extensively (Hopper and Martin, 1992) and has recently been renamed R P M l . The RNA is cotranscribed with the upstream tRNAf-metand the downstream tRNAPro(Biswas, 1991). The 5' end of the RNase P RNA is formed by the 3' tRNA endonuclease, and the 3' end is formed by RNAse P cleavage at the 5' end of tRNAP'". Therefore the RNA component of RNAse P is released from the precursor in part by RNAse P. After cleavage of the 3' extension of the tRNA by a specific 3' endonuclease (Chen and Martin, 1988), CCA is added to the 3' end of the tRNA by an ATP(CTP):tRNA-specific nucleotidyl transferase (Aebi et af., 1990; Chen et al., 1990, 1992). This mitochondrial enzyme is encoded by the same gene that encodes the cytoplasmic enzyme. Differential use of three in-frame AUGs at the beginning of the coding sequence results in the production of longer polypeptides, which are localized to the mitochondria, and shorter polypeptides, which remain in the cytoplasm because they lack the amino-terminal extension that acts as the mitochondrial targeting sequence. Modification of nucleotides within tRNAs occurs both in the nucleus/ cytoplasm and in the mitochondria. Similar to the CCA-addition enzyme, two enzymes, both of which modify tRNAs in the nucleuslcytoplasm and

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151

the mitochondria, are encoded by single genes. MOD5 encodes an enzyme that results in isopentenyl modification of the A that is one nucleotide 3' of the anticodon (i6A) (Laten et al., 1978; Martin and Hopper, 1982). TRMZ encodes an enzyme that dimethylates the G at position 26 of tRNAs (m:G) (Phillips and Kjellin-Strlby, 1967; Hopper et al., 1982). In both cases the enzymes with the longer N-terminal peptide extensions are preferentially localized to the mitochondria (Ellis et al., 1989; Li et al., 1989; Gillman et al., 1991; Slusher et al., 1991). Unlike mutations in RNase P and the CCA addition enzyme, mutations in MOD5 and TRMl do not result in respiratory deficiency. The only phenotype of trmZ mutant strains is the lack of tRNA methylation (Phillips and Kjellin-Strgby, 1967), whereas mod5 strains have attenuated effectiveness of nonsense suppressor tRNAs (Laten er al., 1978). Recently, PET56 has been shown to code for a ribose methylase that modifies G2251 (E. coli numbering system) in the peptidyl transferase center of the large ribosomal RNA (Struhl, 1985; K. Sirum-Connolly and T. L. Mason, personal communication). B. 5'-End Processing of mRNA

Four of the seven mRNAs encoding major components in yeast mitochondria arise from larger precursor RNAs that require 5' processing events (see Table I for nomenclature of yeast mitochondria1 genes and Fig. 1 for 5' and 3' mRNA processing sites). COB mRNA is initially cotranscribed with the upstream tRNAg'". Processing of the 3' end of the tRNA at position - 1098 relative to the COB AUG generates a precursor to the mature mRNA. The precursor is processed further to generate the mature TABLE I Nomenclature of Major Polypeptide Genes

Original gene name

Protein

Common usage ~~

cob

oxi3 oxil oxi2 oli2 aapl olil uarl

COB COXJ cox2 COX3 A TP6 ATP8 ATP9 VARI

~

Apocytochrome b Cytochrome oxidase subunit I Cytochrome oxidase subunit I1 Cytochrome oxidase subunit I11 ATP synthase subunit 6 ATP synthase subunit 8 ATP synthase subunit 9 Small subunit ribosomal protein

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CAROL L. DIECKMANN AND ROBIN R. STAPLES

5’ end of the mRNA at -954. The nuclear gene product CBPl has been implicated in facilitating this cleavage. Mutations in the CBPl gene result in little or no cleavage at the -954 site though precursor RNAs are present, albeit at lower levels than in the wild type (Mittelmeier and Dieckmann, 1993; Staples and Dieckmann, 1993). ATP8 and ATP6 are cotranscribed with the upstream COXZ mRNA. Processing of the 3’ end of the COX1 mRNA at a conserved dodecamer sequence (see later discussion on 3’ mRNA processing) generates a precursor RNA for the downstream ATP8-ATP6 cotranscript (both proteins are translated from the same mRNA). The precursor is further cleaved about 200 nucleotides upstream of the ATP8 AUG to form the mature 5’ end of the mRNA (Cobon et al., 1982; Simon and Faye, 1984). Like CBPl, nuclear gene products may be involved in stabilizing the longer precursor, since it has been found that a cold-sensitive pet mutant has lower levels of the premRNA (Pelissier et al., 1992). VARl mRNA encodes a small subunit ribosomal protein and is initially cotranscribed with the upstream ATP9 mRNA and tRNA”‘. As with COB mRNA, 3’ processing of the upstream tRNA generates an mRNA precursor with an extended 5’ end. Cleavage at - 162 generates the mature 5’ end of VARl mRNA (Zassenhaus et al., 1984). To date, no nuclearly encoded factors have been implicated in this cleavage; however, a 1-bp transversion of an A to a T at - 109 completely blocks processing at the - 162 site (Smooker et al., 1988). The strain is respiratory deficient because the 5‘-extended RNA cannot be translated (Murphy et d . , 1980). Perhaps a nuclear factor cannot bind to the mutated leader to promote processing and/or translation. COX3 is also cotranscribed with an upstream tRNA, tRNAVa’.Both the cleavage at the 3’ end of the tRNA (at -614) and a cleavage at -489 have been reported (Thalenfeld et al., 1983).

C. 3’-End Processing of mRNA The seven major mRNAs and the o transcript are processed near a conserved dodecamer sequence, 5‘ AAUAAUAUUCUU 3’ (Thalenfeld et al., 1983; Osinga et al., 1984) (see Fig. 1). Recently a complex of three proteins has been shown to cross-link to RNA oligomers containing this sequence and to protect the 3’ end of the mRNA from a 3’ to 5’ exoribonuclease (Min and Zassenhaus, 1993). Perhaps the complex marks the site for cleavage by a specific endonuclease and/or protects pre-mRNAs and mRNAs from the 3’ to 5’ turnover enzyme.

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153

0 . Splicing Group I and I1 intervening sequences have different conserved secondary structures (Michel et al., 1982). These two types of introns have been shown to have different mechanisms of excision (Saldanha et al., 1993). For group I organellar introns, which are similar to the nuclear Tetrahymenu rRNA intron, the first of two transesterifications are initiated by nucleophilic attack of the 3’OH of a free guanosine nucleotide (Cech, 1990). Group I1 intron excision is more similar to nuclear intron splicing in that a 2’OH of a nucleotide within the intron is the nucleophile in the first transesterification reaction ( Jacquier, 1990). Three yeast mitochondria1 genes--21s rRNA, COB, and COXl-are split by introns (see Fig. 1). The large ribosomal RNA precursor contains an optional group I intron, w. An open reading frame within this intron encodes an HO-like endonuclease that functions in biased transfer of the intron to most progeny of a cross of a strain without the intron, w - , to one with it, w + (Lambowitz and Belfort, 1993). The gene coding for apocytochrome b, COB, is split by a variable number of introns. The most common configurationsare the “long” strains with five introns, bI1 through bI5, and the “short” strains that only contain b14 and bI5. The first (group 11) and fifth (group I) introns have no coding sequences within them, whereas the middle three (group I) all have open reading frames that are in-frame with the upstream exons. Each of these intronic reading frames codes for a maturase which is required for excision of the encoding intron. Initially, the first and second exons are joined in a reaction that does not require a maturase. The joined exons are then translated as a fusion protein with the downstream adjoining maturase sequence in intron bI2. The maturase aids the excision of bI2, and then the RNA can be translated again as a fusion of exons 1, 2, and 3 to the maturase in bI3. The process is sequentially repeated for removal of b13 and b14. Therefore, blockage of excision of intron 1 by a cis-dominant point mutation also blocks excision of intron 2 because the b12 maturase cannot be translated. The gene coding for the largest subunit of cytochrome oxidase, COXl, is interrupted by seven introns in ‘‘long’’ strains (Hensgens et al., 1983), a11 through aI5a (also named aI5a or aI5), aI5P (aI5b or aI6), and aI5y (aI5c or aI7). “Short” strains are missing aI5a and a15P (Bonitz et al., 1980). Like bI1, a157 is a group I1 intron without an internal open reading frame. a11 and a12 are also group I1 introns; however, each has a maturase encoded within the intron (Carignani et al., 1983, 1986). More recently the proteins encoded in these two introns have been shown to have reverse transcriptase activity (Kennel1et al., 1993).The intron-encoded activities

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CAROL L. DIECKMANN AND ROBIN R. STAPLES

are required generally for both group I and group I1 intron deletion from the genome (Levra-Juillet et al., 1989),and specificallyfor biased transmission of a11 and a12 (Meunier et al., 1990). The a14 group I intron reading frame codes for an inactive maturase that can be activated by a point mutation (Dujardin et al., 1982).In wild-type strains, the maturase encoded by COB b14 is required for the excision of COXl a14. The aI4-encoded protein does, however, act as a “homing” endonuclease very similar to that of the w intron in the large ribosomal RNA (Wenzlau et al., 1989; Delahodde et al., 1989; Wernette et al., 1990, 1992; Sargueil et al., 1990). a13 and aI5a are group I introns that also have been shown to be active in biased intron transmission (Sargueil et al., 1991; Moran et al., 1992; Perea et al., 1993). aI5P has an open reading frame that, like the w reading frame, is not fused to the upstream exon. As yet, neither maturase nor endonuclease function has been proposed for this coding sequence. Besides the maturases, many nuclear gene products have been identified by mutations that affect mitochondrial intron splicing (see Table 11).These proteins fall into two categories-those that are only required for intron splicing and those that have additional functions such as promoting translation. The diagnostic test for categorization is to determine whether mutations in the nuclear gene are suppressed by introducing an intronless mitochondrial genome into the strain (Seraphin et al., 1987). Only CBP2, MRS1, and PET54 fall into the first category. CBP2 is a 76,000-Da protein that is encoded in the nucleus, transported into the mitochondria, and that facilitates the splicing of the group I bI5 intron (McGraw and Tzagoloff, 1983). A respiratory-competent mitochondrial revertant of a cbp2 mutant lacks bI5 (Hill et al., 1985). CBP2 appears then to be involved only in splicing of this one intron. That CBP2 is sufficient for splicing bI5 is suggested by the enhancement of splicing in uitro by the purified protein under physiological conditions (Gampel et al., 1989; Gampel and Cech, 1991). Since this intron can also self-splice in uitro in the presence of 50 mM magnesium ion (Gampel and Tzagoloff, 19871, the protein is presumed to aid in folding the RNA into a conformation active for splicing. The MRSl (PET157) gene product, identified by nuclear pet mutations (Pillar et al., 1983; McEwen et al., 1986), is required for the excision of b13 and aI5P (Kreike et al., 1986, 1987; Bouquet et al., 1990). Since b13 also encodes a required maturase (Lazowska et al., 1989), at least two proteins are needed for the excision of b13. Like CBP2, MRSl is not required for other mitochondrial functions. The third protein in this category is PET54. Although PET54 does have an additional function in initiating translation of COX3 mRNA, the inability of pet54 mutants to express COXl is suppressed by the absence of aI5P in the strain (Valencik et al., 1989). In addition to CBP2 and MRS1, there

155

GENE EXPRESSION IN SACCHAROMYCES CEREVlSlAE TABLE II SplicinglPleiotropicFactors ~

Mutation suppressed by A introns

Nuclear gene

Mitochondria1 intron

CBP2 MRSl

suv3

COB bI5 COB b13 COX1 aI5P COXl aI5p COB, COXl introns COXl aI5p

MSS18 NAMI

COXl aI5P COXl introns

NAM2

COB b14 COXl a14

No

MRS2

COB bI1 COXl a1I , aI2, aI5y

No

PET54 MSSl16

Other function(s)

Yes Yes

No No

Yesb No

Yesb Translation of COXl, COX3, or ATP6 Excised intron turnover Post-translational Transcription, translation General translation (leucyl tRNA syn.) COX2 translation aln3 spectrum

No Not completely

No

References'

(1) McGraw and Tzagoloff, 1983; Hill et al., 1985; Gampel and Tzagoloff, 1987; Gampel et a / . , 1989; Gampel and Cech, 1991. (2) Kreike et a / . , 1986, 1987; Bousquet er al., 1990. (3) Costanzo et al., 1986, 1989; Valencik ef a / . , 1989; Valencik and McEwen, 1991; Burke and McEwen, 1991. (4) Seraphin et al., 1989. ( 5 ) Conrad-Webb et al., 1990; Stepien et al., 1992. (6) Sdraphin et a / . , 1988. (7) Ben Asher et a / . , 1989; Lisowsky and Michaelis, 1989; Lisowsky, 1990; Lisowsky et al., 1990. (8) Labouesse, 1990; Labouesse et a / . , 1987; Zagorski et a / . , 1991; Li e t a / . , 1992. (9) Koll et a / . , 1987; Wiesenberger et al., 1992. The splicing defect is suppressed by intronless mitochondrial DNA, but PET 54 is required for COX3 translation in an intronless strain.

may be several additional splicing factors that do not affect mitochondrial function in other ways. Eleven complementation groups of pet mutants that are rescued by introducing an intronless mitochondrial genome have been identified (Seraphin et al., 1987). Splicing factors in the second category have other functions in addition to their role in intron excision; however, most were first identified by their involvement in intron removal. MSS116 is a PET gene that encodes a protein with a sequence similar to RNA helicases (Straphin et al., 1989). Splicing of several of the COB and COXl introns is inhibited in mssll6 mutant strains, but respiration is not restored by introducing intronless

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CAROL L. DIECKMANN AND ROBIN R. STAPLES

mtDNA. A decrease in labeling of the mitochondrial proteins COXI and either COX111 or ATP6 is seen in a msslZ6 strain without introns, suggesting an additional role in translation. SUV3 is also a PET gene that encodes a putative helicase with similarity to MSS116 (Stepien et al., 1992). The mutation SUV3-1 affects splicing, particularly of the aI5P intron (Conrad-Webb et al., 1990); however, the strain also accumulates excised group I introns, which is suggestive of a defect in RNA turnover. In addition, the strain has lower steady-state levels of COB mRNA, though COB mRNA splicing is not impaired. That this protein has other functions is suggested by the fact that the disruption mutant loses mitochondrial DNA (Stepien et al., 1992). This is a phenotype common to mutant strains defective in DNA replication (Foury , 1989), transcription (Greenleaf et al., 1986), or the general translation machinery (Myers et al., 1985). A disruption of the MSS18 gene in a strain containing aI5P results in respiratory incompetence on lactate medium. An mssl8 strain lacking the intron can grow on lactate, though at a rate 50% slower than the isogenic MSS18 wild-type strain (SCraphin et al., 1988). Thus, lack of this protein may partially inhibit some other mitochondrial function. Most of the factors described above were first identified as pet mutants; however, several other pleiotropic nuclear factors have been identified by selecting for high-copy suppression of point mutations in the mitochondrial genome that block splicing. Four PET genes have been identified in this way. In namZ mutant strains, COXZ intron splicing is deficient and no COXI subunit is synthesized. All other proteins seem to be translated but at a level 5-20% that of the wild type (Ben Asher et al., 1989). The mutant strain loses mtDNA, again suggesting a general role in replication, transcription, or translation. Indeed the NAMl gene was also cloned by rescue of a temperature-sensitive mutation that leads to a severe decrease in transcription of mtDNA (Lisowsky, 1990; Lisowsky et al., 1990). NAM2, which encodes mitochondrial leucyl tRNA synthetase, has also been implicated in splicing, particularly of b14 and a14 (Labouesse et al., 1987; Labouesse, 1990; Zagorski et al., 1991; Li et al., 1992). Like many other synthetases, deletion of the gene leads to loss of mtDNA. One nam2 mutant retains 50% wild-type synthetase activity but is almost completely defective in splicing, and the splicing defect cannot be suppressed by importing b14 maturase from the cytoplasm. Therefore, it is hypothesized that the tRNA charging and splicing functions are separate, as they have been shown to be in Neurospora crassa mitochondrial tyrosyl tRNA synthetase (Cherniack et al., 1990; Kittle et al., 1991). However, in a nam2 intronless strain, COX2 mRNA is not translated, and translation of all other mitochondrial proteins is depressed (Labouesse, 1990). Thus, the protein may have a third function, namely, initiation of translation.

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157

MRS2 is also essential for mitochondrial function. Isolated first as a high-copy suppressor of a group I1 intron mutation (Koll et af., 1987), it is not suprising that the mrs2 disruption strain is defective in excision of all the group I1 introns. However, like the aforementioned factors, introducing intronless mtDNA does not rescue mrs2 mutants. Such strains translate all of the mitochondrial proteins to about 50% of the wild type, but there is no cytochrome ala3 peak in the spectrum and thus no growth on glycerol medium (Wiesenberger et af., 1992). This protein then must have some function other than enhancing splicing of group I1 introns-presumably a post-translational or assembly role. NAM7 was isolated as a high-copy suppressor of an intron mutation. It can suppress mutations in both group I and group I1 introns. Like MSSll6 and SUV3, NAM7 has homology to helicases. Only partial respiratory deficiency is observed for a disruption of the NAM7 gene and the protein does not exhibit an N-terminal targeting signal for the mitochondria. However, the phenotype of the disrupted strain cannot be alleviated by introducing intronless mtDNA, indicating that this protein functions in other critical mitochondrial processes (Altamura et af., 1992). When in high copy, MRS3, MRSI, and NAM8 suppress intron mutations, but disruptions in these three genes have no effect on respiration. MRS3 and MRS4 are 73% identical at the amino acid level and have considerable similarity to the ADP/ATP carrier protein. Overexpression of these two proteins may suppress the bI1 mutation by raising the intramitochondrial concentration of cations (Schmidt et al., 1987; Wiesenberger et al., 1991). Indeed these are mitochondrial proteins, but in wild-type strains they probably have no role in splicing. The NAM8 gene encodes a protein with homology to snRNP RNA-binding domains (Ekwall et af., 1992). This protein does not have a mitochondria1 transit peptide at the N-terminal, but can suppress both group I and group I1 cis-dominant mutations. Perhaps this is normally a nuclear or cytoplasmic protein that when overproduced is transported into mitochondria at a frequency that can suppress the splicing mutations. In addition to these three genes, 10 others have been isolated from a high copy library made from a strain deficient in MRS2, MRS3, and MRS4. Since MRS2 is essential for respiration, these suppressors must suppress the two mrs2 phenotypes, the inability to splice group I1 introns, and the post-translational defect (see earlier discussion). The library was used in two ways: (1) transformation of an intronless strain to obtain suppressors of the nonintron-related function and (2) transformation of a strain with introns to obtain suppressors of the intron-excision defect. Five of the 10 genes can suppress the defect in intronless strains but not in introncontaining strains. The other five genes can suppress the intron-excision

158

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L. DIECKMANN AND ROBIN R. STAPLES

defect as well as the post-translational defect (Waldherr et al., 1993). It is not yet known whether these genes are PET genes, code for non-PET mitochondrial proteins or, possibly, are wayward cytoplasmic factors that when overexpressed find themselves at some frequency in the mitochondrial compartment. It is tempting to try to find a universal explanation for the pleiotropic nature of many of the genes described in this section. The RNA helicases MSSll6, NAM7, and SUV3, and the RNA-binding protein NAM8 may help stabilize or remodel secondary structures in pre-mRNA required for splicing and/or translation. Similarly, the role of NAM1, NAM2, and MRS2 may be to interact with pre-mRNA to stabilize conformations favorable for splicing. E. RNA Turnover

There are two classes of nuclearly encoded proteins that affect mitochondrial RNA stability and turnover. The first class includes general factors that could affect all mitochondrial transcripts, while the second class includes proteins that affect the stability of individual mitochondrial mRNAs. Two enzymes that attack single-stranded RNA have been isolated from yeast mitochondria. In uitro, the NUCl nuclease can digest doublestranded and single-stranded DNA as well as RNA; however, the activity is only seen after detergent treatment of mitochondrial membranes (Dake et al., 1988). Disruption of the NUCl gene does not cause respiratory deficiency (Zassenhaus et al., 1988). Thus, it may not be important for mitochondrial RNA turnover, or its function can be replaced by another enzyme. Recently, a 3’ to 5’ exoribonuclease has been purified from mitochondrial membranes of nucl mutant cells (Min et al., 1993). This enzyme was purified to homogeneity and contains three proteins. Its role in mitochondrial RNA turnover will be evaluated when mutant strains become available. There are now three nuclear genes that encode factors that stabilize individual mitochondrial mRNAs-NCAZ, CBPZ, and PET309. The NCAl gene product has been shown to affect the stability of ATP9 mRNA (Ziaja et al., 1993). The ATP9 message in the ncal mutant strain is undetectable by Northern blotting, but ATP9 is transcribed at the same rate in mutant and wild-type strains. The site at which NCAl may interact with ATP9 mRNA to promote stability is as yet undetermined. The CBPl protein interacts with the 5’ end of COB transcripts. CBPl protects precursor RNAs with 5’ extensions to position - 1098 (the 3‘ end of the cotranscribed tRNA) (Staples and Dieckmann, 1993). The inter-

GENE EXPRESSION IN SACCHAROMYCES CEREVlSlAE

159

action site has been mapped to a region between -948 and -938, just downstream of the - 954 5' end of mature COB mRNAs (Mittelmeier and Dieckmann, 1993). Substitution of the CCG in this interval to AAT results in a cbpZ phenotype, that is, no stable COB mRNA (W. Chen and C. L. Dieckmann, unpublished data). One hypothesis for the action of CBPl was that it might create stable mRNA from unstable precursors by cleaving the RNA at -954, which would allow the RNA to fold into a nucleaseresistant structure. When the sequence between the 3'end of the tRNA and the -954 processing site is deleted, the 5' end of COB mRNA is formed by the endonuclease that matures the 3' ends of tRNAs. COB mRNA in such a strain is still dependent on CBPl for stability, suggesting a role for CBPl in stability of the mRNA after 5' processing (W. Chen and C. L. Dieckmann, unpublished data). The PET309 gene product is necessary for the stability of COXl transcripts that contain introns. In pet309 strains containing intronless mitochondrial DNA, COXl mRNA is detectable by Northern blotting but is in decreased abundance with respect to the wild type. Despite measurable amounts of COXl mRNA in the intronless strain, no protein is translated in pulse-labeling studies, suggesting a defect in translation of the mRNA as well as protection of precursor RNAs containing introns (G. M. Manthey and J. E. McEwen, personal communication). PET309 may indeed have two functions-an association with introns that protects against nuclease attack, and a translation initiation function. Perhaps the role of PET309 in translation initiation also protects the mRNA from degradation. As more becomes known about the functions of NCAl, CBP1, and PET309, these factors may become a subset of the message-specific translation factors described in the next section. Indeed, disruptions of the genes encoding translation factors AEP2 (needed for ATP9 mRNA translation) and PET1 11 (needed for COX2 translation) cause decreased mRNA levels (Payne et al., 1991; Ackerman et al., 1991; Poutre and Fox, 1987), a phenotype similar to that of the pet309 intronless strain. However, disruptions in several other translation factor genes (PET494, PET54, and PET122 are all needed for translation of COX3 mRNA) have no effect on the steady-state levels of the mRNA (Costanzo et al., 1986; Ohmen et al., 1988; Kloeckener-Gruissem et al., 1988). IV. Translation

Several nuclearly encoded factors that are necessary for the translation of individual mitochondria1 messenger RNAs have been characterized (see Table 111). The hypothesis that these factors interact with the 5'

CAROL

160

L. DIECKMANN AND ROBIN R. STAPLES

TABLE 111 Translation Factors

Nuclear gene

Mitochondria1 gene target

CBSJ CBS2(CBP7) CBP6 AEPJ AEP2 PETJ I J PET494 PET54 PET122 MSS5 J PET309

COB COB COB ATP9 ATP9 cox2 COX3 COX3 COX3 COXJ COXJ

Suppressed by 5'UTR substitution

Reference'

Yes Yes No

2

?

4

1

3

?

5

Yes Yes Yesb Yes No

6

10

?

11

7

8 9

' (1) Rodel et al., 1985, 1986; Rodel, 1986; Rodel and Fox, 1987; Korte et a / . , 1989; Michaelis et a / . , 1991. (2) Rodel, 1986; Rodel et a / . , 1986; Muroff and Tzagoloff, 1990; Michaelis and Rodel, 1990; Michaelis e t a / . , 1991. (3) Dieckmann and Tzagoloff, 1985. (4) Payne et al., 1991. (5) Payne e t a / . , 1991; Finnegan et a/., 1991; Ackerman e t a / . , 1991. (6) Poutre and Fox, 1987; Strick and Fox, 1987; Mulero and Fox, 1993. (7) Miiller et a/., 1984; Costanzo and Fox, 1986; Costanzo et a/., 1986. (8) Costanzo et al., 1986, 1989; Valencik et al., 1989; Valencik and McEwen, 1991; Burke and McEwen, 1991. (9) Kloeckener-Gruissem et a/., 1988; Ohmen et a/., 1988; H a t e r et a / . , 1990, 1991; Haffter and Fox, 1992; McMullin et al., 1990; Costanzo and Fox, 1993. (10) Faye and Simon, 1983; Decoster et a / . , 1990. ( I 1) G. M. Manthey and J. E. McEwen, personal communication, 1994. In short form, COXJ strain.

untranslated leaders (5'UTRs) of the mRNAs to promote initiation of translation is supported by the finding that mutations in the nuclear genes can be suppressed by mtDNA rearrangements that fuse a different 5' untranslated leader to the coding sequence of the target gene. For example, the PET1 11 protein is required for the translation of COX2 mRNA. per1 11 mutations can be suppressed by a fusion of the COX3 5'UTR to the COX2 coding sequence (Mulero and Fox, 1993). The COX3-COX2 fusion, however, requires the COX3 translation factors. The products of CBSI and CBS2 are required for translation of the apocytochrome b mRNA. cbsl and cbs2 mutations can be suppressed by a fusion of the ATP9 5'UTR

GENE EXPRESSION IN SACCHAROMYCES CEREVlSlAE

161

to the COB coding sequence (Rodel ef al., 1985; Rodel, 1986), and fusions of the COB 5’UTR to other genes require CBSl function (Rodel and Fox, 1987). Since COB is most commonly split by several introns containing maturases, disruptions in CBSl and CBS2 have a secondary effect on splicing (Rodel, 1986; Muroff and Tzagoloff, 1990). The CBSl and CBS2 proteins have been found to be localized to the mitochondrial inner membrane (Korte et al., 1991; Michaelis et al., 1991; Michaelis and Rodel, 1990). PET494, PET54, and PET122 are required for the translation of COX3 mRNA, and mutations in each of the three can be suppressed by rearrangements that fuse 5’UTRs from other mitochondrial genes to the COX3 coding sequence (Miiller et al., 1984; Costanzo et al., 1986, 1987). As mentioned in the discussion of splicing, PET54 is required for splicing aI5P (Valencik et al., 1989; Valencik and McEwen, 1991); thus pet54 mutations can only be suppressed by 5’UTR rearrangements in “short” strains lacking aISP. The working hypothesis for the function of the translation factors is that they bind to the 5’UTR of the mRNA and act as message-specific translation factors. In support of direct interaction between mRNA and protein, deletions in the COX3 leader causing cold-sensitive respiration can be suppressed by a missense mutation in PET122 (Costanzo and Fox, 1993). That leader-specific proteins in turn interact with the ribosomes to promote initiation of translation is supported by the acquisition of suppressor mutations in several mitochondrial small subunit ribosomal proteins (PET123, M R P l , MRPl7) that suppress a carboxyl terminal truncation of PET122 (Haffter et al., 1990, 1991; McMullin et al., 1990; Haffter and Fox, 1992). That the three proteins needed for translation of COX3 mRNA might form a complex with each other is supported by the finding that PET54 interacts with PET122 and PET494 (N. G. Brown, M. C. Costanzo, and T. D. Fox, personal communication) in the “two-hybrid” system developed to identify protein-protein interactions (Fields and Song, 1989; Chien et al., 1991). A second class of translation factors in which defects cannot be suppressed by 5’UTR swapping, includes CBP6 and MSS51. CBP6 is required for translation of COB, but mutations in the gene cannot be suppressed by the gene rearrangement that suppresses cbsl and cbs2 mutations (Dieckmann and Tzagoloff, 1985). cbp6 mutations also cause a deficiency in succinate dehydrogenase activity (B. D. Lemire, personal communication). Perhaps CBP6 has a second function in posttranslational modification of one or more imported subunits of the succinate dehydrogenase complex. A second function would explain why cbp6 mutations cannot be suppressed by leader rearrangements. MSSS 1 was originally thought to be a splicing factor because mutations in the gene inhibited splicing of

162

CAROL L. DIECKMANN AND ROBIN R. STAPLES

most of the COX1 introns (Faye and Simon, 1983). However, the effect on splicing has been shown to be a by-product of the abolition of translation that inhibits production of the all and a12 maturases (Decoster et al., 1990). rnss51 mutations, like cbp6 mutations, have not been shown to be suppressed by gene rearrangements, although attempts to aquire such rearrangements have been extensive (Decoster et al., 1990). Perhaps MSS5 1 makes contacts with the coding portions of the mRNA that cannot be substituted while retaining the function of the COX1 subunit. Alternatively, MSSS 1 may have some as-yet-unidentified function other than promoting translation of COX1 mRNA. Two factors have recently been described that are necessary for the translation of ATP9. It has not been reported whether mutations in AEPl and AEP2 (ATP13)can be suppressed by a heterologous S’UTR (Payne et al., 1991; Finnegan et al., 1991; Ackerman et al., 1991). What do the translation factors do, and why are they message-specific? As discussed earlier, one hypothesis is that the translation factors recognize particular sequences or structures in the S’UTR and facilitate the association of mRNA and ribosomes. It is not clear why different factors have coevolved with separate 5’ UTRs. However, separate inititiation systems for each mRNA would allow the translation rate of each mRNA to be controlled independently. Selection for such a system could be brought about by the need to regulate the levels of individual subunits strictly and appropriately. An alternative but not exclusive hypothesis is based on the finding that several of the translation factors are membrane bound (Michaelis et al., 1991; McMullin and Fox, 1993). If the factors themselves are organized in some fashion in the membrane, that organization could dictate the insertion of newly synthesized polypeptides in a pattern that would facilitate assembly into the large respiratory chain complexes.

V. Post-translational Factors

Several nuclear gene products have been described that are necessary for post-translational modification or assembly of the respiratory chain complexes (see Table IV). CYC3 codes for cytochrome c lyase (Dumont ef al., 1987) and CYT2 for cytochrome c1 lyase (Zollner et al., 19921, enzymes that attach heme to their respective cytochromes. From the characterization of the BCSZ gene, it appears that this factor may have a role in the insertion of the iron-sulfur center into the Rieske subunit of coenzyme QH2reductase (Nobrega et al., 1992).Alternatively, this protein may aid in the assembly of the Rieske subunit with the other components

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163

TABLE IV Post-translational Factors

Nuclear gene ABCl BCSl CBP3 CBP4 PET117 PET191 cox10 cox1I SCOI ATPIO ATPll ATPl2 c YC3 CI12

Proteinkomplex coQ reductase coQ reductase (Rieske) coQ reductase coQ reductase Cytochrome oxidase Cytochrome oxidase Cytochrome oxidase Cytochrome oxidase Cytochrome oxidase ATP synthase F, ATP synthase F, ATP synthase F, Cytochrome c lyase Cytochrome cl lyase

Reference9 1

2 3 4 5

6 7

8

9 10 11

12 13 14

(1) Bousquet er al., 1 9 9 1 . (2) Nobrega er al., 1992. (3) Wu and Tzagoloff, 1989. (4) Crivellone, 1993. (5) McEwen er al., 1993. (6) McEwen er al., 1993. (7) Nobrega el al., 1990. (8) Tzagoloff er al., 1990. (9) Schulze and Rodel, 1988, 1989; Krummeck and Rodel, 1990. (10) Ackerman and Tzagoloff, 1990a. (1 1) Ackerman and Tzagoloff, 1990b; Ackerman er al., 1992. (12) Ackerman and Tzagoloff, 1990b; Bowman er al., 1991. (13) Dumont er al., 1987. (14) Zollner er al., 1992.

of the complex. abcl mutant strains are defective in electron transfer through the bc, complex, though all of the cytochromes are present (Bousquet et al., 1991). ABCl was first isolated as a high-copy suppressor of a cbs2 translation mutant, so ABCl may have an additional noncritical role in the translation of COB. Many of the proteins in the post-translational category appear to have some function in the assembly of the large inner membrane enzyme complexes involved in electron transfer and ATP synthesis. ATPll and ATP12 code for factors necessary for the assembly of the F, portion of ATP synthase. When a strain is defective for either of these factors, the (Y and p subunits of the F, form large aggregates that are nonfunctional (Ackerman and Tzagoloff, 1990b). It is hypothesized that ATPll and ATP12, which are not themselves part of the ATPase, act as complex-specific chaperones, perhaps keeping the newly imported subunits from aggregat-

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ing before they can be properly assembled with the other subunits. Similarly, ATPlO is necessary for the assembly of the Fo membrane portion of the enzyme (Ackerman and Tzagoloff, 1990a). COX10 and COX11 are thought to act in the assembly of cytochrome oxidase (Nobrega et al., 1990; Tzagoloff et al., 1990). However, instead of the mutations causing aggregation of unassembled subunits, the cox10 and cox1 1 mutant strains contain partially assembled complexes, with the nuclearly encoded subunits 4 and 5 in a low-molecular-weight fraction. Interestingly, homologs of the genes for these two proteins are found in a cytochrome oxidase operon of the bacterium Paracoccus denitriJicans, which suggests that the assembly function of these polypeptides has been conserved during evolution. SCOl first appeared to be required for the translation of COX1 and COX2 mRNAs (Schulze and Rodel, 1988, 1989). However, after a careful examination of '%met labeling of mitochondrial translation products in a pulse-chase experiment, it was shown that COX1 and COX11 are indeed synthesized, but decay very rapidly (Krummeck and Rodel, 1990).Therefore, it is thought that the SCOl function is needed directly after protein synthesis, perhaps to stabilize the individual protein subunits and/or to enhance their assembly into the cytochrome oxidase complex. Recently, two other proteins encoded by PET117 and PET191 have been suggested to have similar assembly-enhancing functions for cytochrome oxidase (McEwen et al., 1993). Complex 111, coenzyme QH2reductase, also requires proteins for assembly. Mutations in CBP3 and CBP4 result in only partial assembly of the complex (Wu and Tzagoloff, 1989; Crivellone, 1993). The function of many of the assembly factors may be to act downstream of mitochondrial hsp60. The heat-shock protein facilitates folding of proteins newly transported from the cytoplasm and then the assembly factors may ensure that the interactive surfaces of individual polypeptides are masked until the proper binding partner is found. These proteins may be needed in the intricate assembly pathway for the inner membrane complexes.

VI. Regulation

What do we mean by regulation of mitochondrial function? We will define it here as the processes that dictate the increase in mitochondrial mass per cell when yeast cells are switched from a fermentative to a respiratory environment. Though a great deal of progress has been made in understanding how nuclear genes coding for components of the respiratory chain are regulated (Forsburg and Guarente, 1989; Zitomer and Lowry,

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1992), much less is known about how mitochondrial gene expression is regulated during these switches. There is some evidence for regulation by increasing the steady-state levels or translation of the mRNAs encoding nuclear factors that directly affect mitochondrial gene expression. Regulated factors include both general factors such as RNA polymerase, ribosomal proteins, and tRNA synthetases, and specific factors such as PET494 and CBPl . As shown by Mueller and Getz (1986b), the steady-state levels of most mitochondrial RNAs increase about 5-fold when yeast cells are grown on a carbon source that requires mitochondrial function compared with growth on glucose medium. This increase in the steady-state level of RNAs could be due to an increase in the rate of transcription and/or a decrease in the rate of RNA turnover. We have investigated how switching from fermentable to nonfermentable medium affects the expression of COB. COB mRNA levels increased 8- to 10-fold over the course of 12 hr, whereas mitochondrial DNA levels fluctuated slightly, but did not change significantly (R. R. Staples and C. L. Dieckmann, unpublished data). That the increase in transcript levels is due to an increase in transcription rate is suggested by Mueller and Getz’s (1986a) pulse-labeling study. One way to increase the rate would be to increase levels of polymerase. Increases in the levels of RP041 mRNA have been observed, but MTFI is expressed constitutively. Furthermore, the polymerase levels do not correlate with the levels of mitochondrial transcripts (T. L. Ulery and J. A. Jaehning, personal communication). Perhaps changes in the level of ABF2 or some other factor that decreases the time constant for promoter recognition and first bond formation is at work during the induction of mitochondrial function. The answer to this question awaits further developments in the analysis of mitochondrial transcription. Certainly an effective method of increasing the levels of mitochondrially encoded proteins is to increase the abundance of general translation machinery components. For every nuclearly encoded mitochondrial ribosomal protein that has been examined, the abundance of the mRNA for the protein or protein levels themselves has been shown to increase. This includes MRPl and MRP2 (Myers et al., 1987),MRP7 (Fearon and Mason, 1988), MRP13 (Partaledis and Mason, 1988), and MRP20 and MRP49 (Fearon and Mason, 1992). The abundance of mRNA for mitochondrial tRNA synthetases has also been reported to increase (Myers et al., 1987). In addition to the ribosomal proteins and tRNA synthetases, an abundant mitochondrial protein, p40, has been shown to bind to specific regions of the 5’UTRs of several mitochondrial mRNAs. The distance of these sites relative to the AUG of the individual mRNAs varies. The sites have no homology to each other but are adjacent to a conserved stem structure (Papadopoulou et al., 1990; Dekker et al., 1991, 1992). Recently it has

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been found that p40 is isocitrate dehydrogenase. Two homologous genes, ZDHl and ZDH2, encode the two polypeptides that make up the heterodimeric enzyme. Disruptions of either gene abolish the RNA-binding activity. The possibility for regulatory cross-talk between citric acid cycle enzyme levels or activity and the stability or rate of translation of the mitochondrial mRNAs is intriguing and awaits further investigation (S. D. J. Elzinga, P. J. T. Dekker, K. van Oosterum, A. Bednarz, and L. A. Grivell, personal communication). There is no evidence for modulation of the transcription rate for individual mitochondrial genes by trans-acting factors. However, as has been outlined in the sections of this review, there are many factors that affect the stability of specific mitochondrial mRNAs, the translation of the mRNAs, and modification and assembly of the encoded proteins. It is possible that the levels of mitochondrial gene products could be regulated in turn by regulation of the nuclear genes encoding these mitochondrial gene-specific factors. What follows is a discussion of a few investigations into the carbon-source regulation of these factors. Why do individual mitochondrial mRNAs have proteins that specifically affect their stability? One argument is that it might be a way to regulate the levels of protein produced. Analysis of a variety of t s cbpl and cob 5'UTR mutants has led to the conclusion that approximately 20% of wildtype COB mRNA is required for wild-type growth on nonfermentable carbon sources (Staples and Dieckmann, 1993; Mittelmeier and Dieckmann, 1993). Clearly, as much as a 5-fold decrease in COB mRNA is not rate limiting for growth. Recent studies show that overproduction of COX2 mRNA results in decreased respiration ( J . L. Pinkham, personal communication). We are curious whether overproduction of COB mRNA would also decrease respiration because the expression of CBPl is downregulated when cells are switched from fermentation to respiration (Mayer and Dieckmann, 1989, 1991). Perhaps this is a mechanism to modulate the levels of COB mRNA while increasing the levels of tRNAd" during the fivefold increase in transcription accompanying the switch to respiration. There are several hints that the message-specific translation factors may be rate limiting for growth on glycerol and therefore would be good candidates for control of protein production from mitochondrial mRNAs. The AUG start codons for COX2 (J. J. Mulero and T. D. Fox, personal communication) and COX3 (Folley and Fox, 1991) have been mutated to AUA. These changes result in a leaky respiratory-competent phenotype. When the abundance of the respective translation factors PETl 11 (COX2) and PET122 or PET494 (COX31 is reduced in a heterozygous PETlpet diploid strain, the AUA mutants do not grow as well as the homozygous PETIPET AUA strain on glycerol medium. This result suggests that the concentrations of PETl 11, PET122, and PET494 are limiting in the cell.

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The translation factor PET494 has been shown to increase in abundance when cells are switched to growth on nonfermentable media (Marykwas and Fox, 1989). Therefore, coupled with the finding that PET494 is rate limiting, the increased level of the PET494 protein during respiration may determine the upper limit of COX111 production. This may also be true for the other rate-limiting translation factors. Assembly of the mitochondrially encoded proteins into the respective enzyme complexes may be the ultimate level of control. Many factors could influence just how many correctly assembled enzymes function in the inner membrane. The abundance of the respiratory chain components that are encoded in the nucleus may be the primary means of controlling how many complexes assemble. As reviewed in Forsburg and Guarente (1989) and Zitomer and Lowry (19921, most of the nuclear genes coding for structural subunits of the large enzyme complexes are regulated at the level of transcription. Whichever component of each complex is of lowest abundance would then restrict the number of complete complexes. It is likely that an excess of subunits may be turned over rapidly, as it has been shown that some subunits are particularly sensitive to degradation if they are not assembled (Crivellone et al., 1988; Nobrega et al., 1990; Tzagoloff et al., 1990). Another way that assembly could be regulated is if the abundance of the complex-specific chaperones is altered. However, neither CBP3 (Wu and Tzagoloff, 1989) nor COX10 (Nobrega et al., 1990) is regulated with respect to carbon source.

VII. Concluding Remarks Our ultimate goal is to understand how mitochondrial gene expression is regulated. However, to do so we first have to understand the function of the nuclear PET genes. In this chapter we have compiled much of the newer information on nuclearly encoded factors that affect mitochondrial transcription; mRNA processing, stability, and translation; and posttranslational modification and assembly. The following paragraphs summarize our review; however we have omitted the citations for the sake of brevity. Transcription of yeast mitochondrial operons appears to be somewhat more complicated than transcription of phage genes but less complex than E. coli transcription. The 9-bp mitochondrial promoter is positioned immediately upstream of the start of transcription, which is reminiscent of the spacing of the polymerase recognition sequence in T3 and T7 phage genes. Though the mitochondrial enzyme contains two subunits, the catalytic subunit RP041 has considerable sequence similarity to the single

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subunit T-odd polymerases. The mitochondrial promoter specificity subunit MTFl has properties that mimic those of E. coli u factor in that it does not bind to DNA, but when assembled with the catalytic subunit allows promoter recognition by the holoenzyme. ABF2 is an abundant mitochondrial DNA-binding protein similar to E. coli HU, which stimulates transcription of mitochondrial genes in uitro and may aid in the initiation of transcription in uiuo by its ability to bend DNA and unwind it in the presence of topoisomerase. With the exception of one transcript, that for a second tRNAthr,mitochondrial transcription units are multigenic. In some cases the initial transcripts contain multiple tRNAs, in other cases combinations of mRNAs and tRNAs or rRNAs and tRNAs (see Fig. 1). Many processing events are needed to free the individual tRNAs and mRNAs before they can engage in translation. Mitochondria1RNAse P precisely cleaves precursor RNAs at the 5’ end of mitochondrial tRNAs. Following cleavage at the 3‘ end by a tRNA-specific endonuclease, a CCA-addition enzyme adds the residues critical for amino acylation. Two modification enzymes, encoded by MOD5 and T R M l , modify specific residues in both nuclear/ cytoplasmic and mitochondrial tRNAs. Methylation of a single ribose in the large ribosomal RNA peptidyl transferase center is necessary for translation. mRNA processing requires several different enzymes, some of which are general factors and others which are message-specific. One general factor is an endonuclease that cleaves mRNA precursors near a conserved dodecamer sequence, forming the mature 3‘ end of the mRNAs. A complex of three proteins can bind to the dodecamer and protect the RNA from a mitochondrial 3’ to 5’ exonuclease in uitro. Four mRNAs are processed at the 5’ end and processing may require message-specific factors. For example, CBPl is required for 5’ processing of COB pre-mRNA, but does not affect processing of other mitochondrial RNAs. Excision of introns from the large ribosomal transcript, the COB transcript, and the COX1 transcript requires several different nuclearly encoded factors and the mitochondrially encoded maturases. Some of these factors only affect the excision of one intron (e.g., b13 maturase, CBP2, PET54) while others affect the excision of many (e.g., MSS116, MRS2). In addition, many of the factors first discovered based on their role in splicing have other functions in the mitochondria; the most common additional roles are in translation (e.g., PET54, NAM2). The RNAs involved in mitochondrial translation are all encoded on the mitochondrial genome, including the large and small ribosomal RNAs and the tRNAs. However, the ribosomal proteins are, with the exception of VARl, nuclearly encoded. All of the mitochondrial tRNA synthetases and EF-Tu are also nuclearly encoded. In addition to these general factors,

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there are several nuclearly encoded factors that are required for the translation of individual mitochondrial messenger RNAs. PET494, PET54, and PET122 are needed for the translation of COX3 mRNA, PETlll for the translation of COX2 mRNA, and CBSl and CBS2 for the translation of COB mRNA. These message-specificfactors are hypothesized to interact with the S’untranslated leader sequences of the mRNAs to promote the initiation of translation. Evidence for this model includes suppression of mutations in the nuclear genes by fusions of heterologous mitochondrial leader sequences to the target coding sequences (all of the earlier mentioned genes), suppression of a leader mutation by a missense mutation in the nuclearly encoded protein (COX3/PET122),and suppression of mutations in the PET122 gene by mutations in several genes coding for mitochondrial ribosomal proteins. Once polypeptides are synthesized, there are several nuclearly encoded factors that are required for posttranslational modification of the proteins or their assembly into enzyme complexes. CYC3 and CYT2 are involved in heme attachment to cytochromes c and c,, respectively. Several proteins are thought to act as complex-specific chaperones that aid in the assembly of the complex enzymes of the inner mitochondrial membrane. These include ATPll and ATP12 (ATP synthase F,), ATPlO (F,,), COX10 and COX11 (cytochrome oxidase), and CBP3 and CBP4 (coQH,cytochrome c reductase). There are many levels at which mitochondrial gene expression could be controlled, including transcription, RNA stability, translation, and posttranslational modification and assembly. An increase in the steady-state level of mitochondrial RNAs is observed when yeast cells are switched to media requiring mitochondrial function. This may very well be due to an increase in the rate of transcription; however, it could also be due in part to a decrease in RNA turnover. Message-specific translation factors PET1 11 (COX2),PET122, and PET494 (COX3) appear to be rate-limiting for translation, and the abundance of PET494 has been shown to be greater under respiratory conditions. Therefore, these translation factors may play an important role in the upregulation of mitochondrial gene expression during the switch to respiration. Perhaps the ultimate level of control may be in the assembly of the ribosomes and large enzyme complexes in the mitochondrial inner membrane. All of the major mitochondrial translation products assemble with nuclearly encoded subunits: COXI, COXII, and COX111 assemble with at least five proteins to form cytochrome oxidase; cytochrome b assembles with eight subunits to form coQH,-cytochrome c reductase; ATP6, ATP8, and ATP9 assemble with several subunits to form ATP synthase, and VARl assembles with many nuclearly encoded ribosomal proteins to form the small ribosomal subunit. An increase in the abundance of the mRNA or

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the encoded polypeptide has been observed for many of the nuclearly encoded subunits of these complexes when yeasts are switched to nonfermentable media. For each complex, the least abundant of the nuclearly encoded subunits could dictate the total number of complexes assembled as long as a sufficient supply of the mitochondrially encoded polypeptides is available. A clear challenge for the near future is to try to determine which of the above-mentioned factors or as-yet-uncharacterizedfactors truly regulate the levels of mitochondrial gene products. With the advent of mitochondrial transformation (Johnston et al., 1988; Fox et al., 1988; Armaleo et al., 1990), which has allowed targeted mutagenesis of the genome (Folley and Fox, 1991; Costanzo and Fox, 1993; Mittelmeier and Dieckmann, 1993), and the use of T7-driven mitochondrial gene transcription (J. L. Pinkham, personal communication), which has allowed the expression of mitochondrial genes at different levels, there are more tools available to analyze the levels of control. We look forward to advances in this area. Acknowledgments The authors thank Telsa Mittelmeier and Alison Adams for critical reading of the manuscript, and members of the laboratory for their efforts in the final editing process. Many thanks to everyone for sending material before publication. Research in the authors’ laboratory is supported by Grant GM34893 from the National Institutes of Health. C. L. Dieckmann is an Established Investigator of the American Heart Association. R.R.S. is supported by Cancer Biology Training Grant CA09213.

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Wernette, C. M., Saldahna, R., Perlman, P. S., and Butow, R. A. (1990). Purification of a site-specific endonuclease, I-Sce 11, encoded by intron 4 alpha of the mitochondrial cox1 gene of Saccharomyces cerevisiae. J. Biol. Chem. 265, 18976-18982. Wernette, C. M., Saldanha, R., Smith, D., Ming, D., Perlman, P. S., and Butow, R. A. (1992). Complex recognition site for the group I intron-encoded endonuclease I-SceII. Mol. Cell. Biol. 12, 716-723; erratum: l2(4), 1903. Wiesenberger, G., Link, T. A., von Ahsen, U., Waldherr, M., and Schweyen, R. J. (1991). MRS3 and MRS4, two suppressors of mtRNA splicing defects in yeast, are new members of the mitochondrial c a m e r family. J . Mol. Biol. 217, 23-37. Wiesenberger, G., Waldherr, M., and Schweyen, R. J. (1992). The nuclear gene MRS2 is essential for the excision of group I1 introns from yeast mitochondrial transcripts in vivo. J . Biol. Chem. 267, 6963-6%9. Winkley, C. S., Keller, M. J., and Jaehning, J . A. (1985). A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae. J. Biol. Chem. 260, 14214-14223. Wu, M., and Tzagoloff, A. (1989). Identification and characterization of a new gene (CBPJ) required for the expression of yeast coenzyme QH2-cytochrome c reductase. J . Biol. Chem. 264, 1 1 122-1 1130. Xu, B., and Clayton, D. A. (1992). Assignment of a yeast protein necessary for mitochondrial transcription initiation. Nucleic Acids Res. 20, 1053-1059. Zagorski, W., Castaing, B., Herbert, C. J., Labouesse, M., Martin, R., and Slonimski, P. P. (1991). Purification and characterization of the Saccharomyces cerevisiae mitochondrial leucyl-tRNA synthetase. J . Biol. Chem. 266, 2537-2541. Zassenhaus, H. P., Martin, N. C., and Butow, R. A. (1984). Origins of transcripts of the yeast mitochondrial var I gene. J . Biol. Chem. 259, 6019-6027. Zassenhaus, H. P., Hofmann, T. J., Uthayashanker, R., Vincent, R. D., and Zona, M. (1988). Construction of a yeast mutant lacking the mitochondrial nuclease. Nucleic Acids Res. 16, 3283-3296. Ziaja, K., Michaelis, G., and Lisowsky, T. (1993). Nuclear control of the messenger RNA expression for mitochondrial ATPase subunit 9 in a new yeast mutant. J . Mol. Biol. 229, 909-916. Zitomer, R. S., and Lowry, C. V. (1992). Regulation of gene expression by oxygen in Saccharomyces cerevisiae. Microbiol. Rev. 56, 1-1 1. Zollner, A., Rodel, G., and Haid, A. (1992). Molecular cloning and characterization of the Saccharomyces cerevisiae CYT2 gene encoding cytochrome-cl-heme lyase. Eur. J . Biochem. 207, 1093-1 100.

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Dynamics of the Calcium Signal That Triggers Mammalian Egg Activation Karl Swann*q’ and Jean-Pierre Ozilt *MRC Experimental Embryology and Teratology Unit, St. George’s Hospital Medical School, London SW17 ORE, United Kingdom and h t i t u t National de la Rechsrche Agronomique, Unite de Biologie de la Fecondation, 78352 Jouy-en-Josas, France

1. Introduction At fertilization in mammals, the incoming sperm makes two essential contributions to the development of the new organism. One contribution is the DNA and all its associated genes, which enables a diploid organism to develop to full term (Surani et al., 1984). The other is the message that triggers egg development. Without this message, delivering the DNA is pointless. In all animal species studied, the message that the sperm delivers is written in the language of cell Ca2+ changes (Jaffe, 1983; Whitaker and Steinhardt, 1982). This chapter focuses on the way that changes of intracellular Ca2+ are generated in mammalian eggs, and describes our knowledge of how these Ca2+changes affect the subsequent development of the embryo. It is shown that the Ca2+ dynamics at fertilization in mammalian eggs are more complex than in nonmammalian systems. We are only beginning to understand this complexity and how it is related to events at fertilization. Most mammalian eggs are ovulated ready to be fertilized while arrested in metaphase I1 of second meiosis. Fertilization is characterized by a number of changes that have been reviewed by others (Austin, 1961;Cran and Esper, 1990; Yanagimachi, 1988). The most evident events are cortical

’

Present address: Department of Anatomy and Developmental Biology, University College London, Gower Street, London WClE 6BT, United Kingdom.

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granule exocytosis, which leads to modification of zona pellucida proteins to prevent other sperm from entering the egg, decondensation of the incoming sperm head, exit from meiotic arrest at metaphase 11, and extrusion of a second polar body. These events are followed by the formation of male and female pronuclei and the onset of cleavage (Cran and Esper, 1990; Yanagimachi, 1988). These changes in the egg can be triggered by the sperm or artificial parthenogenetic agents and this stimulation is generally referred to as egg “activation” (Kaufman, 1983; Whittingham, 1980). The term “activation” is rather poorly defined since any number of the changes in the egg at fertilization have been used as criteria for activation. The most common criteria are the extrusion of the second polar body and pronuclear formation. The formation of a pronucleus, or pronuclei, is the better end-point for early signs of development. In mammals this can take 4-8 hr, depending upon the species and the conditions of stimulation. The paradigm for signaling at fertilization has been set by studies in sea urchinand frog eggs (for reviews, see Jaffe, 1983; Whitaker and Steinhardt, 1982; Busa, 1990; Bement, 1992). In these species, the fertilizing sperm causes a single large transient increase in the cytoplasmic Ca2+concentration. The Ca2+increase lasts about 5-10 min and rises from resting levels of around 100 nM to a peak of 1-2 p M (Whitaker and Steinhardt, 1982; Busa, 1990; Whitaker and Swann, 1993). The origin of the Ca2+in each transient increase is an internal store, probably the endoplasmic reticulum (Whitaker and Steinhardt, 1982). Preventing the increase in intracellular Ca2+blocks cortical granule exocytosis, pH changes, metabolic increases, and the development of the embryo. The artificial induction of a similarsized Ca2+transient triggers essentially all of the early events normally activated by the sperm (Whitaker and Steinhardt, 1982). It is quite clear that Ca2+plays the key role in signal transduction at fertilization in these species because Ca2+changes are induced by the sperm, are essential, and are sufficient for activating the egg (Jaffe, 1983: Whitaker and Steinhardt, 1982; Bement, 1992). The evidence for this in frogs and sea urchin eggs was largely established before much of the work began on mammals. There may have been an expectation in the field of fertilization biology that the early events at activation in mammals would be very similar to those in other species. However, the discovery that mammalian eggs show sustained Ca2+ oscillations at fertilization, as opposed to a monotonic change, makes them distinctly different from many nonmammalian eggs (Miyazaki, 1991; Whitaker and Swann, 1993). The presence of oscillations provides an opportunity to reach a deeper understanding of what the sperm does to Ca2+homeostasis in the egg, as well as to explore how a new range of dynamic parameters affects subsequent development.

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II. A Calcium Signal in Eggs a t Fertilization The first indication that there were Ca2+changes during fertilization in mammals came indirectly from studies of membrane potentials. Miyazaki and Igusa measured membrane potentials in zona-free hamster eggs during in uitro fertilization (Miyazaki and Igusa, 1981a). They found that after sperm-egg interaction there was a series of hyperpolarizations in egg membrane potential from a resting level of -40 mV to troughs of - 70 mV. Although the frequency of repetitive hyperpolarizing responses (HRs) was higher in cases of polyspermy, repetitive HRs clearly occurred in cases where single sperm fused with the egg (Miyazaki and Igusa, 1981a). This membrane response is in contrast to many other eggs where the membrane potential becomes transiently more positive at fertilization (Hagiwara and Jaffe, 1979). In hamster eggs, the membrane potential becomes more negative and the responses are repeated for over an hour after penetration by the sperm. The membrane potential changes in hamster eggs do not have any obvious role by themselves. They are not involved in blocking polyspermy, as in many nonmammalian eggs (Miyazaki and Igusa, 1981a). They are also not a general feature of mammalian fertilization; mouse eggs show only very small changes in membrane potential at fertilization (Jaffe et al., 1983). The HRs in hamster eggs are of interest because they are caused by the activation of a Ca’+-activated potassium conductance in the plasma membrane (Igusa and Miyazaki, 1983, 1986). This clearly implies that the repetitive HRs reflect repetitive Ca2+increases that occur during hamster egg fertilization. Shortly after Miyazaki’s report, another study suggested that there are also repetitive Ca2+ increases during mouse fertilization. Cobbold and colleagues monitored Ca2+ more directly in mouse eggs during in uitro fertilization by measuring light output from eggs injected with the Ca’+sensitive photoprotein, aequorin (Cuthberston et al., 1981). Penetration by sperm was associated with repetitive increases in light output, suggesting that repetitive Ca2+ increases were occurring about once every 20 min. Later studies showed that these repetitive Ca2+transients lasted for 2-4 hr (Cuthbertson and Cobbold, 1985). Since these early studies, the field has been revolutionized by the availability and relative ease of use of a range of fluorescent dyes, such as fura2, to measure intracellular Ca2+(Tsien, 1992). The original observations of prolonged Ca2+oscillations in hamster and mouse fertilization have been confirmed (Kline and Kline, 1992a; Shiina et al., 1993; Miyazaki et al., 1992). Figure 1 shows Ca2+oscillations in mouse eggs measured with the fluorescent indicator dye, indol , Many studies have confirmed the findings of the original work on mouse and hamster eggs, although the frequency

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FIG. 1 Recording of CaZ+oscillations from single mouse eggs after in uirro fertilization. Shown are two different eggs that displayed different frequency responses under the same conditions. Intracellular CaZ+was monitored by the fluorescence ratio of indol loaded into eggs using the membrane-permeable form, indol-AM. The fluorescence emission measurements were taken at 490 nm and 420 nm; the scale is an arbitrary ratio of the 420-nml 490-nm signals. Further experimental details can be found in Swann (1990) and Ozil and Swann (1993).

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of oscillations in mouse eggs has been shown to be as high as one transient per 2-3 min (Kline and Kline, 1992a). Figure 1 indicates that there is even some variability in the frequency of oscillations among individual mouse eggs under similar conditions. Ca2+oscillations have now been reported during fertilization of pig, cow, rabbit, and human eggs (Sun et al., 1992; Fissore et al., 1992; Fissore and Robl, 1993; Taylor et al., 1992). The oscillations in domestic animals are generally similar in pattern to rodent eggs, but of lower frequency (Sun et al., 1992; Fissore et al., 1992). Apart from frequency differences, it is clear that Ca2+oscillations are a characteristic of mammalian fertilization. The sperm-induced oscillations in mammalian eggs generally consist of a long-lasting series of approximately 10-fold changes in free Ca2+ levels, with each transient lasting for about 1 min. Although external CaZ+ is needed to maintain oscillations, each individual Ca2+ transient is the immediate result of release from an internal store (Igusa and Miyazaki, 1983). Given that the length of the first cell cycle in mammalian embryos is about 20 hr, these oscillations appear to represent one signal package that occurs at a single cell-cycle control point (Whitaker and Patel, 1990). This means that they represent a different class of phenomena from Ca2+ oscillations that occur after fertilization in marine invertebrate eggs. In invertebrate eggs, the oscillations consist of a series of Ca2+transients that occur at a distinct control point during the course of about a 1-hr cell cycle (Poenie et al., 1985; Steinhardt, 1990; Speksnijder et al., 1989; Whitaker and Patel, 1990). As well as the frequency of Ca2+oscillations, another notable feature of the fertilization response is the delay, or latent period, for the onset of Ca2+release (Whitaker and Swann, 1993). The latent period has been extensively studied in sea urchins and is equivalent to the delay between sperm-egg fusion and the onset of regenerative Ca2+release (McCulloh and Chambers, 1992; Whitaker and Swann, 1993). The exact timing of sperm-egg membrane fusion has not been measured in mammals. However, judging from data on sea urchin fertilization and from observations of sperm-egg interaction in zona-free hamster eggs, it seems likely that sperm-egg membrane continuity is established around the time an attached sperm tail stops beating (Yanagimachi, 1988; Whitaker and Swann, 1993). This delays the first Ca2+ transient in hamsters by 1-10 sec, whereas in mice the time delay is 5-10 min (Miyazaki and Igusa, 1981a; Jaffe et al., 1983). This suggests very different latent periods, depending upon the species, between sperm-egg fusion and the onset of Ca2+ release. The differences appear to be mainly due to the sperm. If zona-free hamster eggs are fertilized with mouse sperm, there is a delay of about 10 min before the onset of Ca2+oscillations (Igusa et al., 1983).

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So far we have considered measurement of Ca2+taken as an average across the whole cell. In eggs of sea urchins and frogs, the single Ca2+ transient consists of a propagating wave that crosses the egg from the point of sperm entry (Jaffe, 1983; Swann and Whitaker, 1990). In hamster eggs, the spatial distribution of Ca2+ transients has been measured by imaging the light output from injected aequorin (Miyazaki et al., 1986). After fusion of a single sperm, the first Ca2+increase travels as a wave moving away from the point of fusion, crossing the egg in about 6 sec. The subsequent Ca2+increases start from a more diffuse area near the point of sperm-egg interaction and then spread into the remainder of the egg. After several responses have occurred, the Ca2+increases are initiated over the entire egg volume and no wave is detectable within the time resolution of the measuring system (Miyazaki et al., 1986).Thus in hamster eggs the Ca2+waves turn into near-synchronous "pulses," and it appears that some agent from the sperm irreversibly changes the egg so that Ca2+increases are initiated over its entire volume synchronously. Ca2+sensitive electrode measurements also show that each Ca2+transient occurs throughout the depth of the egg cytoplasm (Igusa and Miyazaki, 1986). Associated with the change in the spatial Ca2+dynamics is the sensitivity of the egg to Ca2+-inducedCa2+release (CICR). Injecting Ca2+can trigger Ca2+release in hamster eggs, but the response requires a relatively large injection of Ca2+and can generally be seen only once in any one egg (Igusa and Miyazaki, 1983; Swann, 1991). In contrast, after fertilization injections of Ca2+repeatably cause full Ca2+release responses (Igusa and Miyazaki, 1983). This postfertilization increase in CICR is also seen with the Ca2+release response to voltage-gated Ca2+influx (Swann, 1990), or in response to Ca2+ influx after electrical field pulses (Ozil and Swann, 1993). Overall, the increase in sensitivity of CICR is at least 10-fold (Igusa and Miyazaki, 1983). The enhancement of CICR is the most critical point in explaining how the sperm causes Ca2+oscillations, as illustrated by the following experiments. If fertilized eggs undergoing oscillations are perfused with Ca2+-free media, the oscillations cease to occur (Igusa and Miyazaki, 1983). However, if Ca2+is then injected into the egg in a series of small iontophoretic pulses, Ca2+oscillations are restimulated (Igusa and Miyazaki, 1983). This small amount of Ca2+ required to maintain oscillations in fertilized eggs in Ca2+-freemedia does not actually cause any oscillations in unfertilized eggs (Igusa and Miyazaki, 1983; Swann, 1991). This suggests that the sensitization of eggs to CICR is such that a level of Ca2+influx that may well occur in an unfertilized egg is sufficient to maintain oscillations at fertilization. A Ca2+influx pathway is necessary at fertilization (Miyazaki, 19911, but a requirement for increased Ca2+influx over prefertilization

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levels remains to be demonstrated and may not be required. Sensitization of CICR, on the other hand, is both necessary and sufficient to explain oscillations (Igusa and Miyazaki, 1983; Swann, 1991). Whatever messengers are invoked to explain Ca2+ oscillations in mammalian eggs, they must account for the phenomena of enhanced CICR.

111. Generation of the Calcium Signal

A remaining problem to be solved in fertilization is how the sperm generates the Ca2+ changes in the egg cytoplasm. This problem remains as unresolved in nonmammalian eggs as in mammals. As in most other animals, the immediate source of Ca2+for each transient in mammalian eggs appears to be an intracellular store (Igusa and Miyazaki, 1983,1986; Kline and Kline, 1992b). Consequently, there is a transduction problem in which messengers must be found to mediate a message from the site of initial sperm-egg interaction at the plasma membrane to the intracellular Ca2+ store. At least three main ideas have been put forward for the signaling systems used by a sperm to trigger Ca2+release in eggs at fertilization. The three ideas we will discuss are illustrated schematically in Fig. 2. A. The Calcium “Bomb” Hypothesis

One of the most straightforward suggestions of how the sperm triggers Ca2+release in eggs is that after the fusion of the sperm and egg plasma membranes, the sperm introduces Ca2+itself (see Fig. 2a) (Jaffe, 1983; Yanagimachi, 1988). The “calcium bomb” hypothesis suggested that the injected Ca2+could act as a trigger for further calcium release in a manner analogous to Ca2+-inducedCaZ+release described in muscle cells (Fabiato, 1983). Support cited for the Ca2+ bomb idea is that sperm from many species, including mammals, take up Ca2+ prior to fusing with the egg (Jaffe, 1983, 1991). The idea that the sperm “injects” Ca2+also requires that sperm-egg fusion be a primary event. This is the case in sea urchin eggs (McCulloh and Chambers, 1992), and there is no indication that any Ca2+release occurs without fusion in mammalian systems. However the “calcium bomb” idea is flawed, as revealed by studies of Ca” homeostasis in the unfertilized egg. Injecting Ca2+ions with an iontophoretic pulse can trigger a regenerative Ca2+transient in hamster eggs, suggesting the existence of CICR (Igusa and Miyazaki, 1983; Swann, 1991). However, the regenerative response is rarely seen more than once in any one egg and a single injection pulse

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FIG. 2 The three main classes of schemes for how a sperm causes CaZt release in mammalian eggs. The ideas are that: (a) The sperm fuses and acts as a conduit for CaZt influx into the egg, which then overloads with Ca2+.(b) The sperm acts as an agonist on surface receptors which act via G-proteins or tyrosine kinases to stimulate production of InsP,. (c) The sperm fuses with the egg and introduces a specific factor or oscillogen that releases Ca2+ from internal stores.

never triggers oscillations. Generating action potentials to cause Ca2+ influx across the plasma membrane can also trigger regenerative Ca2+ release in hamster eggs (McNiven er al., 1988), but again it only does this once in any egg and it does not cause oscillations. Electrical field pulses in mouse, pig, and cow eggs trigger a single Ca2+transient that is caused by Ca2+influx analogous to Ca2+injection, but again oscillations cannot be triggered by a single pulse alone (Fissore and Robl, 1992; Sun er al., 1992; Ozil and Swann, 1993). These findings clearly argue against the simplest version of the “calcium bomb” hypothesis. A more recent adaption of the calcium injection hypothesis is that sperm acts as a conduit to inject Ca2+more gradually (see Fig. 2a) (Jaffe, 1991). After fusing with the egg, it is suggested that the sperm mediates Ca2+ influx across its own membrane into the egg cytoplasm. This spermdirected Ca2+influx could cause a gradual filling of an internal Ca2+store, eventually leading to overloading and subsequently a large Ca2+ release into the cytoplasm. The idea is analogous with “Ca2+-overload”-induced release that has been described in sarcoplasmic reticulum of muscle, and in liver endoplasmic reticulum (Fabiato, 1983; Missiaen er al., 1991). It

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seems an attractive explanation of Ca2+ oscillations since this type of Ca2+-overload-inducedrelease is thought to be responsible for oscillations of Ca2+ release seen in skinned cardiac muscle cells (Fabiato, 1983). However, the Ca2+conduit idea is again insufficient for mammalian eggs. Sustained injection of Ca2+into unfertilized hamster or mouse eggs fails to mimic the repetitive Ca2+transients seen at fertilization, and at best Ca2+injection causes a series of small and critically damped oscillations (Igusa and Miyazaki, 1983; Swann, 1992). Repetitive action potentials in high Ca2+ media also fail to cause oscillations (McNiven et a / . , 1988; Swann, 1990). In fact, all procedures to artificially increase influx of Ca2+ into the egg lead to a decrease in the sensitivity of CICR (Igusa and Miyazaki, 1983; Swann, 1991). This is the exact opposite of what the sperm does at fertilization since it causes an order of magnitude increase in the sensitivity of CICR (Igusa and Miyazaki, 1983; Swann, 1990). We conclude that whatever messenger the sperm uses to trigger Ca2+oscillations, it is not Ca2+by itself.

6. The G-Protein-lnositol Phosphate Production Hypothesis

The polyphosphoinositide messenger system (PI system) is responsible for triggering hormone-induced Ca2+ oscillations in many somatic cell types (Berridge and Galione, 1988). This system has been suggested to operate at fertilization, giving the sperm the status of a giant hormone (see Fig. 2b and Jaffe, 1990). It is envisaged that molecules, presumably on the plasma membrane of the sperm, bind to receptors on the egg surface; these couple to guanosine triphosphate (GTP)-binding proteins (or G-proteins), which stimulate a phospholipase C. Phospholipase C then produces inositol( 1,4,5)-trisphosphate (InsP,), which releases Cat+, and diacylglycerol (DAG), which activates protein kinase C (Jaffe, 1990;Miyazaki, 1991). It has been shown biochemically that PI turnover occurs at fertilization in sea urchin eggs (Turner et a / . , 1984). Such methods require thousands of eggs and are not suitable for mammalian studies. Nevertheless, there is indirect evidence that protein kinase C is stimulated during fertilization of mouse eggs (Colonna et al., 1989), which suggests that some PI turnover does occur. However, as suggested in sea urchin and frog eggs, it is likely that PI turnover is stimulated by a Ca2+increase and so it is difficult to know if PI turnover and DAG production are the result or the cause of Ca2+release (Swann and Whitaker, 1990; Bement, 1992). The principal evidence for the G-protein-InsP, hypothesis at fertilization in mammalian eggs has come from physiological experiments. It has been demonstrated that injecting nonhydrolyzable analogs of GTP, such

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as GTP[g]S [guanosine-5'-0-(3-thiotriphosphate] to stimulate G-proteins, or sustained injection of InsP,, will both cause repetitive Ca2+releases in hamster and mouse eggs (Miyazaki, 1988; Swann er al., 1989). Figure 3 shows examples of Ca2+oscillations and HRs generated in hamster eggs injected with InsP, and its nonhydrolyzable derivative. Applying serotonin to hamster eggs, or acetylcholine to mouse eggs, will also cause similar Ca2+oscillations, apparently via a G-protein pathway (Miyazaki er al., 1990; Swann, 1992). In addition, it is possible to activate eggs (exocytosis and pronuclear formation) either by injecting InsP,, or expressing extra acetylcholine receptors and adding acetylcholine (Cran and Esper, 1990; Williams er al., 1992). All these data show that there is a G-protein-InsP,

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FIG. 3 InsP, and its nonhydrolyzable derivative trigger damped CaZt oscillations in hamster eggs. Micropipettes containing 1 m M InsP, (a) or 10 m M of its nonhydrolyzable derivative InsPS, (b) were inserted into unfertilized zona-free hamster eggs. Membrane potentials (mV) and fluorescence (FL) from the Cazt-sensitive intracellular dye flu03 were recorded from single zona-free eggs. Hyperpolarizing responses and CaZt transients occurring simultaneously show a decline in magnitude with time. Records are typical of 26 eggs injected with InsP, and 3 eggs injected with InsPS,. The intervals between Caz' transients varied from 20 to 160 sec and the duration of the Cazt increases after the initial response was 1020 sec. Methods are given in Swann (1991) and Swann et al. (1989).

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messenger system in eggs that can release Ca2+.The question remains as to whether this system is used at fertilization. The evidence that the sperm stimulates the G-protein-PI messenger system at fertilization is the finding that injection of the nonhydrolyzable guanosine diphosphate (GDP) analog (which inhibits G-proteins) blocks fertilization-associated HRs in hamster eggs (Miyazaki, 1988). The GDP analog did not inhibit subsequent Ca2+release stimulated by InsP, injection (Miyazaki, 1988). This was a control for the possible nonspecific effects of the GDP analog, although it is only relevant if the sperm actually uses InsP, to release Ca2+.The evidence that InsP, is involved at fertilization is that injecting a monoclonal antibody to the InsP, receptor blocks sperminduced Ca2+oscillations (Miyazaki et al., 1992).This result is the strongest argument for the whole of the G-protein-InsP, story, but if we accept that the antibody is entirely specific, then it shows that the InsP, receptor is involved in Ca" release. Whether InsP, is the messenger that the sperm uses to activate the InsP3 receptor is another matter. These points are elaborated further in Section 111. Despite the suitability of the G-protein-InsP, system for explaining Ca2+release in mammalian eggs, several problems arise if one believes it operates at fertilization. Although GTP[g]S can trigger repetitive Ca" release, the response to GTP[g]S in hamster eggs is variable and oscillations are always high frequency and critically damped (Swann et al., 1989). There appears to be an in-built desensitization to GTP[g]S in hamster eggs. The same is also true of serotonin-induced Ca2+oscillations (Miyazaki et al., 1990). Even the Ca2+oscillations set up by sustained injection of InsP,, or its nonhydrolyzable derivative are high-frequency, critically damped responses (see Fig. 3). This is in complete contrast to fertilization, where the Ca2+transients show a large range of frequencies but very little damping or decrease in amplitude over 2 hr (Miyazaki, 1991). The full reasons for the rundown of responses in the G-protein-InsP, pathway are not clear, but for G-proteins at least the problem is associated with protein kinase C. The G-protein-mediated oscillations can be completely blocked by prior treatment with phorbol esters, which activate protein kinase C, and enhanced by sphingosine, which blocks protein kinase C (Swann et al., 1989). This suggests that the hamster egg has a negative feedback loop in the PI messenger system in which DAG production and protein kinase C activation inhibit Ca2+release set up by Gproteins and InsP, (Swann et al., 1989; Miyazaki, 1991). However, the same treatments with phorbol esters and sphingosine have very little effect on fertilization-associated Ca2+oscillations (Swann et al., 1989). Since the ability of the sperm to cause Ca2+ oscillations is not affected by protein kinase C, it is assumed that either the sperm excludes this negative

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feedback loop, or else it does not mediate Ca2+release via the G-protein pathway. As yet, the G-protein hypothesis lacks its most important elements: an agonist on the sperm and a receptor on the egg. Molecules on the surface of sperm that bind them to eggs have been identified and sequenced in guinea pigs (Primakoff et al., 1987; Blobel et al., 1992). The proteins consist of two subunits that have sequence homology to viral fusion proteins and to a class of molecules known as disintegrins that couple to cell-surface receptors called integrins (Blobel er al., 1992). The sequence homology with integrins may implicate a different kind of transmembrane signaling system for generating InsP, ; integrins appear to mediate phosphoinositide turnover via tyrosine kinase activation (Schwartz, 1992). A tyrosine kinase pathway for InsP, production might avoid some of the problems with the G-protein negative feedback view. However, it has yet to be shown that these sperm surface molecules cause Ca2+release, activate G-proteins, or increase InsP, production by any other means in any type of egg. In sea urchin eggs, the relevant molecule on the sperm is bindin, which is known to cause membrane fusion but not egg activation or Ca2+release (Glabe, 1985). C. The Soluble Sperm Factor Hypothesis

Another explanation of how the sperm causes Ca2+ release is that it introduces a soluble factor after fusion of the gamete membrane and this factor generates Ca2+release within the egg (see Fig. 2C) (Dale et al., 1985; Swann and Whitaker, 1990). As with the Ca2+ bomb hypothesis, this idea requires that the sperm fuse with the egg before Ca2+oscillations are started and, as discussed above, this is a reasonable assertion. The soluble sperm factor hypothesis also implies that an egg-activating and Ca2+-releasingfactor should be found in the sperm. This point has been its principal focus and source of controversy. Original evidence for a soluble sperm factor came from sea urchins. Injecting cytosolic extracts of sperm was shown to cause exocytosis of cortical granules (Dale et al., 1985). However, since the extracts contained Ca2+,which can cause exocytosis, it was not clear that the effect was entirely due to sperm components. Similar experiments were carried out in rabbit and mouse eggs, where the effect of extracts was shown to be trypsin sensitive (Stice and Robl, 1990). Again, however, the extracts contained more than 100 p M Ca2+and since Ca2+injection can activate eggs, it was not clear whether there was a specific factor in the sperm. A more definitive demonstration of the existence of a soluble sperm factor is from experiments that measure Ca2+after injection of extracts

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and compare the response with that at fertilization. The injection of cytosolic extracts from boar and hamster sperm has been shown to cause sustained Ca2+oscillations in hamster and mouse eggs (Swann, 1990, 1992). Examples of hamster and mouse eggs injected with sperm extracts are shown in Fig. 4. The sperm extracts that cause oscillations contain a low (<1 p M ) Ca2+and are only effective when injected into the egg and not when applied to the outside of the egg (Swann, 1990). In these experiments it is irrelevant if the extracts contain Ca2+because Ca2+oscillations are not stimulated by injection of Ca2+-containingsolutions, or injection of any other protein-containing solutions (Igusa and Miyazaki, 1983; Swann, 1990). These data clearly suggest that the sperm contains a specific factor which causes Ca2+release in eggs. Preliminary data showed that the active factor in sperm extracts is high molecular weight and trypsin sensitive, suggesting it is protein based (Swann, 1990). It has been possible to purify the active factor to some degree by using ion exchange and affinity columns, which again suggests that the factor is protein in nature (K. Swann, J. Parrington, and F. A. Lai, unpublished data). This excludes the idea that

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FIG. 4 Sperm oscillogen triggers Caz' oscillations in hamster and mouse eggs. In each case the egg was injected (at the time indicated by the arrows) with -5 pl of different cytosolic sperm extracts (Swann, 1990). Conditions are the same as those in the legend to Fig. 3. (a). Record obtained from an unfertilized hamster egg with simultaneous recordings of membrane potentials (mV) and flu03 fluorescence (FL).(b). Recording made from an unfertilized mouse egg injected with sperm oscillogen. Only the flu03 fluorescence changes are shown for the mouse egg, Records are typical of 8 hamster eggs and more than 50 mouse eggs injected with sperm oscillogen.

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the sperm factor is InsP, (Tosti et al., 1993). The soluble sperm factor is defined by its ability to cause Ca2+oscillations, so we refer to it as an oscillogen. Sperm oscillogen not only mimics fertilization in causing sustained Ca2+ oscillations but it also enhances CICR in exactly the same way as the sperm does (Swann, 1990). It mimics fertilization in hamster and mouse eggs better than any other agent in causing oscillations that remain of fairly fixed amplitude, but that vary in frequency, depending upon the amount injected (see Fig. 4). Its existence suggests a fairly simple scheme for fertilization in which a protein diffuses into the eggs after gamete fusion, enhances CICR, and triggers oscillations. If different types of sperm contained different amounts of the oscillogen, then it might explain why the latencies and frequencies of oscillations are to some extent spermspecies specific (Igusa et al., 1983; Swann, 1990). Sperm oscillogen may represent a novel type of Ca2+-releasingagent since it triggers inward current oscillations that are Ca2+-dependentand enhances CICR when injected into dorsal root ganglion (DRG) neurons (Currie et al., 1992). Interestingly, the same DRG neurons were largely insensitive to the injection of InsP, (Currie et al., 1992). The main problems for sperm oscillogen are confirming its identity and showing exactly how it releases Ca2+from intracellular stores. The simplest hypothesis is that it is a protein that binds to and opens some type of Ca2+release channels (Swann, 1992). It may act as an allosteric modulator of CaZfrelease channels, making them more sensitive to Ca", and hence enhance CICR (see later discussion). There is a precedent for this idea in muscle cells where caffeine or a peptide oscillogen causes oscillations in Ca2+release in skeletal muscle by acting upon the sarcoplasmic reticulum Cazf-releasechannel (HerrmanFrank, 1989; Herrmann-Frank and Meissner, 1989). D. Mechanisms of Calcium Oscillations

In addition to the question of what messengers the sperm uses to trigger Ca2+oscillations is the issue of the basic oscillation mechanism in mammalian eggs. For mammalian eggs, the most important point for understanding the underlying mechanism of oscillations is CICR enhancement. The reasons for this are perhaps best illustrated by the effects of the sulfhydryl reagent, thimerosal. Applying thimerosal to eggs causes the same degree and type of enhancement of CICR that occurs at fertilization and, after sperm oscillogen, thimerosal is the next-best agent for mimicking the Ca2+ oscillations caused by the sperm (Swann, 1991;Fissore et al., 1992; Fissore and Robl, 1993). The site of action of thimerosal in unclear, but it can sensitize Ca2+ release from internal stores in many cell types (Bootman

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et al., 1992; Missiaen et al., 1991). Its effects emphasize that CICR and Ca2+oscillations are interlinked. Once CICR is sensitized to a sufficient degree, it seems inevitable that the Cat+ levels in the cell will become unstable and regenerative release will occur. Explaining why CICR is enhanced is, in this sense, the same as explaining why oscillations occur. CICR is a phenomenological term that describes a property of a Ca2+ release mechanism. It implies that release is triggered by Ca2+.We stress this point because there is some confusion generated by reference to CICR as implicating a particular Ca2+channel in the release process. The term CICR does not a priori mean that any particular Ca2+release channel is involved. It simply means that the Ca2+release system has regenerative properties and it turns out that this regenerative property is a common feature of Ca2+release channels. Specific explanations of CICR have been made in terms of the behavior of two classes of specified Ca2+release channels (Endo et al., 1977; Rousseau and Meissner, 1989; Taylor and Marshall, 1992). One class of channel binds InsP, with high affinity and is referred to as the InsP, receptor (IP-R) (Furuichi et al., 1989; Ferris and Snyder, 1992);the other is identified by specific binding by the alkaloid ryanodine and is called the ryanodine receptor (Ry-R) (Lai et al., 1988). Most attention has been focused on an IP-R's involvement in fertilization. As described above, injections of InsP, into mouse, hamster, and bovine eggs can cause Ca2+release and oscillations, so there is little doubt that InsP, receptors exist in mammalian eggs (Miyazaki, 1991; Kline and Kline, 1992b; Swann, 1992; Fissore et al., 1992). Furthermore, a monoclonal antibody, 18A10, raised against the cerebella Purkinje cell IP-R (classified as type I) has been shown to block Ca2+release in response to InsP, or Ca2+injection and fertilization in hamster eggs (Miyazaki et al., 1992). The 18AlO antibody binds to the Ca2+channel-forming domain of the IP-R and inhibits CICR and all forms of Ca2+release in hamster eggs. This suggest that all forms of Ca2+release involve only the IP-R. However, it should be noted that the 18AlO monoclonal antibody used in this study cross-reacts with a number of unknown proteins in the cerebellum and its specificity of action in intact cells needs to be proven (Miyazaki et al., 1992). It is also not clear why the antibody gives different results from another IP-R antagonist. Heparin inhibits InsP,-induced Ca2+ release in many different systems and acts by competitive binding to the InsP, binding site (Ferris and Snyder, 1992). Injecting the IP-R antagonist heparin is not effective at blocking HRs at fertilization in hamster eggs, and yet heparin injection is as effective at inhibiting InsP,-induced HR as the 18A10 antibody (Miyazaki et al., 1993). Therefore, the effects of the antibody may not be definitive proof that the IP-R is the only Ca2+release channel needed to cause oscillations in eggs. If it is selectively acting on the IP-R, then the difference between the results with the 18A10 antibody

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and heparin implies that the InsP, binding site is not the target of a physiological regulator at fertilization. There is a separate problem with invoking only InsP, and the IP-R for explaining oscillations and CICR in eggs. The best explanation of the enhancement of CICR at fertilization, involving the IP-R, is that production of InsP, activates the IP-R to change it to a state where opening is highly sensitive to Ca2+.This means that when it is bound by InsP,, the InsP, receptor becomes a CICR channel (Bezprovzanny et al., 1991). This idea has received direct experimental support in smooth muscle cells and frog oocytes (Iino and Endo, 1992; DeLisle and Welch, 1992). However, in this case it is difficult to explain why sustained injection of InsP, does not sensitize CICR to anywhere near the same extent as the sperm, thimerosal, or sperm oscillogen (Swann, 1991). Hamster eggs always irreversibly desensitize to pulses of InsP, injection after the first full-blown Ca2+response (Swann et al., 1989; A. Galione, K. Swann, and M. J. Whitaker, unpublished observations). Hence the egg loses its regenerative response to InsP,, that is, the ability to have its CICR affected by InsP, . The poor ability of InsP3 to enhance CICR might explain why sustained injection of InsP, or its nonhydrolyzable derivative only triggers notably damped Ca2+oscillations (Swann et al., 1989; also see Fig. 3). Whatever way InsP, triggers release in hamster eggs, it does not appear to mimic the sperm because its effects desensitize and it fails to be a sufficient CICR enhancer. This implies that either another Ca2+ channel is involved in CICR, or alternatively, the IP-R is involved but there is some other factor (the sperm oscillogen?) which is an InsP,-independent activator an the IP-R at fertilization. If another Ca2+release channel is needed, the ryanodine receptors (RyRs) represent a distinct class of Ca2+ release channels that have some sequence homology to IP-Rs. The type I and I1 subtypes of the Ry-Rs are associated with CICR in muscle and nerve cells and can be activated by the drug caffeine (Lai et al., 1988; McPherson et al.. 1991). The type I1 Ry-R may be the target of the recently discovered Ca2+-releasingmessenger, cyclic-ADP ribose (Galione et al., 1991; Lee, 1993; Meszaros et al., 1993). Neither cyclic ADP-ribose nor caffeine cause Ca2+oscillations, or enhance CICR in mammalian eggs (Swann, 1990; Miyazaki et al., 1992). So it seems unlikely that these channels account for Ca2+release. However, the type I11 Ry-R is not sensitive to caffeine, and other uncharacterized Ry-Rs in lobster muscle, liver cells, and brain also appear to be caffeine insensitive (Gianni et al., 1992; Seok et al., 1992; ShoshanBarmatz et al., 1991; Takeshima et al., 1993). Since ryanodine itself has been shown to cause Ca2+release and inhibit Ca2+oscillations in mouse eggs, it is possible that these eggs possess one of these other Ry-R subtypes

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(Swann, 1992). It is also possible that eggs possess other types of Ca2+ release channels (Salama et al., 1992; Wang et af., 1992). In addition to Ca2+release channels, the egg must have a specific Ca2+ store and Ca2+ pumps for recovery from transients. The Ca2+ store is probably the endoplasmic reticulum, as in other cell types (Cran and Esper, 1990). The inhibition of Ca2+homeostasis by the inhibitor thapsigargin suggests that the endoplasmic reticulum has a Ca2+-ATPasesimilar to that found in other cells (Kline and Kline, 1992b). The plasma membrane appears to have both a Ca2+-ATPaseand a Na+/Ca2+exchanger for extruding Ca2+(Georgiou et al., 1988; Igusa and Miyazaki, 1983). The plasma membrane must also have some kind of Ca2+channel for an influx pathway that permits the maintenance of oscillations (Miyazaki, 1991). Eggs possess voltage-gated Ca2+channels, but these do not appear to play a significant role in fertilization because voltage-gated Ca2+channels are mostly inactivated at steady-state membrane potentials (Peres, 1986; Miyazaki and Igusa, 1982). In fact, hyperpolarizing the membrane potential appears to increase the degree of influx at fertilization (Miyazaki and Igusa, 1982). This suggests that neither voltage-gated Ca2+channels nor reversal of the Na+/Ca2+exchanger could account for the influx pathway. E. Models for Explaining Calcium Oscillations

Given Ca2+channels, pumps, and stores, there are various models of Ca2+ oscillations that might be used to explain the response at fertilization in mammalian eggs. Nearly all models have been proposed to explain how Ca2+oscillations are generated by sustained InsP, production (Berridge and Galione, 1988). One popular model suggests that sustained InsP, production causes sustained Ca2+release from one Ca2+store in the cell. This then leads to cycles of Ca2+ uptake and release via CICR from a separate Ca2+store (Goldbeter et al., 1990). It was originally proposed that this second oscillating store was InsP,-insensitive, but recently it has been suggested that the second store has InsP, receptors that are instead less sensitive to InsP, (Bootman et a/., 1992). Another model suggests that InsP, production is regenerative by virtue of a Ca2+-activatedphospholipase C (Meyer and Stryer, 1991. The regenerative production of InsP,, combined with a nonlinear dependence of the release channel opening to InsP, concentrations, gives rise to regenerative Ca2+ release that is effectively CICR (Bezprozvanny et al., 1991). This model has been modified to include the observed Ca2+-inducedsensitization and desensitization of the IP-R (DeYoung and Keizer, 1992). These models all have the potential to explain oscillations and CICR. However,

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there is still a lack of data on the localization of Ca2+stores, or whether Ca2+-stimulatedInsP, production exists in mammalian eggs. It is rather difficult to experimentally distinguish the various models of Ca2+oscillations because they tend to make the same predictions, which are variably suited to the findings in mammalian eggs. For example, all the models predict that increased Ca2+influx leads to increased frequency of oscillations (Kuba and Takeshita, 1981; Berridge and Galione, 1988; Goldbeter et al., 1990; Meyer and Stryer, 1992). This is certainly true of mammalian eggs after fertilization (Igusa and Miyazaki, 1983).The models also suggest that the frequency of oscillations is determined by the steadystate InsP, concentrations (Goldbeter et al., 1990; Meyer and Stryer, 1992). Some models predict that the refilling of Ca2+stores is the determinant of frequency of oscillations (Kuba and Takeshita, 1981; Goldbeter et al., 1990), which was not found in bovine eggs (Fissore et al., 1992). In contrast to models with similar predictions is a recent theoretical comparison between Ca2+oscillations generated by either one or two Ca2+ stores (Dupont and Goldbeter, 1993). Whether a cell has one or two Ca2+ stores involved in oscillations does lead to some predictive differences. However, both the Dupont and Goldbeter models are designed for explaining how InsP, causes Ca2+oscillations, and this has yet to be firmly established in mammalian eggs. Whatever the case, it should perhaps be remembered that all the various models suggested so far are tailormade for understanding oscillations in somatic cells. They have not as yet been designed to addressed specific problems in mammalian eggs, such as the way unfertilized eggs desensitize to InsP,-induced Ca2+ release and to CICR, or the way eggs show such a marked increase in CICR sensitivity after fertilization.

IV. Effects of the Calcium Signal on the Egg As we have seen, fertilization signals in mammalian eggs differ from signals in eggs of lower species in that the Ca2+oscillates after fertilization for several hours. The precise function and consequences of these Ca2+oscillations remain as unknown in mammalian eggs as they do in many somatic cell types that undergo prolonged Ca2+oscillations (Berridge and Galione, 1988; Miyazaki, 1991; Sun et al., 1992). The dynamics of oscillatory Ca2+ signaling makes the understanding of signal transduction more difficult because any of a variety of features associated with the oscillations might be encoded by the cell and involved in organizing early activation events which, in turn, might then affect later stages. Two approaches have been used to evaluate the importance of Ca2+in the process of activation. The

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first is to inhibit the Ca2+transients after fertilization and to see if this inhibits further development. The second is to increase Ca2+artificially to reproduce changes normally caused by fertilization to see how if and how this affects parthenogenetic development. These approaches seek to establish that Ca2+oscillations are both essential and sufficient to trigger development of the mammalian embryo. In this section we examine the data supporting a role for Ca2+ in mammalian egg activation before discussing how the egg may decode the Ca2+ signal at fertilization. A. Requirement of Calcium Oscillations for Activation of the Egg

In principle, two methods for blocking CaZ+increases at fertilization may be used in mammals. The first is based on the observation that Ca2+ oscillations can be halted by perfusing oscillating eggs with Ca2+-free media (Igusa and Miyazaki, 1983). This approach cannot be employed to try and stop all the oscillations at fertilization because external Ca2+ is required for the sperm and egg to fuse and, in hamster eggs at least, Ca2+ increases appear to start within seconds of gamete fusion (Miyazaki and Igusa, 1981a; Yanagimachi, 1988). Nevertheless, it has been shown that when hamster eggs were transferred into a Ca2+-freemedium 15 min after insemination, the eggs went on to develop pronuclei 3 hr later (Yanagimachi, 1982). This result suggests that exit from mitosis and pronuclei formation does not require any more than a half dozen or so Ca2+transients that would likely have occurred before transfer to a Ca2+-freemedium. This method is limited for looking at the later role of Ca2+ oscillations because when the culture in Ca2+-freemedium is prolonged, the eggs die (Yanagimachi, 1982). A more direct way to block Ca2+transients in the egg at fertilization is to introduce cytoplasmic Ca2+buffers. For example, Ca" chelators such as EGTA or 1,2-bis(o-aminophexy)ethane-N,N,Nf ,N'-tetraacetic acid (BAPTA) can be used to block Ca2+increases in eggs. This approach was applied to mouse eggs by incubating eggs in BAPTA-AM, which is a membrane-permeable form of the Ca2+chelator BAPTA (Kline and Kline, 1992a). Incubating eggs in BAPTA-AM for different durations or concentrations can be used to trap different (although generally undefined) concentrations of BAPTA in the egg cytoplasm. BAPTA loading of mouse eggs blocks Ca2+oscillations at fertilization, or after artificial activation (Kline and Kline, 1992a). Levels of BAPTA loading sufficient to completely knock out Ca2+oscillations completely blocked cortical granule exocytosis at fertilization, and eggs remained arrested in metaphase (Kline

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and Kline, 1992a). Sperm penetrated such eggs but no sperm nuclear decondensation or pronuclear formation occurred. Interestingly, if BAPTA loading was such that only the first Ca2+transient was seen, then in some cases cortical granule exocytosis occurred, but metaphase arrest was still seen. This result shows that in mouse eggs, the first Ca2+transient is not sufficient to cause exit from metaphase arrest and that cortical granule exocytosis requires fewer Ca2+transients than cell cycle resumption. This may be because exocytosis is stimulated by diacylglycerol production and subsequent stimulation of protein kinase C as well as by a Ca2+increase (Gallicano et al., 1993).

6. Monotonic Calcium Increase and Activation of the Egg In 1974 Steinhardt and colleagues demonstrated that addition of the Ca2+ ionophore, A23187, to sea urchin, frog, or hamster eggs triggered all the early signs of activation in these species. About 70% of unfertilized hamster eggs treated with A23187 formed pronuclei. This study helped set the paradigm for Ca2+ being a universal trigger for egg activation. Using a different approach, Fulton and Whittingham showed that iontophoretic injection of Ca2+ into mouse eggs induces cortical granule breakdown, release of the oocyte from the block at metaphase 11, and pronuclear formation in the majority of eggs (Fulton and Whittingham, 1978). In some cases development to the blastocyst stage was also seen. Iontophoretic injection of other ions such as Mg2+was ineffective in triggering development (Fulton and Whittingham, 1978). These two studies suggested that a Ca2+increase was sufficient to trigger resumption of the cell cycle and development of mammalian eggs. It has now been found that many different agents or methods which elicit a rapid rise in Ca2+cause some degree of parthenogenetic development in rodent eggs (Whittingham, 1980). Conversely, many agents previously known to cause parthenogenetic development are known to increase Ca2+ in the egg (Cuthbertson, 1983; Cuthbertson et al., 1981; Colonna et al., 1989;Kline and Kline, 1992a).The stimuli used for parthenogenetic activation seem to increase intracellular free Ca2+ levels either by promoting Ca2+influx, by mobilizing intracellular Ca2+,or by a combination of both. For example, exposure to 5-10% ethanol for a few minutes is an efficient and popular protocol for activating mouse eggs (Cuthbertson et al., 1981; Kaufman, 1983). This is known to cause a single, large Ca2+increase that lasts about as long as the exposure to ethanol is maintained (Cuthbertson et al., 1981; Shiina et al., 1993). Figure 5a shows the Ca2+ increase in mouse eggs caused by exposure to ethanol. This Ca2+increase is greatly reduced in eggs bathed in Ca2+-freemedium, thus suggesting that ethanol causes a Ca2+influx (Shiina et al., 1993). Ca2+ionophores such as A23187

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ratio of fluorescence

(culhwemedie, Ml6

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I

ratio of fluorescence

k

1-2,

[CulturemediiM16]

+

Z+

.9 mMCa

[

Culhrre medie, M16

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FIG.5 Recordings of Ca2' increases in unfertilized mouse eggs exposed to two commonly used parthenogenetic stimuli. Caz' is monitored using indol fluorescence ratio measurements under conditions similar to those in Fig. 1 . (a) Perfusing the egg with 7% ethanol causes a large Ca2' increase that is well maintained until ethanol is washed out. (b) When an egg is exposed to a single large electrical field pulse, there is an initial rapid Ca2+increase followed by an asymptotic decline to prestimulation levels. The conditions for electrical stimulation were those used by Rickords and White (1993).

induce a monotonic Ca2+increase similar to that of ethanol, but in this case the increased cytosolic Ca2+is mostly due to release from internal stores (Miyazaki and Igusa, 1981b; Colonna et al., 1989). Another method for parthenogenetic activation that is increasingly being adopted is electrical field stimulation. Exposure of eggs to a single electrical field (EF) pulse (>I kV/cm) in the presence of media containing Ca2+ induces a transient Ca2+influx and triggers egg activation in all species

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examined (Rossignol et al., 1983; Collas et al., 1989, 1993a; Ozil, 1990; Didion et al., 1990; Sun et al., 1992; Rickords and White, 1993; Fissore and Robl, 1992). As alluded to earlier, the EF pulse causes a transient permeabilization of the egg plasma membrane, and Ca2+ in the external medium flows in through these transient pores (Rossignol et al., 1983). Ca2+measurement after electropermeabilization shows that a single electrical pulse causes a single large Ca2+transient (Fissore and Robl, 1992; Rickords and White, 1993; Sun et al., 1992, and see Fig. 5b). The E F pulse triggers an immediate Ca2+increase followed by a decrease which generally has a negative exponential curve shape (see Fig. 5 ) . Owing to the simplicity of the electroporation technique and the fact that the strength, duration, and external Ca2+levels are readily adjusted, electroactivation is now one of the most widely used methods for inducing parthenogenetic development. It is invariably used for activation in techniques designed for cloning embryos (Prather and First, 1990). All the methods of parthenogenetic stimulation discussed so far cause a single monotonic Ca2+ increase; they do not induce a series of Ca2+ oscillations as seen at fertilization (see Section I). This suggests that a single, long-lasting Ca2+increase is sufficient for triggering development. In this case one might well ask why the sperm causes sustained oscillations. Since mammals appear to have evolved a dynamic signaling mechanism that is not seen in other animal eggs, it seems inevitable that they have some purpose. It would be useful to establish if there was any aspect of the egg response that is better suited to an oscillatory stimuli. Before discussing the response of eggs to oscillatory stimuli, we outline some of the drawbacks of stimuli that cause a monotonic Ca2+increase. A main feature of the mammalian egg's response to most parthenogenetic stimuli is that it improves as the eggs age (Kaufman, 1983; Whittingham, 1980). Freshly ovulated eggs are very resistant to artificial activation. Sometimes when freshly ovulated eggs are exposed to parthenogenetic stimuli they do not enter interphase but instead extrude a second polar body, and the remaining haploid set of chromosomes fails to decondense and form a new metaphase, referred to as metaphase I11 (Kubiak 1989). All the above parthenogenetic stimuli are alike in that low levels of activation (<20%) are generally obtained when freshly ovulated eggs are treated with ethanol, electrically stimulated, or injected directly with Ca2+ (Robl and Stice, 1989; Collas and Robl, 1990; Onodera and Tsunoda, 1989; Marcus, 1990; Fulton and Whittingham, 1978). In contrast, nearly any parthenogenetic stimuli can activate >90% of eggs that are aged with respect to the time of ovulation. In mouse eggs, the optimum timing between ovulation and effective parthenogenetic activation has been found to be 8 hr (i.e., 20 hr post-HCG, Webb et al., 1986).

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This aging of oocytes before activation leads to practical problems. Changes associated with postovulatory aging lead to an increase in the incidence of chromosomal segregation errors at the second meiotic division in embryos (Eichenlaub-Ritteret al., 1986; Kaufman, 1983; Kaufman and Surani, 1974). Aging also creates a problem because aged eggs are less viable (Kaufman, 1983; Hunter, 1967). Parthenogenetic activation of aged eggs produces a variety of activation types with different nuclear configurations. There are reports of activated eggs that immediately cleave, or else contain one pronucleus and one second polar body, or two pronuclei and no second polar body, or one pronuclei with no polar body (Whittingham, 1980; Kaufman, 1983). It has also been shown that there is a spontaneous central migration of the second meiotic spindle in response to postovulatory aging, and that the proportion of activated oocytes that have aged, which belong to the two pronuclei or immediate cleavage type, dramatically increase (Kaufman, 1983). The reasons why eggs become more susceptible to activation with age is not clear. It may be that eggs buffer Ca2+less easily with age and hence Ca2+transients may last longer in older eggs (Vincent et al., 1992), but a decrease in buffering capacity is not always seen in mammalian eggs (Fissore and Robl, 1992). Another explanation may be that the increased susceptibility to activation is due to a gradual loss of the proteins that are needed to arrest metaphase. It is known that protein synthesis is necessary for maintenance of the meiotic block at metaphase I1 in mouse oocytes because eggs will complete meiosis when protein synthesis is inhibited by treatment with cycloheximide or puromycin (Siracusa et al., 1978; Whittingham, 1980). A Ca2+signal may become more effective in aged eggs if there is reduced synthesis of proteins that arrest the cell cycle. In this context it should be noted that where better activation rates (-50% pronuclear formation) have been observed on recently ovulated eggs with a monotonic Ca2+stimulus, it involved activation of mouse eggs with Ca2+ionophore A23187 applied in Ca2+-freemedia (Vincent et al., 1992). It was also found that applying Ca2+ionophores in Ca2+-freemedia greatly enhanced activation rates of hamster eggs (Steinhardt et al., 1974). The use of Ca2+-freemedia does not appear to enhance the ionophoreinduced Ca2+transient (Miyazaki and Igusa, 1981b; Eusebi and Siracusa, 1983). So, it is possible that the ionophore treatment works better in Ca2+-freemedia because the combination has other effects. Notably, the combination of Ca2+ionophore plus Ca2+-freemedia is a potent inhibitor of protein synthesis in mammalian somatic cells (Brostom and Brostom, 1990), and as just mentioned, inhibition of protein synthesis is an effective way to enhance activation in mammalian eggs (Whittingham, 1980). The effects of aging on activation of eggs are also seen in more recent studies using second messengers to cause Ca2+release. A single bolus

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injection of I ~ S Pinto , mouse eggs causes a single large Ca2+transient (Kline and Kline, 1992b), which is not sufficient to cause pronuclear formation in mouse eggs despite the fact that exocytosis of cortical granules occurs (Kurusawa et d . , 1989). Injecting InsP, or GTP analogs was not even very effective in causing exocytosis in hamster and sheep eggs unless the pH of the medium was raised (Cran et al., 1988). Elevation of media pH probably enhances the duration of InsP,-induced Ca2+transients by inhibiting plasma membrane Ca2+-ATPase(Georgiou et al., 1988). Whatever the full explanation for the need for aging of the egg, it is generally found that fertilization is more efficient than artificial stimuli (Whittingham, 1980). This general feature is constant among mammalian eggs since it is seen in rabbits (Onodera and Tsunoda, 1989), cows (Nagai, 1987; Collas et al., 1993b), and pigs (Prather et al., 1991), as well as mice. Human eggs also appear to be difficult to activate with the conventional treatments that cause monotonic Ca2+ increases (Balakier and Casper, 1993). Simply increasing the amplitude of single Ca2+changes by increasing the strength of the parthenogenetic stimuli does not appear to alleviate the problem of poor and variable rates of activation. Some experiments suggest that repetitive Ca2+stimulation is different. C. An Oscillatory Calcium Signal and Activation of the Egg

To investigate the function of repeated Ca2+ increases on mammalian embryo development, one really needs an efficient method of triggering Ca2+oscillations. Adding the sulfhydryl reagent, thimerosal, to unfertilized mammalian eggs is the simplest and most effective way of artificially causing Ca2+oscillations (Swann, 1991, 1992; Fissore et al., 1992). However, thimerosal appears to have many side effects and is of little practical use in activation studies (K. Swann and A. Bos Mikich, unpublished observations). Instead, another simple parthenogenetic treatment that causes Ca2+ oscillations is incubation in strontium medium. Incubating mouse eggs in standard culture media with Ca2+replaced by equimolar or higher concentrations of strontium activates mouse eggs and triggers several Ca2+spikes (Kline and Kline, 1992a) (see Fig. 6a). The exact way in which strontium works to release Ca2+in mammalian eggs is unknown, but it has been shown to trigger Ca2+release from isolated sea urchin egg endoplasmic reticulum, and it may work comparably on mammalian egg reticulum (Lee, 1993). As with many other parthenogenetic treatments, the postovulatory age at the time of the treatment influences the proportion of eggs activated (Whittingham, 1980). However, strontium causes high rates (-90%) of pronuclear formation of mouse egg treated 15 hr post-HCG injection (A. Bos Mikich, unpublished data).

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a fluorescence

ratio Indo-1

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FIG. 6 Recordings of Ca2+changes in unfertilized mouse eggs using indol fluorescence as in Fig. 1 and Fig. 5 . (a) An egg exposed to media containing 10 m M strontium instead of Ca2+.(b) An egg that was exposed to a series of electrical field pulses ( E F pulses) once every 4 min. E F pulses (0.75 kV/cm, 1 msec) were applied in media containing glucose and 40 p M Ca*+,with normal culture media reperfused during the interval between pulses (see Ozil and Swann, 1993, for further methods).

Given the reliability of activation types, strontium is arguably the best simple activation treatment for mouse eggs. This may be because it causes oscillations in CaZ+rather than a monotonic increase. However, a problem with strontium is that the number of Ca2+ oscillations seen in any one egg, or batches of eggs, tends to vary and it is not clear if this slight variability correlates with some of the variable degree of development. To answer this question, we need a more precise method of controlling the Ca2+transients.

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Another novel method to artificially generate repetitive Ca2+is to use a series of repetitive electrical field pulses. For eggs to survive this kind of treatment, they have to be exposed to electropermeabilization in a specially designed chamber in which the washing procedure for replacement of media is highly efficient (Ozil, 1990; Ozil and Swann, 1993). This technique enables one to cause distinct patterns of Ca2+ spikes in eggs, as can be seen in Fig. 6B. Using such a system, it has been shown that repetitive electropermeabilization for 2 hr in Ca2+-containingmedium can trigger pronuclear formation in freshly ovulated mouse eggs at rates of up to 100% (12 hr post-HCG) (Vitullo and Ozil, 1992). Figure 7 shows examples of batches of mouse eggs exposed to repetitive electrical field pulses and illustrates the uniformity of response that is obtained by any one treatment protocol. In these experiments, the rate of pronuclear formation was also shown to be strictly dependent upon the precise pattern of pulses or range of Ca2+in the pulsating media (Vitullo and Ozil, 1992). These data suggest that all the problems with poor activation rates with parthenogenetic stimuli could be overcome if a repetitive Ca2+signal is applied. The same general result using repetitive stimulation was also seen in freshly ovulated rabbit eggs (Ozil, 1990). Furthermore, it was also shown that these rabbit eggs parthenogenetically activated by repetitive stimulation went on to develop after implantation in foster mothers. Although development arrested at around 10-1 1 days after activation, it was found that the morphology of rabbit embryos depended upon the pattern of stimulation in the first few hours of day 1 (Ozil, 1990). The extent and uniformity of development in these studies was considerably greater than that seen using other parthenogenetic stimuli in rabbits or mice. As well as emphasizing that a repetitive Ca2+stimulus is better at activating eggs, these results showed that when the pattern of stimulation is controlled and uniform across a population of eggs, then the response (in terms of development) is fairly uniform. It is an attractive idea that development of mammalian embryos is affected by the nature of the repetitive Ca2+ signal at activation, not least because it has the potential to explain some of the variability in development after fertilization. As mentioned earlier, there is considerable variation in the frequency of Ca2+oscillations at fertilization, even with a single species and with similar methods for measuring Ca2+(Cuthbertson and Cobbold, 1985; Kline and Kline, 1992a; Fissore et al., 1992). In many species there is also considerable variation in the speed of early cell cycles and in the number of embryos arresting in early development (Fissore et al., 1992). It is possible that some parameters associated with oscillations at fertilization, such as frequency or number of Ca" spikes, are encoded by the egg in a manner that determines whether and how rapidly the

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FIG. 7 Freshly ovulated (12 hr post-HCG) mouse eggs parthenogenetically activated by repeated EF pulse stimulation (exact conditions given in Vitullo and Ozil, 1992). (a) Mouse eggs were exposed to 33 EF pulses in the presence of 12 pM Ca2+and all eggs treated formed pronuclei 4 hr later. (b) Mouse eggs were exposed to the same protocol except EF pulses were of decreasing strength (as in Vitullo and Ozil, 1992) and all eggs extruded second polar bodies but did not form pronuclei and arrested between metaphase I1 and interphase. Magnification x 154.

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embryo develops. As yet, there have been no attempts to correlate variations in Ca2+signals at fertilization with variations in subsequent development. D. Decoding the Calcium Signal

The primary effect of Ca2+at fertilization in vertebrate eggs is the resumption of meiosis and pronuclear formation. In frog eggs it is known that the resumption of these events in the cell cycle is achieved by the disappearance of maturation-promoting factor (MPF) as well as destruction of a cytostatic factor (CSF) involved in the maintenance of MPF activity (Masui, 1991;Whitaker and Patel, 1990).MPF is a stoichiometric complex between cyclins and the cdc2 +-encoded protein kinase whose activity can be measured using exogenous histone H1 as a substrate (Labbe et al., 1989; Gauthier et al., 1990). The principal component of CSF appears to be the c-mos oncogene protein product (Masui, 1991; Watanabe et al., 1991). In frog eggs, a single Ca2+spike at fertilization is sufficient to cause cyclin degradation and loss of MPF activity in extracts prepared from metaphase II-arrested Xenopus eggs (Lorca et al., 1991). Recent experiments show that the Ca2+increase causes MPF activity to fall by binding to and stimulating a calmodulin-dependent protein kinase I1 (CAM-I1kinase) (Lorca et al., 1993). The calmodulin-dependent pathway leads to a loss of MPF activity and inactivation of and subsequent destruction of CSF (Watanabe et al., 1991). The results in frog eggs provide clues as to how Ca2+oscillations may be transduced in mammalian fertilization. The experiments using repetitive EF pulses in mouse eggs show that cell cycle reinitiation rates are driven by the pattern of Ca2+ spikes. In this case the destruction of cyclins and loss of MPF activity could be an integral function of Ca2+transients in mammalian eggs. This is supported by measurements in bovine eggs of HI kinase that is used as an assay of MPF activity (Collas et al., 1993b). It was shown that a single electrical pulse, which causes a single Ca2+ spike, led to a rapid decrease in HI kinase activity, but that H1 kinase activity returned after a few hours. However, if six pulses and hence six Ca2+spikes were induced, there was a loss of H1 kinase for at least 8 hr (Collas et al., 1993b). The finding that MPF activity may return to preactivation levels after only a single Ca2+transient occurs may explain why sometimes extrusion of the second polar body occurs but a female pronucleus fails to form (Kubiak, 1989). It remains to be established if an isoform of CAM-I1 kinase is responsible for the loss of H1 kinase activity in mammalian eggs. It is also unclear what role a c-mos-associated CSF plays in mammals

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since c-mos protein levels remain high after meiosis has resumed in mouse eggs (Weber et al., 1991). CAM-I1kinase is a good candidate for signal transduction in mammalian eggs because it is thought to be a Ca2+binding target protein implicated in transducing cell Ca2+oscillations. Meyer and colleagues (1992) have outlined a scenario for how CAM-I1 kinase may sense Ca2+spikes. Ca2+calmodulin formed during each Ca2+ increase binds to CAM-I1 kinase. This is followed by autophosphorylation of the CAM-I1 kinase, which slows down the release of bound calmodulin even after Ca2+levels have declined. The calmodulin is therefore trapped on CAM-I1 kinase and maintains its activity for some time after Ca2+levels have declined. This makes CAM-I1 kinase an effective target for single brief Ca2+ spikes, as suggested for controlling entry into mitosis in sea urchin embryos (Steinhardt, 19901, and in exiting from meiosis in frog eggs (Lorca et al., 1993). Over long periods CAM-I1 kinase-induced phosphorylation may also provide a mechanism for counting Ca2+spikes or else responding to the frequency of Ca2+oscillations (Meyer et al., 1992; Meyer and Stryer, 1992). Since CAM-I1 kinase isoforms are found in many cell types, they may be the targets for decoding Ca2+oscillations seen in somatic cells, as well as in eggs. Although we suggest that a CAM I1 kinase is involved in mammalian egg activation, there is little direct evidence. In contrast. there is more direct evidence for a role for protein kinase C in mammalian egg activation. Phorbol esters and diacyglycerols stimulate protein kinase C and can cause some exocytosis, second polar body emission, and pronuclear formation in mouse and hamster eggs (Cuthbertson and Cobbold, 1985; Colonna et al., 1989; Endo et al., 1987; Gallicano et al., 1993). Phorbol esters at high concentrations have been shown to cause small Ca2+oscillations in mouse eggs (Cuthbertson and Cobbold, 1985). However, a Ca2+increase is not necessary for protein kinase C simulators to activate mouse or hamster eggs, or for phorbol esters to trigger patterns of protein phosphorylation similar to those seen after fertilization (Colonna et al., 1989). It is likely that the protein kinase C stimulation is downstream of a Ca2' transient and therefore protein kinase C may be a primary target of the oscillations at fertilization (Gallicano et al., 1993). Since Ca2+ ionophores induce patterns of phosphorylation similar to those of protein kinase C activators (Colonna et al., 1989), Ca2+ may cause DAG production, which then stimulates protein kinase C, as suggested in frog and sea urchin eggs (Bement, 1992;Whitaker and Aitchison, 1985; Swann and Whitaker, 1990). It is not clear how this method of stimulating protein kinase C would respond to specific series of Ca2+spikes, but the level of stimulation at fertilization has to be modulated since prolonged overstimulation of pro-

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tein kinase C causes reabsorption of the second polar body (Gallicano et al., 1993). Experiments using repetitive electrostimulation of rabbit eggs suggest that, in addition to affecting the very earliest events in development, such as cell cycle resumption, the Ca2+ oscillations have subtle effects on later pre- and postimplantation development. It is known that there is a developmental program set off at fertilization that involves changes in synthesis of proteins and in protein phosphorylation (McConnell, 1990; Howlett and Bolton, 1986). These changes ultimately lead to the expression of the embryonic genome, which then determines later development (Telford et al., 1990). In mice the expression of the embryonic genome is switched on at the two-cell stage strictly according to a phosphorylation pattern clock that is started at fertilization (Poueymirou and Schultz, 1990). This clock is probably started by the Ca2+ signal so it is not impossible that the pattern of oscillations may influence the later expression of the embryonic genome. However, the Ca2+oscillations at fertilization appear to last for only a few hours and the embryonic genome starts days later (Telford et al., 1990). Also, the available evidence suggests that phosphorylation events are similar in fertilized eggs, which undergo Ca2+oscillations, and in parthenogenetically activated ones, which have a single Ca2+ transient (Colonna et al., 1989; Sun et al., 1992). If Ca2+oscillations at fertilization are to influence later development, then small initial differences in the signal must become increasingly significant during early development. Since providing the egg with a repetitive parthenogenetic stimulus improves the extent to which they undergo postimplantation development, it is worth considering the developmental potential of mammalian parthenogenotes. Studies using monotonic Ca2+stimuli show that although preand postimplantation development of diploid parthenogenetic embryos is possible, normal organogenesis does not progress beyond the early somite stage. Eggs with two female pronuclei can sometimes develop as far as the 25-somite stage, but they die because the extraembryonic tissues are poor (Surani and Barton, 1983; Surani et al., 1984). Parthenogenotes fail to develop to term because mammalian development is influenced by parental genome imprinting, which results in differences in the expression of some homologous maternal and paternal alleles; the presence of both parental pronuclei is required for normal development (Surani et al., 1984). Ethanol-activated mouse eggs that receive either one additional male pronucleus from a fertilized egg (Surani and Barton, 1983) or both male and female pronuclei from another fertilized egg after being enucleated (Mann and Lovell-Badge, 1984) are able to develop to term and provide viable young. Therefore, the cytoplasm of activated eggs which is associated with a single large Ca2+is fully competent to support development to term

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after activation. This in turn implies that the extent to which parthenogenetic development occurs is set by the effects of paternally imprinted genes. Repetitive Ca2+stimulation can be expected to improve the rate and degree of parthenogenetic development, but there are good reasons for never expecting live young.

V. Summary and Future Directions We have reviewed many aspects of Ca2+changes at fertilization in mammals that have been discovered in the past decade or so. One of the main points that we have emphasized is that mammalian eggs are different from other eggs in that they undergo Ca2+oscillations, or repetitive Ca2+spikes, during activation at fertilization. One of the central problems to be solved is the triggering mechanism for the oscillations at fertilization. Ca2+oscillations have been described in a vast range of somatic cell types. In many cases the basic components of the signaling systems have been established. It is remarkable that the problem of signal transduction involving Ca2+ release in eggs at fertilization has not been solved in any species. There is still a split between the idea that the sperm either acts upon cell-surface receptors that somehow stimulate InsP, production, and the idea that the sperm introduces a soluble factor into the egg after fusion (Whitaker and Swann, 1993). Mammalian eggs can offer special advantages for resolving this debate because their Ca2+signal at fertilization represents a fingerprint response that is not matched by parthenogenetic agents or injected Ca". This is unlike virtually all other types of egg, where the sperm's single Ca2+ increase cannot be distinguished from a range of imitators. For example injecting Ca2+buffers, InsP3, cyclic GMP, GTP analogs, or cyclic adenosine diphosphate ribose (ADPR) all cause a similar Ca2+transient to fertilization in the sea urchin egg (Whitaker and Swann, 1993; Shen, 1992). None of these agents precisely mimics the sperm in hamster and mouse eggs. Whatever messenger is proposed for fertilization in mammals, it has to be able match the fingerprint response pattern of fertilization. Examining the fingerprint requires measurement of the Ca2+dynamics. The observation that mammalian eggs undergo Ca2+oscillations when stimulated also suggests that they are better models for somatic cell proliferation than the more commonly studied sea urchin and frog eggs. Ca2+ oscillations occur in many somatic cells in response to growth factors or serum (Metcalf et al., 1986; Polverino et al., 1991). These oscillations are often correlated with the triggering of cell replication (Metcalf et al., 1986). Ca2+oscillations that appear to be autonomously generated have also been detected at key control points during the cell cycle (Kao et al., 1990;

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Whitaker and Patel, 1990). In some cases the presence of sustained Ca2+ oscillations in response to hormones has been linked to cell transformation (Fu et af., 1991). Despite these correlations, the precise function of Ca2+ in growth stimulation is not clear. One aspect of this problem that is often overlooked is that the Ca2+is signal is oscillatory. Experiments to investigate how Ca2+is involved in somatic cell proliferation have used Ca2+ionophores, which cause monotonic changes in Ca2+,not oscillations (Metcalf et af., 1986). There do not appear to have been any attempts to look at the specific effects of causing different patterns of repetitive Ca2+ stimulation on somatic cell cycle progression. Some studies in somatic cells suggest that there is frequency tuning of responses to Ca2+spikes. For example, in isolated pituitary cells, artificially generated CaZ+spikes have to be tuned to a specific frequency to obtain maximal secretion of prolactin (Law et al., 1989). The use of electrical field pulses, as pioneered in mammalian eggs, may be a novel approach to see how defined Ca2+ spikes affect somatic cell proliferation. The studies that examine the role of repetitive Caz+stimulation in mammalian eggs suggest a feature of cell Ca2+ signaling that has not been evident from studies in any other cell type. It appears that when all eggs are exposed to the same pattern of Ca2+ pulses, with defined electrical pulses, there is a uniform response in the population (Ozil, 1990; Vitullo and Ozil, 1992). Hence, within limits, the same starting conditions give rise to the same type of development and in this sense the CaZ+stimulation pattern is deterministic. In addition, relatively small differences in the pattern of Ca” stimulation affect the first cell cycle only slightly by altering its length, but have much greater effects on the gross morphology and size of embryos 10 days later (Vitullo and Ozil, 1992; Ozil, 1990). The implication is that the system is deterministic but that small differences in starting conditions have increasingly greater consequences with time, as development proceeds. This behavior is a common feature of dynamic systems that are chaotic or highly complex, in which two closely related variables can diverge exponentially with time (Grebogi et al., 1987; Wolfram, 1984). This sensitivity to initial conditions is commonly known as the “butterfly effect.” It is possible that a parameter associated with the CaZ+signal can change exponentially with time during development. If this is the case, then the difference between the Ca2+signals during parthenogenetic activation and fertilization may result in only very small differences in the initial patterns of protein synthesis, protein phosphorylation, or whatever else is targeted by Ca2+.This may explain why there have been no immediately detectable differences in protein phosphorylation, or protein synthesis pattern between eggs that are activated by monotonic Ca2+ stimuli and fertilized eggs that are activated by oscillations (Colonna et af., 1989; Sun et al.,

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1992). Instead of looking for small differences at the start of development, it may be more productive to try and understand and model how Ca2+induced changes alter with time. We anticipate that significant further progress in our understanding of how Ca2+oscillations activate mammalian eggs will come from studying changes in eggs in real time. For example, it will be invaluable to monitor Ca2+changes simultaneously with an associated parameter such as protein phosphorylation, at the single-cell level. This would allow us to examine directly how oscillations are transduced. There is an increasing range of fluorescent probes and agents being designed that can be used for monitoring and modulating second messengers and their effectors in living cells (Tsien, 1992). The large size and relative ease of microinjection into eggs suggests that these new tools may be fruitfully applied to problems of understanding the initiation of mammalian development.

Ac knowledgrnents We thank David Whittingham for supporting and encouraging our work, Adriana Bos Mikich for discussions and letting us present some of her unpublished data, and John Carroll for comments and discussion of the manuscript. This work was funded by the Medical Research Council, Institut National de la Rechikche Agronomique, and a traveling fellowship awarded to J. P. Ozil by the British Council and Ministtre de la Rech&rche et de I’Espace.

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Regeneration of Mammalian Retinal Pigment Epithelium Gary E. Korte,* Jay I. Perlman,t and Ayala Pollackt *Departments of Ophthalmology and Visual Sciences, and Anatomy and Structural Biology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 10467; ?Department of Ophthalmology, Loyola University Medical Center, Chicago, Illinois; and fKaplan Hospital, Rehovot, Israel, and Hadassah Medical School, Hebrew University, Jerusalem, Israel

I. Introduction

The retinal pigment epithelium (RPE) is a simple cuboidal epithelium that lies on a basement membrane. To appreciate the importance of repair, or regeneration, of the RPE, consider some of its functions in the embryonic and mature eye. In the embryonic eye, the RPE is an organizer. It influences the formation of the sclera, the differentiation of photoreceptor outer segments and the choroidal capillary bed (the choriocapillaris),which is the main source of nutrition for the photoreceptors (Mann, 1950; CoulombrC, 1979). It elicits the layering of embryonic retinal cells and differentiation of photoreceptor outer segments in culture (Liu et af., 1988; Spoerri et af., 1988; Wolburg et a!., 1991). In cold-blooded animals and embryonic warm-blooded ones, the RPE can transdifferentiate to replace neural retina (CoulombrC and CoulombrC, 1965; Coulombrt, 1979; Stroeva and Mitashov, 1983; Park and Hollenberg, 1991; Hitchcock and Raymond, 1992). In the mature eye the RPE influences two tissues that flank it-the photoreceptors and the choriocapillaris (Bok, 1985; Korte et af., 1989a). For example, the photoreceptors atrophy when the neural retina is detached from the RPE, and the atrophy stops and even reverses with reapposition of the retina to the RPE (Hollyfield and Witkovsky, 1974; Fisher and Anderson, 1989; GuCrin et al., 1989,1993; Kaplan et al., 1990). Experimental evidence and pathologic observations attest to the apparent interdependence between RPE and choriocapillaris in the mature eye. For example, the choriocapillarisatrophies next to dysfunctional RPE in eyes International Review of Cytology, Vol. IS2

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from patients with retinitis pigmentosa (Henkind and Gartner, 1983) and after the RPE is selectively damaged in rabbits (Korte ef al., 1984a). These poorly understood trophic interactions are complemented by better-understood contributions by RPE cells to the blood-retinal barrier, turnover of photoreceptor outer segment membrane, the vitamin A cycle, elaboration of a special extracellular matrix (the interphotoreceptor matrix) in the tissue space containing the outer segments of the photoreceptors (the subretinal space), and retinal adhesion (Bok, 1988; Hewitt and Adler, 1989; Berman, 1991). Disease or injury to the RPE upsets these functions. Unless the RPE can heal, that is, regenerate its normal structure and function, the photoreceptors and choriocapillaris atrophy. Dysfunctional RPE may contribute to the photoreceptor atrophy seen in retinal diseases, such as age-related macular degeneration and central serous retinopathy (Morris and Henkind, 1979; Eagle, 1984; Spitznas, 1986; Young, 1987). This chapter focuses on the RPE regeneration seen in several experimental situations, as a model of what happens in human retinal disease. By regeneration we mean the replacement of the damaged RPE sheet by a new one, involving cell proliferation and migration to produce new cells that differentiate into epithelium with the structural and functional characteristics of normal RPE. This is opposed to the recovery of surviving but functionally compromised RPE cells, for example, which may produce retinal edema (Spencer, 1985; Spitznas, 1986) The emphasis is on the mammalian RPE. Observations in mammals most directly elucidate the RPE response to injury during human retinal disease and the biology of RPE and photoreceptors transplanted into diseased retinas (Bok, 1993 for review). The success of these efforts depends on the reestablishment of normal interactions between RPE, photoreceptors, and choriocapillaris. For example, normal RPE cells may exert a trophic effect that helps “rescue” photoreceptors when they are transplanted into the retina of rats with dysfunctional RPE (Li and Turner, 1988; Lopez et al., 1989).

II. RPE Regeneration in the Mammalian Eye

Several animal models regenerate the RPE after it is destroyed or its environment altered. A. Laser Lesions

Many studies, mainly in monkeys, have documented the ability of RPE to repair itself after laser damage (for example, Tso et al., 1972; Ishikawa

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et al., 1973; Biilow, 1978; Wallow, 1984; Miller et al., 1990; Pollack and Korte, 1990). These studies show that new RPE replaces necrotic RPE at a laser site. The lesion heals by a combination of spreading and proliferation of surrounding RPE cells. Some of the cells migrate into the lesion along the RPE basement membrane, which is left bare after macrophages remove necrotic RPE cells. The cells often pile up on each other, and extracellular matrix accumulates among them, disappearing as the lesion heals (e.g., Pollack and Korte, 1990). If the wound is not too large, a complete reepithelialization occurs, with re-formationof normal morphology and function by the RPE cells, for example, intact barrier function due to re-formation of tightjunctions. If the wound is too large for complete reepithelialization, a scar forms in the central portion of the lesion and restricts epithelial regeneration to its periphery. The interactions between RPE and choriocapillaris previously mentioned appear active at laser sites. For example, in studies of the RPE and choriocapillaris response in lasered rats, Pollack and Korte (1990, 1992a) observed that the initial regeneration of these tissues occurs simultaneously, and that secondary atrophy of the choriocapillarisas it remodels during regeneration occurs next to RPE with a characteristic phenotype.

B. Other Models

Excess light has detrimental effects on photoreceptors that extend over large areas of the fundus (Lanum, 1978). In rats with phototoxic retinopathy, some of the responses seen at laser sites are also seen at sites of focal hyperplasia and where retinal capillaries enter the epithelium as photoreceptors atrophy. In phototoxic rats, the photoreceptor atrophy thins the outer neural retina to such an extent that the retinal capillary bed becomes focally apposed to the RPE. At these sites the retinal capillaries become intraepithelial in position and undergo remarkable changes (Bellhorn et al., 1980; Korte et al., 1983; Shiraki et al., 1983; Korte et al., 1986a). For example, they form fenestrae, which retinal capillaries normally do not have. The RPE at these sites spreads out along the capillaries and uses them as avenues of migration into the neural retina. Some of these migrating cells, which probably arise from proliferating RPE cells, appear immature and have lost the structural and functional polarity of normal RPE. They thus exhibit similarities to RPE cells at repairing laser sites. Similar changes in the phenotype of RPE cells occur in rats with urethane-induced retinopathy and hypertensive retinopathy (Korte et al., 1984b; Mancini et al., 1986). Where RPE cells exhibit focal hyperplasia in rats with phototoxic retinopathy, the cells proliferate and form a mound between the RPE base-

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ment membrane and the neural retina. The cells in the mound are immature, with poorly developed apical processes and basal infoldings, and lack the structural polarity of normal RPE. Yet, the cells directly facing the neural retina can have structural and functional specializations such as apical villi and tight junctions that maintain the blood-retinal barrier at these sites (Korte and Hirsch, 1986). A similar migration of cells occurs where proliferating RPE covers subretinal neovascularizations at laser sites (Miller et al., 1990; Pollack and Korte, 1993) and at sites of retinal detachment (Anderson et al., 1983). When the neural retina detaches from the RPE, the epithelium dedifferentiates. For example, in cats, apical processes of RPE cells retract and proliferation produces clusters or layers of undifferentiated cells. These can redifferentiate to the point of being able to phagocytose the shed outer segments of reapposed photoreceptors and produce new basement membrane (Anderson et al., 1986; Fisher and Anderson, 1989). This also occurs in monkeys with detached retinas and in cultured human RPE cells transplanted onto Bruch’s membrane (Kroll and Machemer, 1968, 1969; Gouras et al,, 1985). In a study of mechanically wounded RPE in rabbits, Heriot and Machemer (1992) also describe cell division and spreading to close the wound, and the production of mounds of undifferentiated cells that can express some of the polarized structure of normal RPE cells. For example, the cells in the mound that face the subretinal space have apical villi and tight junctions that reestablish some phagocytosis and barrier functions. The authors point out that similar events probably occur at healing tears and rips in the human retina. Ophthalmoscopic observations support this (Kanno et al., 1993). C. Overview of Cytologic Changes during RPE Regeneration

The above examples reveal some basic changes that occur in RPE during regeneration. Spreading and proliferation produce motile, undifferentiated cells that close the wound. Barrier function breaks down due to disruption of tight junctions, with re-formation of tight junctions as the immature cells differentiate. The cells elaborate extracellular matrix and basement membrane. They pile up and form mounds, the cells within the mound appearing immature and lacking the structural polarity of normal RPE. These observations suggest that changes in the plasma membrane, cytoskeleton, and extracellular matrix accompany RPE regeneration. To begin studying these processes, we have used an animal model in which RPE regeneration occurs with a consistent time course and pattern-rab-

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bits injected intravenously with sodium iodate (Korte et al., 1984a, 1989a). This chemical destroys large expanses of RPE throughout the fundus, leaving foci of spared cells that serve as sources of a regenerating epithelial sheet. The cells in such sheets exhibit a gradient in their differentiation. Immature, undifferentiated cells are at the leading edge of the regenerating epithelial sheet. They grade into more mature cells that appear similar to normal RPE. This has been a useful model for examining the ability of RPE cells to re-form the structural and functional polarity seen in normal RPE. D. Polarity of Normal RPE

The RPE of all vertebrates exhibits a striking structural and functional polarity. This is manifested as differences in organization, molecular composition, and function along the basolateral and apical plasma membranes (Fig. IA). The lateral and basal domains of the basolateral plasma membrane differ significantly in RPE cells. The basal plasma membrane directly facing the basement membrane on which the RPE lies has myriad folds and tubules (Hogan et al., 1971; Korte, 1984; Korte and Goldberg, 1986). Punctate attachment sites connect it to the basement membrane and it has numerous coated pits that are active sites of endocytosis for the RPE cell (Miki et al., 1975; Orzalesi et al., 1982; Perlman et al., 1989). Several molecules involved in attaching RPE cells to the basement membrane and in signaling between the RPE cells and the microenvironment of Bruch’s membrane are localized here. The basement membrane and the connective tissue of Bruch’s membrane contain fibronectin; types I, 111, and IV collagen; laminin; heparan sulfate; and chondroitin sulfate proteoglycans. The attachment sites contain p l integrin, talin, and vinculin (Turksen et al., 1985; Campochiaro et al., 1986; Opas and Kalnins, 1986; Philp and Nachmias, 1987; Sramek et al., 1987; Lin, 1989; Turksen et al., 1989; Das et al., 1990; Philp et al., 1990; Rizzolo and Heiges, 1991; Lin et al., 1992b). The lateral plasma membrane is flat, that of adjacent cells connected only by infrequent punctate attachments and, apically, by the intercellular junctional complex. Unlike the basal plasma membrane, the lateral membrane has virtually no coated pits (Perlman et al., 1989). Smooth endoplasmic reticulum and mitochondria focally appose the lateral plasma membrane. A junctional complex separates the basolateral and apical plasma membrane domains. The latter faces the tissue space (subretinal space) containing the outer segments of the photoreceptors. The junctional com-

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plexes are similar to those seen in other transporting epithelia; they consist of a zonula adherens, zonula occludens (tight junctions), and gap junctions in close proximity to each other (Hudspeth and Yee, 1973). The tight junction seals off the tissue space of the neural retina from that of the choroid and is the site of the so-called “outer” blood-retinal barrier (the inner one being located at the tight junctions between retinal capillary endothelial cells; Raviola, 1977). The zonula adherens is composed of a circumferential bundle of actin filaments associated with vinculin, myosin, tropomyosin, and a-actinin (Owaribe and Masuda, 1982; Opas and Kalnins, 1985a; Opas et al., 1985; Turksen and Kalnins, 1987; Turksen et al., 1989). The nearby cytoplasm contains vimentin intermediate filaments, ankyrin, and fodrin (Matsumoto et al., 1990; Opas and Kalnins, 1985b; Gundersen et al., 1991). The apical plasma membrane has many villar or lamellar folds that contain longitudinal arrays of actin filaments (Burnside and Laties, 1979). The folds are involved in one of the most important functions of the RPE-the phagocytosis of shed photoreceptor outer segment membrane. Their plasma membrane contains a mannose receptor that may recognize plasma membrane from photoreceptor outer segment or lysosomal enzymes released into the subretinal space (Mishima and Kondo, 1981; Wilcox, 1987; Tarnowski et al., 1988a,b; Shepherd et al., 1991; but see Hall and Abrams, 1991). The basolateral and apical plasma membranes of RPE differ in their molecular constituents (Table I). While the functional significance of the polarized distribution of some plasma membrane components is unknown (e.g., the enrichment of alkaline phosphatase in the basolateral plasma membrane; Korte et al., 1991), others are important in transport of molecules, water, and ions into and across the RPE. A range of ion transporters,

FIG. 1 Normal and regenerating RPE of the rabbit. (A) Electron micrograph of normal RPE. Apical villi (V; with photoreceptor outer segments abutting them) are at the top and basal folds (F) are at the bottom of the cell. Note the mitochondria in the cytoplasm above the basal folds and black, oval melanosomes in the cytoplasm below the apical villi. The arrow denotes a junctional complex. The arrowheads denote basement membrane, with fenestrated endothelium of the choriocapillaris beneath it. x 6500. (B) Light micrograph of normal rabbit RPE flanked by photoreceptors (os, outer segments; is, inner segments). The choriocapillaris runs along the bottom of this and subsequent pictures. x 160. (C,D) Light micrographs of regenerating RPE (arrow) I week after administration of sodium iodate. Cells removed from the leading edge of the regenerating epithelial sheet (in C) still contain scattered melanosomes, while those near the leading edge (in D) are smaller, flatter, and contain no melanosomes. Inner and outer segments of photoreceptors are shorter due to atrophy secondary to RPE damage (cf. B). Macrophages (m) occupy the subretinal space between the photoreceptors and RPE. x 160.

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Location Apical a-1 adrenergic receptor Aminopeptidase Ankyrin Fibronectin Fodrin Glucose transporter pl integrin Mannose receptor Mn-dependent nucleotidase Na-bicarbonate transporter Na+ K+-ATPase Na-K-CI transporter Transfemn receptor Basolateral Alkaline phosphatase Fibronectin Glucose transporter Integrin, 120 kDa RBP receptor Transfemn receptor

(12) (13) (14) (15) (16)

(17) (18)

References" 1

2 3 4

5 6 7 8

9 10 I1 12 13 14 15 16 17 18 19

See Lin et a / . (1992a). Gundersen e r a / . (1991). Gundersen er a/. (1991). Pino, (1986); Fisher and Anderson, (1989). Gundersen et al. (1991). Takata er a / . (1992). Anderson er al. (1990); Rizzolo, (1991); Hergott e r a / . (1993). Shepherd e r a / . (1991). Irons (1987). See Lin er al. (1992a). Bok, (1982); Mircheff er a / . (1990); Korte and Chandra-Wanderman (1993). See Lin er al. (1992a). Hunt e r a / . (1989). Mircheff et al. (1990); Korte et al. (1991). Philp and Nachmias (1987). Takata er al. (1992). Philp and Nachmias (1987). Bok and Heller, (1976); Pfeffer er al.

(1986). (19) Hunt et al. (1989).

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including Na+K+-ATPase,reside in the apical plasma membrane (see Lin et al., 1992a). They are major determinants of the RPE's control of fluid transport from the retina to the choroid, and the volume of the RPE and the subretinal space. Clathrin-coated pits, indicative of endocytosis, are preferentially located on the basal and apical plasma membranes, the lateral plasma membrane being almost devoid of them (Orzalesi et al., 1982; Perlman et al., 1989). Discrete plasma membrane zones-the bases of apical microvilli and the basal plasma membrane directly facing the basement membrane-contain the coated pits in rats (Perlman et al., 1989). Some important molecules that are located on all plasma membrane domains, such as the glucose transporter (Takata et al., 1992),may require morphometry to uncover their polarized distributions in normal or reactive RPE. The RPE also synthesizes and releases a host of molecules in vitro (collagenases, tissue plasminogen activator, acid hydrolases, proteases, retinol binding proteins, transthyretin, glycosaminoglycans, and other extracellular matrix components) about whose secretion in vivo, polarized or nonpolarized, nothing is known (Haddad and Bennett, 1987; Wilcox, 1987; Hayes et af., 1989; Tripathi et af., 1989, 1990; Cavallaro et al., 1990; Herbert et al., 1991; Hirata and Feeney-Burns, 1992). The cytoplasm of the RPE cells is also polarized, in that many organelles preferentially occupy the apical or basal cytoplasm or an intermediate zone in between (Figs. IA, 2A). When the RPE responds to its environment or to injury, its polarized structure and function can change. Some examples illustrate this: (1) in rats with experimentally induced phototoxic or urethane retinopathies, the RPE rearranges its polarity in relation to the intraepithelial capillaries. The lateral plasma membrane facing the capillary basement membrane forms the specializations normally restricted to the basal plasma membrane, such as folds, intracytoplasmic tubules, and attachment sites (Korte et al., 1986a). (2) In dystrophic rats, RPE cells that have lost their intercellular junctions express the Na+K+-ATPaseon the basolateral as well as apical plasma membrane, to which it is normally restricted (Caldwell and McLaughlin, 1984). (3) In embryonic RPE, the Na+K+-ATPaseand p l integrin are initially present over the entire surface, but become restricted to specific plasma membrane domains as the cells mature (Rizzolo, 1991; Rizzolo and Heiges, 1991). Changes in the structural and functional polarity of RPE are especially profound in rabbits treated with sodium iodate. In these animals, an immature epithelial sheet replaces normal RPE. Its cells must reestablish the polarity characteristic of normal RPE if the tissues flanking it, in particular the choriocapillaris and photoreceptors, are to remain functional.

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E. Sodium Iodate Retinopathy as a Model of RPE Regeneration

Sodium iodate selectively damages the retinal pigment epithelium (Zinn and Marmor, 1979; Korte et al., 1989a). This model provided the first experimental evidence that damaged RPE regenerates (Orzalesi et al., 1965). Within a day of intravenous administration, sodium iodate elicits necrosis of the RPE throughout the fundus in rabbits. Patches of spared cells, however, consistently survive near the ora serrata (the most peripheral part of the retina) and the optic disc. The RPE cells at these sites appear normal or only slightly affected when examined by electron microscopy, compared with the necrotic RPE cells in the intervening fundus (Fig. 2A,B). Macrophages that enter the retina from the choroid remove the necrotic cells (Fig. lC,D) and slowly disappear over the next several weeks. The spared RPE cells adjacent to the necrotic ones spread and lose their apical processes and basal folds, and start to migrate into the wound area (Fig. 2C). Thus, the result of sodium iodate administration in the rabbit is a retina whose outer border is partly occupied by a regenerating RPE sheet arising from spared RPE, mainly located near the ora serrata and optic disc. A scar derived from Miiller glial cells occupies the outer border of the retina between these two sites. The new RPE and the glial cells are oriented toward the remnant RPE basement membrane. The regenerating RPE migrates on it, and the Miiller cells extend their processes choroidally to form a glia limitans along it (Korte et al., 1992). Scar formation along the remnant RPE basement membrane blocks further advancement of the regenerating epithelial sheet.

FIG. 2 Electron micrographs of RPE and choriocapillaris from rabbits 2 days (A-C) and 1 day (D,E) after sodium iodate. (A,B) In some regions (e.g., near the ora serrata and optic disc) RPE cells are spared (in A) and look normal or only mildly affected; they contain intact organelles with their characteristic distribution in the cytoplasm (cf. Fig. 1A). Necrotic RPE cells with broken membranes and disrupted organelles (in B) occur throughout the rest of the retina of the same animal. (A) x 6800. (B) x 5600. (C) Micrograph showing spared cells spreading out along the basement membrane (arrowhead), undercutting some cellular debris (*) due to RPE cell necrosis. Although intercellular junctional complexes are still present (arrow), the cell has flattened and retracted its surface specializations. x 15,200. (D,E) As early as 1 day after administration of sodium iodate, the endothelium of the choriocapillaris adjacent to necrotic RPE begins to thicken, and only small patches of fenestrated cytoplasm remain (arrows in D). Endothelial cytoplasm adjacent to the spared RPE (in E) remains thin and fenestrated. Basement membrane and basal folds of an RPE cell are along the upper half of E. (C,D) x 12,000.

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The photoreceptors next to necrotic RPE atrophy (Fig. lB,C). Their outer and inner segments shorten and the outer nuclear layer becomes thinner. Where RPE regenerates, the photoreceptor atrophy appears to stop, and the inner and outer segments begin to recover (Figs. 3,4). This also occurs when a detached neural retina is reattached to the RPE (Fisher and Anderson, 1989 and GuCrin et al., 1993). The tissue on the choroidal side of the RPE is affected as well. The choriocapillaris adjacent to necrotic RPE atrophies. As early as 1 day after administration of sodium iodate, the endothelium begins to thicken and lose its fenestrae (Fig. 2D,E). Subsequently, many of the endothelial cells die, producing a shrunken capillary bed with reduced permeability (Korte et al., 1986b; Korte and Pua, 1988a; Korte et al., 1989b). The capillary atrophy appears to result from apoptosis. Endothelial cells exhibiting its hallmarks (dark, shrunken cytoplasm and nuclei; cytoplasmic blebbing into the lumen) are seen throughout the capillary network next to necrotic RPE (see Figs. 4 and 5 of Korte and Pua, 1988a and compare them with Azmi and O’Shea, 1984 and Walker et al., 1989). Similar endothelial cells occur in remodeling subretinal neovascularizations at laser lesions in rats (see Fig. 7 of Pollack and Korte, 1990; and Fig. 5 of Pollack and Korte, 1992a). The choriocapillaris regenerates next to the regenerating RPE (Korte and Pua, 1988b; Korte, 1989), the endothelial cells of the regenerating choriocapillaris again becoming fenestrated and permeable. Unlike normal or atrophic choriocapillaris, they exhibit plasma membrane staining for alkaline phosphatase (Korte, 1989; Korte et al., 1989b; Andracchi and Korte, 1991). The tandem nature of RPE and choriocapillaris regeneration provides additional evidence of an interaction between the two tissues. In the course of these studies, some cytologic features like those seen in regenerating choriocapillaris were observed that suggest remodeling occurs in normal choriocapillaris (such as the presence of endothelial sprouts) (Korte and Chase, 1989; Manche and Korte, 1990). F. Cytology of Regenerating RPE during Sodium Iodate Retinopathy

The regenerating RPE cells exhibit a gradient in morphology within the epithelial sheet that makes for convenient comparison of cells at different stages of differentiation. The cells near and at the leading edge of the epithelial sheet are immature, and many of them have the appearance of migrating cells. They grade into cells that resemble normal RPE. Some of these differences are evident with scanning electron microscopy after the neural retina is peeled off to expose the RPE sheet (Fig. 5). Table I1 summarizes the changes.

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FIG. 3 In the area adjacent to regenerated RPE (identified by its lack of melanosomes or large lipid droplets) in a rabbit obtained 8 weeks after sodium iodate, photoreceptor atrophy stops and some regrowth of inner segments (is) and outer segments (0s) may occur. N , photoreceptor nuclei of outer nuclear layer. x 7200.

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FIG. 4 Where RPE regeneration does not occur in the same rabbit seen in Fig. 3, a subretinal scar forms (s), photoreceptor atrophy continues, and only short inner segments (is) remain. The cilium (arrows) from which outer segments develop, however, is still located at their distal tips. Photoreceptor nuclei in the remnant outer nuclear layer are seen along the top of the picture. The arrowheads denote remnant RPE basement membrane, along which subretinal glial scars form. ~ 5 1 0 0 .

The immature cells near the leading edge are flat and lose most of the surface specializations of normal RPE, such as apical microvilli and basal folds (Fig. 6A), nor do they have the “layering” of organelles seen in normal RPE cytoplasm. Only some punctate adherent junctions connect the cells to each other and to the basement membrane (Fig. 6B). Many of these cells have prominent stress fibers that insert into focal contacts with the basement membrane (Fig. 6C) (see Korte and Song, 1990, 1991). The morphologic characteristics of normal RPE cells gradually return as undifferentiated cells mature (Fig. 7A-C). These cells have numerous

FIG. 5 Scanning electron micrographs of regenerating RPE just behind the leading edge (in A) and at the leading edge (in B), from a rabbit obtained 1 week after administration of sodium iodate. Cells are flat, irregular in shape, tend to overlap each other, and have

numerous slender processes. Those at the arrow in A are detailed in the inset. At the leading edge of the epithelial sheet (in B), cells have more elongate processes (P)that branch and emit filopodia (arrows). (A) x 7200; inset x 24,700. (B) x 6500.

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TABLE I1 Summary of RPE Cell Morphology during Regeneration

At and near leading edge Cells flat with stress fibers inserting into focal contacts with basement membrane. Few intercellular junctional complexes. Loss of plasma membrane domains, polarity, and surface specializations. Little endoplasmic reticulum, few mitochondria, small and infrequent lipid droplets. Away from leading edge Cells gradually become cuboidal, and plasma membrane domains, polarity, and surface specializations are gradually restored. Stress fibers disappear as circumferential bundle and intercellular junctional complexes are restored. Organelles characteristic of normal RPE-abundant endoplasmic reticulum, small lipid droplets, mitochondria.

focal basement membrane contacts and well-developed intercellular junctions that reestablish the barrier function of the RPE (Fig. 8A,B; 9A-D). Cytoplasmic and nuclear changes accompany these plasma membrane changes. Immature RPE do not have an abundance or concentration of any organelle, except perhaps free ribosomes. As they mature, the organelle complement and disposition characteristic of normal RPE cells returns. For example, mitochondria, endoplasmic reticulum, lysosomes, and lipid drops increase (cf. Figs. 6A and 7A). These morphological changes may accompany functional changes. Mitochondria supply ATP for the Na+K+ATPase, the activity of which is also increasing as the cells mature. Endoplasmic reticulum contains enzymes involved in retinoid metabolism and is a site of calcium sequestration (Berman, 1991;Lytton and Nigam, 1992). Lipid droplets are storage sites for retinoids (Berman, 1991).The nucleolus of many of the regenerating RPE cells often appears more complex than that of normal RPE cells, suggesting increased RNA and ribosome production (e.g., Fig. 7A). A major difference between immature cells at and near the leading edge of the regenerating epithelial sheet and the more mature ones removed from the leading edge is the presence of discrete apical and basolateral plasma membrane domains in the latter cells. The disposition of plasma

FIG. 6 Electron micrographs of RPE cells near the leading edge of the epithelial sheet, 1 week after administration of sodium iodate. (A) A flat, undifferentiated cell (note absence of surface specializations and organelles characteristic of normal RPE; cf. Fig. 1A) lies on the basement membrane (arrowheads) and abuts remnant photoreceptor inner segments, above. x 11,500. (B)Small, poorly differentiated punctate junctions connect cells to each other (arrow) and to the basement membrane (arrowheads). From tissue mordanted with tannic acid in the primary fixative. ~ 4 5 , 0 0 0 .(C) Many of the cells in this location have numerous stress fibers (sf) that extend out into their processes and insert into large focal basement membrane contacts (arrows). x 14,300.

FIG. 7 Electron micrographs of regenerating RPE cells that are more mature than those seen in Fig. 6, from rabbits obtained I week after administration of sodium iodate. (A,B) These cells have some apical villi and foci of basal folds (arrow in A, detailed in B) that in this specimen bear some black reaction product denoting alkaline phosphatase activity (which returns in conjunction with basal fold formation; see Korte er al., 1991). Numerous mitochondria and some small lipid droplets that are clear due to extraction of their contents are also seen. The nucleolus (n) in many of these cells is very complex. (A) x 5200. (B) x 10,500. (C) More mature cells than the ones seen in A and B (e.g., apical villi and basal folds are better developed). The arrow denotes a junctional complex, detailed from comparable cells in Fig. 8. x 6000.

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FIG. 8 Re-formed intercellularjunctions among RPE, from a specimen obtained 1 week after administration of sodium iodate. (A) The junctional complex (arrow) retards intravenously injected horseradish peroxidase, seen as the black deposit in the intercellular space beneath it and in Bruch’s membrane (below) after leaking out of the fenestrated endothelium of the choriocapillaris. Some tracer is also endocytosed and accumulates in conspicuous endosomes (e). ~ 3 2 , 5 0 0 .(B) Tight junctions with sites of close membrane apposition (arrows) are closely associated with the zonula adherens. From tissue mordanted with tannic acid in the primary fixative. x 105,000.

membrane molecules normally associated with these domains also changes. For example: (1) the basolateral plasma membrane of normal RPE has intense alkaline phosphatase activity. However, there is less enzyme activity in the immature RPE cells near the leading edge of the regenerating epithelial sheet (Korte et al., 1991). (2) Na+K+-ATPaseactiv-

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FIG. 9 Re-formation of the blood-retinal banier during RPE regeneration, in rabbits obtained 1 week after administration of sodium iodate and 15 min after intravenous injection (A,B) or 30 min after intraocular injection (C,D)of horseradish peroxidase (HRP). (A,B) The

tracer is retarded where RPE has regenerated to the extent that intercellular tight junctions have re-formed (arrow in A, detailed in B). (A) x5OOO. (B) x30,OOO. (C,D) At and near the edge of the regenerating epithelial sheet, the cells (flanked here by melanosome-laden macrophages, m) have not re-formed tight junctions (see Fig. 6B). Horseradish peroxidase injected intraocularly freely crosses between them (area at arrow in C, detailed in D), filling the tissue compartment containing the basement membrane (arrowheads). Tissue beneath this area does not contain tracer (in C),since this was an intraocular injection, and it did not diffuse that far before euthanasia. The arrowheads in D denote uptake of tracer by coated pits and vesicles, which are scattered over the surface of the immature RPE cells. (C) ~ 7 5 0 0 (D) . x 18,OOO.

ity is high on the apical plasma membrane of normal RPE cells, but in immature regenerating RPE cells its activity, too, is reduced and is scattered over the plasma membrane (Korte and Chandra Wanderman, 1993). As the RPE cells mature and re-form discrete apical and basolateral plasma membrane domains, they reestablish the normal distribution of alkaline phosphatase and Na+K+-ATPase activity (Korte er al., 1992;

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Korte and Chandra Wandermann, 1993). The intensity of the cytochemical staining for these enzymes also increases with maturation, suggesting increased synthesis and insertion of enzyme into the plasma membrane (Fig. IOA-C). Enzyme activity in coated vesicles and endosomes in these more mature cells suggests that endocytosis may remove enzyme from plasma membrane domains that are not destined to contain it (see Fig. 3C of Korte et al., 1991 and Fig. 3A of Korte and Chandra Wanderman, 1993). These observations on alkaline phosphatase and Na+K+-ATPase suggest that transport capabilities return with the re-formation of a patent epithelial sheet. The Na+K+-ATPase,for example, is important in controlling ion and water passage across the retina-choroid interface and may even provide an asymmetric ion current involved in establishing and maintaining cell polarity (Nuccitelli, 1983; Bok, 1988; Lin et al., 1992a). Two other transport-related morphologic features also gradually return: immunostaining for the cytoplasmic enzyme, carbonic anhydrase type 11, increases (Smith and Korte, 1992; Korte and Smith, 1993); and the barrier properties of the epithelium return as tight junctions form (Fig. 9A-E). This probably contributes to the re-formation of barrier function at laser lesions (Zweig et al., 1981; Negi and Marmor, 1984; Pollack and Korte, 1993). The distribution of coated pits also changes during RPE regeneration. In normal rabbit RPE cells, these organelles are preferentially located at the bases of apical processes and on the basal folds of plasma membrane facing the basement membrane (Perlman et al., 1989). However, they are scattered over the surfaces of immature cells in the regenerating epithelial sheet (Korte and Song, 1990, 1991). With maturation of the cells, they regain their normal, restricted location. The functional significance of the changes in coated pit distribution is unknown, owing to the lack of knowledge about receptor-mediated endocytosis in RPE cells. Only one molecule, the transferrin receptor, has actually been localized to coated pits in normal RPE (Hunt et al., 1989), although the mannose receptor probably also resides here (Tarnowski el al., 1988a,b; Pontow et al., 1992). Regenerating RPE cells, like normal RPE cells, contain transferrin, suggesting that receptor-mediated endocytosis is active in regenerating as well as normal RPE (Fig. 11A-E). Observations of endocytosis of the fluid-phase tracer, horseradish peroxidase, by regenerating RPE, which is especially prominent in more mature cells, support this (Fig. 12). An increase in the ability to phagocytose photoreceptor outer segments parallels the increase in endocytosis as RPE cells mature. More mature cells contain scattered phagosomes, but these are rare in immature cells at and near the leading edge of the epithelial sheet. Examination of tissue

FIG. 10 Distribution of alkaline phosphatase (A,B) and Na' K+-ATPase (C) in regenerating RPE cells. Pictures are from specimens processed in the course of cytochemical studies of the loss and reexpression of these enzymes (Korte er a/., 1991; Korte and Chandra Wanderman, 1993). (A,B) Alkaline phosphatase activity (at sites of black precipitate) lines the basolateral plasma membrane of RPE in A. The area at the arrow is detailed in B. In 9, reaction product fills intercellular space and is seen in uncoated vesicles (arrows) and endosomes (e) clustered near the plasma membrane. Note the absence of precipitate on the apical plasma membrane in 6, at upper right. (A) ~ 8 5 0 0 (B) . ~50,000.(C) Precipitate denoting Nat Kf-ATPase activity is seen on apical plasma membrane and in some nearby coated vesicles, coated pits, and uncoated vesicles, all denoted by arrows. The Golgi apparatus saccules exhibit some enzyme activity (arrowheads). x 25,500.

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FIG. 11 Direct immunostain for transferrin, using a peroxidase-labeled antibody to human transferrin (Biodesign International, Kennebunkport, ME, USA) applied overnight to sections of paraffin-embedded rabbit retina, at a dilution of 1 :50. (A,B) Both regenerating RPE (in A, obtained 7 days after iodate) and normal RPE (in B) stain. Unstained oval structures in normal RPE are nuclei or lipid droplets. ~ 2 1 6 (C,D) . In control sections without antibody exposure, regenerating ( C ) and normal (D) RPE do not stain. ~ 2 1 6 . (E) Regenerating RPE examined by confocal laser scanning microscopy to show additional details, such as staining of choriocapillaris endothelium (arrow). Unstained oval structures in RPE are nuclei. Bar = 10 p.

stained for acid phosphatase activity to identify lysosomes and phagosomes supports this, and shows that the return of phagocytic activity accompanies the re-formation of the apical processes that effect phagocytosis (Fig. 13A-C). Other plasma membrane changes occur during RPE regeneration that have not been correlated with the reexpression of polarized structure: (1) the binding of ferritin-labeled wheat germ agglutinin is greater in regenerating RPE than in normal RPE (Korte, 1991). Similar observations have been made with ferritin-labeled con A (unpublished observations). (2) The cell coat of regenerating RPE cells appears different from normal RPE when stained with ruthenium red [much of the staining is apparently due to hyaluronate and chondroitin sulfate (Fig. 14A-E)] and light microscopic histochemistry supports this (Fig. 15A-D). Changes in extracellular matrix accompany the changes in the plasma membrane and its associated cell coat. For example, redundant basement membrane accumulates under immature RPE cells near the edge of the

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FIG. 12 Endocytosis by regenerating RPE cells, from a rabbit obtained 1 week after administration of sodium iodate and 15 min after intravenous injection of horseradish peroxidase. Fluid phase endocytosis of tracer occurs at coated pits and coated vesicles (arrows) along basolateral plasma membrane. Tracer accumulates in large endosomes (e). x 23,000.

regenerating sheet, and pockets of fibronectin-rich extracellular matrix occur among the cells (Fig. 16A-E). These accumulations are similar to those seen among cells of regenerating corneal endothelium (Sabet and Gordon, 1989).

FIG. 13 Regenerating RPE obtained 1 week after administration of sodium iodate and processed for acid phosphatase cytochemistry by the procedure of Lewis and Knight (1977). (A) In immature cells near the leading edge, infrequent lysosomes are stained (arrow, detailed in inset). x 13,500. Inset x62,OOO. (B,C) In more mature cells, phagosomes (P) containing partially digested outer segments stain for acid phosphatase. Some lysosomes are seen in the lower part of the field in C. Phagosomes and lysosomes did not stain in control tissue incubated in the absence of substrate. (B) x 8400. (C) x 22,000.

GARY E. KORTE, JAY I. PERLMAN, AND AYALA POLLACK

FIG. 14 Cell coat (glycocalyx) and basement membrane of normal and regenerating RPE cells. The cell coat was stained with ruthenium red by the procedure of Luft as modified by Iozzo er al., (1982) and applied to the RPE surface after neural retina was peeled off to expose it. All pictures are x 54,000. (A) In normal RPE cells, ruthenium red stains the cell coat of apical plasma membrane intensely but evenly. (B) In regenerating RPE, the staining is also intense but with focal accumulations (arrowheads). (C,D) Ruthenium red stain is reduced in control tissue exposed to bovine testicular hyaluronidase (C: 2000 U/ml buffer for I hr at 37°C.) or chondroitinase ABC (D: 10 U/ml buffer, overnight at room temperature). (E). Patches of redundant basement membrane (arrows) are found on the RPE side of the basement membrane beneath immature cells. These disappear as cells mature.

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FIG. 15 Hale’s colloidal iron stain (Barka and Anderson, 1963) of normal and regenerating RPE in paraffin sections. All pictures are x 490. (A) In normal eyes there is intense staining of Bruch’s membrane (arrow), the apical RPE surface with associated photoreceptor outer segments (0s. artifactually split) and extracellular matrix surrounding inner segments (is). (B) In regenerating RPE, there is intense staining of the apical RPE surface (arrow) and remnant photoreceptor inner segments (is). (C,D) After exposure to bovine testicular hyaluronidase, colloidal iron stain associated with normal RPE (C) and regenerating RPE (D) is reduced, indicating that hyaluronate contributes to the extracellular matrix around RPE cells.

The extracellular matrix elaborated during regeneration must also remodel with maturation of the epithelial sheet, since the accumulations of matrix among immature cells and the redundant basement membrane patches beneath them are no longer seen where the RPE cells have matured. Their removal may occur by secretion of tissue plasminogen activator (Tripathi et al., 1989, 1990).

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FIG. 16 Indirect immunostain for fibronectin, applied to pieces of tissue immersed in 4% formaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer for 1 hr after the neural retina was peeled off. Tissue pieces were then soaked in the reagents of a commercial staining kit (Histogen PAP, from Biogenex Corp.,San Ramon, CA: prediluted primary antibody overnight; 1 hr in secondary antibody and PAP); reacted for peroxidase localization with the diaminobenzidine procedure (Graham and Karnovsky, 1966); immersed in osmium tetroxide fixative; and dehydrated and embedded in epoxy resin. Unstained sections 2-3 p thick were examined. All pictures are x500. (A) Normal RPE exhibits intense stain in the Bruch’s membrane area (arrow) and on basement membrane around blood vessels (arrowheads). A lipid droplet, characteristic of normal RPE cells, is seen on the left. (B) In regenerating RPE (obtained 7 days after sodium iodate) the immature cells (*) are surrounded by extracellular matrix rich in fibronectin. (C,D) In control tissue (unexposed to primary antibody), staining associated with normal (C) and regenerating (D)RPE is lost. Red blood cells stain due to their endogenous peroxidase.

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G. Similarities t o Developing Rabbit RPE

The morphology of regenerating RPE in rabbits treated with sodium iodate resembles that of developing rabbit RPE, where the same gradual expression of structural changes occurs as the epithelial sheet matures. In RPE of rabbits examined at various times during gestation (16 and 23 days; newborn), the centrally located RPE cells are consistently more mature than peripheral ones in the expression of Na+Kf-ATPase,the formation of apical and basal plasma membrane specializations, and type I1 carbonic anhydrase activity (Fig. 17A,B). These observations resemble those seen in developing chick retinas, where the Na+K+-ATPaseand integrins gradually change from zones of immature to more mature cells (Rizzolo and Heiges, 1991). Similar central-to-peripheral gradients also occur in the expression of other molecules during retinal development (see Hauswirth et al., 1992).

FIG. 17 The gradient in type I1 carbonic anhydrase staining of differentiating RPE in the retina of newborn rabbit. Tissue was fixed and processed as for transfemn immunohistochemistry (see Fig. 11) and exposed to a peroxidase-linked antibody to human CAI1 (Biodesign International, Kennebunkport, ME, USA) at a dilution of 1 :500 before being incubated for localization of peroxidase by the procedure of Graham and Karnovsky (1966). Immature RPE cells located near the ora serrata (in A) stain less intensely than more mature RPE cells located centrally (in B). Red blood cells in choriocapillaris beneath the epithelium stain intensely in both regions. The photographic parameters were identical for both pictures. x 520.

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111. Concluding Remarks

The tissue flanking the RPE-the choriocapillaris and photoreceptors-atrophies when it is damaged (see Section I). Studies of RPE regeneration have extended our understanding of the interactions between the RPE and the adjacent tissues by showing that the recovery of photoreceptors and choriocapillaris is linked to RPE regeneration. Apart from these interactions between RPE and choriocapillaris, and RPE and photoreceptors, which are still poorly understood, the regeneration of the RPE itself tells us about the response of RPE cells to changes in their environment and their ability to re-establish function after injury. The RPE response includes changes in the plasma membrane, cytoskeleton, and extracellular matrix. The plasma membrane reorganizes, or remodels, during RPE regeneration, This is seen as changes in surface specializations, the binding of plasma membrane probes such as lectins and ruthenium red, and the redistribution of plasma membrane components as the cells mature, such as the gradual enrichment of the apical plasma membrane in Na+K+ATPase . As the RPE cells form new tight junctions, the Na+K+-ATPaseon the choroidal side of the junctions (that is, on the newly established basolateral plasma membrane domain) must be removed. Similarly, alkaline phosphatase on the apical plasma membrane must be removed as the cells mature and the enzyme accumulates in the basolateral plasma membrane. Both events require changes in the cellular apparatus targeting the enzymes to the plasma membrane. This probably involves the Golgi apparatus and vesicles derived from it, such as uncoated vesicles with alkaline phosphatase and Na+K+-ATPaseobserved in regenerating RPE. Coated vesicles and endosomes with enzyme activity suggest that endocytosis removes enzyme from RPE plasma membrane during maturation. Plasma membrane components anchoring these enzymes in the plasma membrane, such as the glycophosphatidyl inositol-anchoring protein associated with alkaline phosphatase and the ankyrin-fodrin complex associated with Na+K+-ATPase(Gundersen et al., 1991; Louvard et al., 1992; RodriguezBoulan and Powell, 1992) may also change during RPE regeneration. The redistribution of coated pits to specific regions of the plasma membrane as cells mature, and differences between immature and mature cells in the ability to phagocytose outer segments of rods suggest that changes in receptor-mediated plasma membrane functions occur during RPE regeneration. The range of changes associated with transport across the RPE-changes in coated pits and endocytosis, reestablishment of the blood-retinal barrier, gradient changes in the activity of transport-related

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enzymes such as Na+K+-ATPaseand carbonic anhydrase-suggest a major reorganization of the transport apparatus as the cells mature within the regenerating RPE sheet. Other changes suggesting alterations in plasma membrane constituents during RPE regeneration include the differences in the cell coats of normal and regenerating RPE. Changes in lectin labeling and ruthenium red staining may reflect changes in plasma membrane glycoproteins. Beaudoin et al. (1978) have suggested that the uniform distribution of ruthenium red stain (like that on normal RPE) and patchy distribution of stain (like that on regenerating RPE) reflect changes in functional status in pancreatic acinar cells. Basement membrane and extracellular matrix components influence plasma membrane polarity of RPE cells. For example: (1) the application of collagen to the apical surface of cultured RPE cells elicits its transformation into a surface with the morphology of the basal plasma membrane (Crawford, 1983). (2) The normally undifferentiated lateral plasma membrane of RPE cells aquires basal plasma membrane morphology when in contact with the basement membrane of intraepithelial capillaries (Korte et al., 1986a). (3) Regenerating RPE cells that are not in contact with the basement membrane do not exhibit polarization of Na+K+-ATPaseon their plasma membrane (Korte and Chandra Wandermann, 1993). Recently, Rizzolo (1991) has shown that basement membrane influences the polarized distribution of integrins in developing chick RPE. The differentiation of several types of epithelia (e.g., intestinal epithelia; Louvard et al., 1992) is correlated with increases in laminin, hyaluronate, and heparan sulfate proteoglycan (Bernfield et al., 1992). The changes in the extracellular matrix suggest that its molecules may influence RPE regeneration. For example, the secretion of extracellular matrix containing fibronectin by regenerating RPE cells and the occurrence of patches of redundant basement membrane recall changes seen in explants of embryonic chicken RPE and other epithelia (Garbi and Wollman, 1982; Turksen et al., 1984). The presence of hyaluronate, heparan sulfate, and chondroitin sulfate proteoglycans and other extracellular matrix molecules in the RPE basement membrane, cell coat, and interphotoreceptor matrix, suggest candidates that may signal changes during RPE regeneration (Porrello and LaVail, 1986; Tawara et al., 1989; Hageman and Johnson, 1991; Iwasaki et al., 1992; Turksen et al., 1985). One future challenge is to identify the extracellular matrix components and extraepithelial growth factors that influence RPE regeneration. For example, fibroblast growth factors, platelet-derived growth factor, epidermal growth factor, insulin-like growth factors, and transforming growth factor-p occur in normal RPE (Schweigerer et al., 1987; Sternfeld et al., 1989; Leschey et al., 1990; Gaur et al., 1992; Wiedemann, 1992).

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In this regard two considerations should be kept in mind: (1) The RPE normally faces two separate tissue compartments-the choroidal compartment containing the connective tissue of Bruch’s membrane and the subretinal space containing the specialized interphotoreceptor matrix. The disrupted tight junctions of injured RPE permit these compartments access to each other, and extracellular matrix components or growth factors normally restricted to one of them may act as signals on parts of the RPE plasma membrane not usually exposed to them, or on other types of cells nearby, such as choriocapillaris endothelium or choroidal fibroblasts. (2) RPE can synthesize and secrete some of the molecules that are candidates for signaling changes in its environment and controlling its re-differentiation during regeneration (RPE cells secrete bFGF and also have receptors for it; Schweigerer et al., 1987; Sternfeld et al., 1989). This creates the possibility of paracrine and autocrine control of RPE regeneration and the response of adjacent tissues, as by secretion of basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF) (Faktorovich et al., 1991; Gross-Jendroska, 1992; see Gaur et al., 1992 and Wiedemann, 1992 for discussion). Thus, besides identifying what extracellular matrix molecules change during RPE regeneration, the source of such changes must be identified as well. The RPE cell is but one of several cell types at the retina-choroid interface that secrete extracellular matrix molecules and growth factors and also interact with each other via these same molecules. These interactions may influence the course of RPE regeneration, like the epithelial-mesenchyme interactions working during development (Bissell et al., 1982; Louvard et al., 1992; see Wiedemann, 1992 for one discussion of this in the retina). Control of the cytoskeleton certainly contributes to the changes in cell shape and motility seen during RPE regeneration. Although detailed observations on regenerating RPE are lacking, instructive examples are seen in (1) the diffuse distribution of talin and integrin in migrating corneal epithelial cells versus their association with attachment sites in stationary cells (Philp et al., 1990), (2) in the accumulation of spectrin in more differentiated cells of RPE explants from chick embryos (Opas and Kalnins, 1985a), and (3) the requirement for pl integrin for RPE cell migration (Hergott et al., 1993). While the factors controlling RPE cytoskeletal changes in uiuo are unknown, they are most likely similar to changes described in explanted and cultured RPE cells, particularly those seen in response to wounding in organ cultures, where the RPE cells are migrating on a normal substratum (Hergott et al., 1989, 1993). (For example, stress fibers are prominent in cells near the edge of the epithelial sheet, and they disappear with the formation of circumferential bundles as RPE cells mature; Crawford, 1980; Turksen and Kalnins, 1987; Hergott et al., 1989.)

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The myriad changes that take place in regenerating RPE occur gradually and must be controlled. Although virtually nothing is known of the mechanism, it is likely that some of the second messenger systems and transcription factors are involved. For example, the inositol phosphate system influences the organization of the cytoskeleton as well as cell-cell adhesion (Stossel, 1989; Geiger and Ayalon, 1992). In the eye, changes in protein kinase C occur during corneal reepithelialization after wounding and influence cytoskeletal changes during epithelial migration to close the wound (Hirakata et al., 1993). In RPE, changes in protein kinase C, CAMP, and calcium are associated with changes in phagocytosis and transport (Koh and Chader, 1984; Frambach et al., 1990; Hall et al., 1991; Gregory et al., 1992). The control path through the nucleus that leads to the changes in phenotype seen during RPE regeneration probably involves the activation or suppression of transcription factors encoded by, for example, c-fos and c-jun (Herrlich and Ponta, 1989). The conspicuous differences in the structure of the nucleolus between regenerating and normal RPE cells supports this notion, in that a more prominent nucleolus is associated with increased production of RNA and increased protein synthesis (Ghosh, 1987). The similarity of some of the experimental observations described earlier with observations in the human retina suggests that the same cellular events and control mechanisms work in human regenerating RPE. For example, the basic responses of damaged RPE that lead to its regeneration in animal models and in uitru (such as cell spreading, migration, and proliferation to produce undifferentiated cells that must redifferentiate; production of extra extracellular matrix; focal hyperplasia; re-formation of barrier function by re-formation of intercellular tight junctions) are seen after human retinal detachments, at sites of subretinal neovascularization, during the healing of laser lesions, and in retinal diseases such as agerelated macular degeneration (Morris and Henkind, 1979; Tso, 1979; Eagle, 1984; Spencer, 1985; Young, 1987; Miller et al., 1990; Pollack and Korte, 1990; Lopez et al., 1991). Ophthalmoscopic observations suggest that some of these events are at work in monkey and human eyes (Kanno et al., 1993; Kaplan et al., 1993). Continued observations on regenerating RPE in animal models in uiuo and in uitru should advance our understanding of the response of human RPE to injury.

Acknowledgment This work was supported by grants from the National Eye Institute (EY08284 to G . Korte) and Research to Prevent Blindness, Inc. to the Department of Ophthalmology, Albert Einstein College of Medicine.

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Mann, I. (1950). “The Development of the Human Eye.” Cambridge Univ. Press, London. Matsumoto, B.. Gukrin, C., and Anderson, D. (1990). Cytoskeletal redifferentiation of feline, monkey and human RPE cells in culture. Invest. Ophthalmol. Visual Sci. 31, 879-889. Miki, H., Bellhorn, M., and Henkind, P. (1975). Specializations of the retinochoroidal juncture. Invest. Ophthalmol. Visual Sci. 14, 701-707. Miller. H., Miller, B., Ishibashi, T., and Ryan, S. (1990). Pathogenesis of laser-induced choroidal subretinal neovascularization. Invest. Ophthalmol. Visual Sci. 31, 899-908. Mircheff, A., Miller, S., Farber, D., Bradley, M., O’Day, W., and Bok, D. (1990). Isolation and provisional identification of plasma membrane populations from cultured human retinal pigment epithelium. Invest. Ophthalmol. Visual Sci. 31, 863-878. Mishima, H., and Kondo, K. (1981). Extrusion of lysosomal bodies from apical mouse retinal pigment epithelium. Greafes Archiv. Ophthalmol. 216, 209-2 17. Moms, D., and Henkind, P. (1979). Pathological responses of the human retinal pigment epithelium. In “The Retinal Pigment Epithelium” (M. Marmor and K. Zinn, eds.), pp. 247-266. Harvard. Cambridge. Nuccitelli, R. (1983). Transcellular ion currents: Signals and effectors of cell polarity. Mod. Cell Biol. 2, 451-481. Opas, M., and Kalnins, V. (1985a).Spatial distribution of cortical proteins in cells of epithelial sheets. Cell Tissue Res. 239, 451-454. Opas, M., and Kalnins, V. (1985b). Distribution of spectrin and lectin binding materials in surface lamina of RPE cells. Invest. Ophrhalmol. Visual Sci. 26, 621-627. Opas, M., and Kalnins, V. (1986). Light microscopical analysis of focal adhesions of retinal pigmented epithelial cells. Invest. Ophthalmol. Visual Sci. 27, 1622-1633. Opas, M., Turksen, K., and Kalnins, V. (1985). Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro. Dev. Biol. 107, 269-280. Orzalesi, N . , Calabria, G., and Castellazzo, R. (1965). Possibilita di sdifferenziamento delle cellule dell’epitelio pigmentato della retina. Accad. Med. 3-4, 223-231. Orzalesi, N . , Fossarello, M., Carta, S., Del Fiacco, G . , and Diaz, G. (1982). Identification and distribution of coated vesicles in the retinal pigment epithelium of man and rabbit. Invest. Ophthalmol. Visual Sic. 23, 689-696. Owaribe, K., and Masuda, H. (1982). Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells. J . Cell Biol. 95, 310315. Park, C., and Hollenberg, M. (1991). Induction of retinal regeneration in vivo by growth factors. Deu. Biol. 148, 322-333. Perlman, J., Piltz, J., Korte, G., and Tsai, C. (1989). Endocytosis in the rat retinal pigment epithelium. Acta Anaf. 135, 354-360. Pfeffer, B., Clark, V., Flannery, J., and Bok, D. (1986). Membrane receptors for retinolbinding protein in cultured human retinal pigment epithelium. Invest. Ophthalmol. Visual Sci. 27, 1030-1040. Philp, N., and Nachmias, V. (1987). Polarized distribution of integrin and fibronectin in retinal pigment epithelium. Invest. Ophfhalmol. Visual Sci. 28, 1275-1280. Philp, N., Yoon, M., and Hock, R. (1990). Identification and localization of talin in chick retinal pigment epithelium. Exp. Eye Res. 51, 191-198. Pino, R. (1986). Immunocytochemical localization of fibronectin to the retinal pigment epithelium of the rat. Invest. Ophthalmol. Visual Sci. 27, 840-844. Pollack, A., and Korte, G. (1990). Repair of retinal pigment epithelium and its relationship with capillary endothelium after krypton laser photocoagulation. Invest. Ophthalmol. Visual Sci. 31, 890-898.

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Pollack, A,, and Korte, G. (1992a). Vascular remodelling after laser photocoagulation. Concurrent regeneration and atrophy. Acta Anat. 143, 151-159. Pollack, A., and Korte, G. (1992b). Choroidal vascular repair. Scanning and transmission electron microscopy. Experientia 48, 219-225. Pollack, A., and Korte, G. (1993). Restoration of the outer blood-retinal bamer after krypton laser photocoagulation. Ophthalmic Res., in press. Porrello, K., and LaVail, M. (1986). Histochemical demonstration of spatial heterogeneity in the interphotoreceptor matrix of the rat retina. Invest. Ophthalmol. Visual Sci. 27, 1577- 1586. Raviola, G . (1977). The structural basis of the blood-ocular barriers. Exp. Eye Res. Suppl. 24, 27-63. Rizzolo, L. (1991). Basement membrane stimulates the polarized distribution of integrins but not the Na’ K+-ATPase in the retinal pigment epithelium. Cell Regul. 2, 939-949. Rizzolo, L., and Heiges, M. (1991). The polarity of the retinal pigment epithelium is developmentally regulated. Exp. Eye Res. 53, 449-553. Rodriguez-Boulan, E., and Powell, S. (1992). Polarity of epithelial and neuronal cells. Ann. Rev. Cell Biol. 8, 395-428. Sabet, M., and Gordon, S. (1989). Ultrastructural immunocytochemical localization of fibronectin deposition during corneal endothelial wound repair. Evidence for cytoskeletal involvement. Biol. Cell 65, 171-179. Schweigerer, L., Malerstein, B., Neufeld, G., andGospodarowicz, D. (1987). Basic fibroblast growth factor is synthesized in cultured retinal pigment epithelial cells. Biochem. Biophys. Res. Comm. 143, 934-941. Shepherd, V., Tarnowski, B., and McLaughlin, B. (1991). Isolation and characterization of a mannose receptor from human pigment epithelium. Invest. Ophthalmol. Visual Sci. 32, 1779-1784. Shiraki, K., Bums, M., and Bellhorn, R. (1983). Abnormal vessel patterns in phototoxic rat retinopathy studied by vascular replicas. Curr. Eye Res. 2, 545-551. Smith, J., and Korte, G. (1992). Changes in carbonic anhydrase activity during regeneration of the retinal pigment epithelium in rabbits. Invest. Ophthalmol. Visual Sci. Suppl. 33, 911. Spencer, W. (1985). “Ophthalmic Pathology. An Atlas and Textbook,” Vol. 2. Saunders, Philadelphia. Spitznas, M. (1986). Pathogenesis of central serous retinopathy: a new working hypothesis. Graefes Arch. Ophthalmol. 224, 321-324. Spoem, P., Ulshafer, R., Ludwig, H., Allen, C., and Kelley, K. (1988). Photoreceptor cell development in vitro: Influence of pigment epithelium conditioned medium on outer segment differentiation. Eur. J . Cell Biol. 46, 362-367. Sramek, S., Wallow, I., Bindley, C., and Sterken, G . (1987). Fibronectin distribution in the rat eye. An immunohistochemical study. Invest. Ophthalmol. Visual Sci. 28, 500505. Sternfeld, M., Robertson, J., Shipley, G., Tsai, J., and Rosenbaum, J. (1989). Cultured human retinal pigment epithelial cells express basic fibroblast growth factor and its receptor. Curr. Eye Res. 8, 1029-1037. Stossel, T. (1989). From signal to pseudopod. J . Biol. Chem. 264, 18261-18264. Stramm, L. (1987). Synthesis and secretion of glycosaminoglycans in cultured retinal pigment epithelium. Invest. Ophthalmol. Visual Sci. 28, 618-627. Stroeva, O., and Mitashov, V. (1983). Retinal pigment epithelium: Proliferation and differentiation during development and regeneration. I n t . Rev. Cytol. 83, 221-298. Takata, K., Kasahara, T., Kasahara, M., Ezaki, O., and Hirano, H. (1992). Ultracytochemical localization of the erythrocyte/HepG2-type glucose transporter (GLUT I ) in cells of the blood-retinal barrier. Invest. Ophthalmol. Visual Sci. 33, 377-383.

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Tarnowski, B., Shepherd, V., and McLaughlin, B. (1988a). Mannose 6 phosphate receptors on the plasma membrane of rat retinal pigment epithelial cells. Invest. Ophthalmol. Visual Sci. 29, 291-297. Tarnowski, B., Shepherd, V., and McLaughlin, B. (1988b). Expression of mannose receptors for pinocytosis and phagocytosis on rat retinal pigment epithelium. Invest. Ophthalmol. Visual Sci. 29, 742-748. Tawara, A., Varner, H., and Hollyfield, J. (1989). Proteoglycans in the mouse interphotoreceptor matrix. 11. Origin and development of proteoglycans. Exp. Eye Res. 48, 815-839. Tripathi, B., Park, J., and Tripathi, R. (1989). Extracellular release of tissue plasminogen activator is increased with the phagocytic activity of the retinal pigment epithelium. Invest. Ophthalmol. Visual Sci. 30, 2470-2473. Tripathi, R., Tripathi, B., and Park, J. (1990). Localization of urokinase-type plasminogen activity in human eyes: An immunocytochemical study. Exp. Eye Res. 51, 545-552. Tso, M. (1979). Developmental, reactive and neoplastic proliferation of the retinal pigment epithelium. I n “The Retinal Pigment Epithelium” (M. Marmor and K. Zinn, eds.), pp. 267-276. Harvard, cambridge. Tso, M., Wallow, I., Powell, J., and Zimmerman, L . (1972). Recovery of the rod and cone cells after photic injury. Trans. Am. Acad. Ophthalmol. Otolaryngol. 76, 1247-1255. Turksen, K., and Kalnins, V. (1987). The cytoskeleton of chick retinal pigment epithelial cells in situ. Cell Tissue Res. 248, 95-101. Turksen, A,, Aubin, J., Sodek, J., and Kalnins, V. (1984). Changes in the distribution of laminin, fibronectin, type IV collagen and heparan sulfate proteoglycan during colony formation by chick retinal pigment epithelial cells in vitro. Collagen Relat. Res. 4,413-426. Turksen, A., Aubin, J., Sodek, J., and Kalnins, V. (1985). Localization of laminin, type IV collagen, fibronectin and heparan sulfate proteoglycan in chick retinal pigment epithelium basement membrane during embryonic development. J . Histochern. Cytochem. 33, 665-67 1. Turksen, K., Opas, M., and Kalnins, V. (1989). Cytoskeleton, adhesion and extracellular matrix of fetal human retinal pigment epithelial cells in culture. Ophthalmic Res. 21, 56-66. Walker, N., Bennett, R., and Kerr, J. (1989). Cell death by apoptosis during involution of the lactating breast in mice and rats. Am. J . Anat. 185, 19-32. Wallow, I. (1984). Repair of the pigment epithelial barrier following photocoagulation. Arch. Ophthalmol. (Chicago) 102, 126-135. Wiedemann, P. (1992). Growth factors in retinal disease: Proliferative vitreoretinopathy, proliferative diabetic retinopathy and retinal degeneration. Surv. Ophthalrnol. 36,373-384. Wilcox, D. (1987). Extracellular release of acid hydrolases from cultured retinal pigment epithelium. Invest. Ophthalmol. Visual Sci. 28, 76-82. Wolburg, H., Willbold, E., and Layer, P. (1991). Miiller glial endfeet, a basal lamina and the polarity of retinal layers form properly in vitro only in the presence of marginal pigmented epithelium. Cell Tissue Res. 264, 437-451. Young, R. W. (1987). Pathophysiology of age-related macular degeneration. Surv. Ophthalmol. 31,291-306. Zinn, K., and Marmor, M. (1979). Toxicology of the human retinal pigment epithelium. I n “The Retinal Pigment Epithelium” (M. Marmor and K. Zinn, eds.), pp. 395-412. Harvard, Cambridge.

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Habituation as a Tumorous State That Is Interchangeable with a Normal State in Plant Cells Kunihiko Syhno and Tomomichi Fujita Department of Pure and Applied Sciences, University of Tokyo, Tokyo 153, Japan

1. Introduction

Plant cells cultured in vitro normally need an exogenous supply of plant hormones, namely an auxin and/or a cytokinin, for continued growth. Sometimes the cells lose this requirement during subculturing and become able to grow on hormone-free medium or on medium that lacks one or other of the hormones. This phenomenon was discovered by Gautheret and named “accoutumance a I’auxine” (Gautheret, 1955) and, later, “anergie a l’auxine,” which is usually translated into English as auxin habituation. Since its discovery, habituation has been noted in cultures of many species, for example, Crepis (Sacristan and Wendt-Gallitelli, 1971), carrots, potatoes (Widholm, 1977), parsley (Masuda et d . , 1977), sugar beets (Kevers et al., 1981), sunflowers (Sogeke and Butcher, 1976), lilies (Sheridan, 1968), and corn (Hawes et ul., 1985). In tobacco, cytokinin habituation, auxin habituation, and both auxin and cytokinin habituation (referred to as full habituation or simply as habituation in this chapter) have also been reported and extensively analyzed. In nature, tumorous outgrowths are induced by various kinds of biological agents, such as insects, nematodes, fungi, bacteria, and viruses, or they develop spontaneously on plants with a specific genetic constitution as so-called genetic tumors (Beiderbeck, 1977). Among such outgrowths, crown galls induced by infection with the soil bacterium Agrobacterium tumefuciens and genetic tumors do not require phytohormones, namely an auxin and a cytokinin, in the nutrient basal medium. As in the case of normal tissues, tumorous tissues induced by other biological agents require these hormones in the medium when they are cultured in vitro after removal of the biological agents. Hormonal autonomy is a unique Internarional Review, of Cytology. Vol. 152

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characteristic of the cells of crown galls and genetic tumors and is one of the criteria used to define a “tumor” in this chapter. In the case of crown gall disease, a portion of the tumor-inducing (Ti) plasmid (T-DNA, transferred DNA) is transferred from the responsible bacterium, A. tumefaciens, to the host cells. Genes on T-DNA, having a eucaryotic gene structure, are expressed in the host cells after integration into the nuclear genome. Tumorous growth of the transformed cells requires the expression of genes for the biosynthesis of phytohormones in the tms and tmr regions of the T-DNA. The tmr region encodes a key enzyme in the biosynthesis of cytokinin, and two genes in the tms region are involved in the biosynthesis of auxin. Cells transformed by the integration of T-DNA produce sufficient amounts of these hormones to support the proliferation of cells and as a result, they lose their requirement for exogenous auxin and cytokinin for growth in culture (Zambryski et al., 1989). Habituation tissue bears a striking resemblance to crown gall tissue, which grows independently of exogenous hormones. However, habituation is a form of neoplastic transformation that involves heritable, progressive changes in cell phenotype that result in phytohormone-autonomous growth. The significance of this phenomenon lies in the fact that habituation occurs in the absence of a recognizable infectious agent and is sometimes reversible at high frequency. The heritable conversion of normal, hormone-requiring cells to the habituated phenotype is a gradual and progressive process which, unlike mutation, is strongly influenced by the physiological and developmental state of the cells. The induction of habituated cells occurs at rates that are much higher than normal rates of mutation, (they are often higher than 1 in lo3). Once established, the habituated state is extremely stable and usually is difficult to reverse, although reversion has been reported in a specific experimental system (Meins, 1983). Different states of habituation and different degrees of competence with respect to habituation have been demonstrated in different tissues of tobacco plants and even in a single type of tissue at different stages of development. Thus, cells with different degrees of competence with respect to habituation may exist in the intact plant and such competence may change during the differentiation of cells. Some evidence for the hypothesis that habituation may be a normal characteristic of development has been presented (Jackson and Lyndon, 1990). Genetic tumors develop spontaneously from hybrid plants that are derived from sexual or somatic crosses between two specified species. The most numerous reported examples of genetic tumors have been those that resulted from crosses of various combinations of species in the genus Nicotiana. Genetic tumors in Nicotiana were the first plant tissues to be grown continuously on artificial nutrient medium in uitro (White, 1939).

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The plants regenerated from such tumors appear to be normal, although they are very susceptible to the induction of tumors. As in the case of habituation, interconversion of the normal (morphologically normal) and tumorous state can be observed in genetic tumors (Smith, 1965). In this chapter, a discussion is presented of the phemomena of habituation and genetic tumors as systems that allow the interconversion of two different states (normal and tumorous) without any apparent genetic modification. For further information on other aspects of this subject, the reader is referred to previously published reviews (Meins, 1982, 1983; Jackson and Lyndon, 1990;Bayer, 1982; Kung, 1989;Ichikawaand Sydno, 1991; Sekine et al., 1993).

II. Habituated Cells and Reversal of the Habituated Phenotype

A. Induction of Habituation

1. Changes in Concentrations of Hormones Although habituation is a well-known phenomenon, it has been considered to be induced spontaneously by prolonged subculturing and there are relatively few reports of external agents that favor the induction of habituated cells. A change in the concentration of the growth substance in question in the culture medium brings about a change from normal (hormone-requiring) to tumorous (hormone-nonrequiring) cells. Two auxin-requiring lines of tobacco cells (T22 and XD6S2), subcultured on a medium containing 1 mg/liter indole-3-acetic acid (IAA, the most widely distributed auxin in nature) as a plant growth regulator, were found to require quite different treatments for induction of habituated cells. From callus T22, derived from Nicotiana tabacum cv. ‘Bright Yellow,’ habituated calluses were induced by treatment with low concentrations of auxin [0.01-0.1 mg/liter of IAA or a-naphthaleneacetic acid (NAA)] (Fig. I), but not with high concentrations of synthetic auxins [ 10-100 mg/ liter of NAA or 1-10 mg/liter of 2,4-dichlorophenoxyaceticacid (2,4-D)]. In the case of callus XD6S2, derived from N. tabacum cv. ‘Xanthi,’ habituated calluses were induced by treatment with high concentrations of synthetic auxins but not with low concentrations (Sydno and Furuya, 1974). Habituated cells of callus G89, derived from N. glauca, were induced by treatment similar to that used for callus T22 (K. Sydno, unpublished data). From the results of experiments with other separately isolated cell lines, it has been shown that cell lines can be divided into two types

a

a

a auxikrequiring cell line T22

n

+=

n

+=on+=

I

n + n I

induction of hathation (IAA: 0.01-0.1 mgl)

a 0 a

0

habituated state

D

n

I

+ growth 0 n

0

transfer inOculum (30 mg FW/&on) inOculurn (100 mgFW/section)

0: basalmedium K: kinetin (1 mgl)medium I: IAA (1 mgl) medium

FIG. 1 Effects of high concentrations of cytokinins and the size of inoculum on growth and induction of habituation. Relatively high concentrations (1 mghter) of kinetin permitted continued growth of cytokinin-habituated cells (T22), but the tissue required auxin when it was returned to a cytokinin-free medium (upper parts of the figure). Habituated cells are induced from callus T22 by treatment with low concentrations of auxin. When the fresh weight of the inoculum was less than 30 mg, an exogenous supply of auxin was required for growth. The resultant cells, however, reverted to an auxin-requiring phenotype (lower parts of the figure).

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in terms of their requirements for the induction of habituation; namely, those susceptible to induction by treatment with low concentrations of auxin (T22 type) and those susceptible to induction by treatment with high concentrations of auxin (XD6S2 type) (Syono and Furuya, 1974). BY-2 cells, derived from N. tabacum cv. Bright Yellow, became habituated cells after a change to a 40-fold lower than normal concentration of auxin (K. Syono, unpublished data), even though this line had been selected to grow at a very high rate (ca. 100-fold increase in cell number per week) (Nagata et al., 1992) and was quite different from line T22 derived from the same cultivar. These results show that requirements for the induction of habituation can be maintained stably during prolonged subculture and depend on the plant species (or cultivar) from which the callus originated. This conclusion suggests that the conditions favorable for the induction of habituation might be determined genetically. Cytokinin-habituated cells were also induced by changes in the concentrations of cytokinin similar to those that were 1000 times lower than the optimum for growth (Meins and Lutz, 1979). Tissues cultured for one passage on the medium that contained N,N’-diphenylurea, which is a cytokinin-active compound different from other cytokinins, which are derivatives of adenine, acquired the ability to proliferate in the absence of N,N’-diphenylurea and cytokinins that are derivatives of adenine (Mok et al., 1979),as in the case of the treatments with synthetic auxin described earlier.

2. Antiauxins and Other Chemicals

Antiauxins supplied briefly to soybean callus provoked habituation for auxin. It was suggested that this result was consistent with the ability of these compounds to lower concentrations of endogenous auxins (Christou, 1988). These results were confirmed in tobacco using triiodobenzoic acid (TIBA), known as a competitive inhibitor of the polar transport of auxin. Habituated cells of N. glauca were induced from auxin-requiring callus G89 by treatment with 0.1 mg/liter TIBA in the presence of 1 mg/liter of IAA (K. Sybno, unpublished data). Two morphactins and three aminofluorenes were reported to induce the formation of compact tissue nodules on cultures of hormone-dependent tobacco callus. These nodules could be subcultured on cytokinin-free media, while untreated control callus and nonnodule tissues still required an exogenous supply of cytokinin. From these results it was suggested that substituted fluorenes, including carcinogenic aminofluorenes, can cause a neoplastic growth response in cultured tobacco tissues (Bedner and Linsmaier-Bedner, 197I).

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3. Interactions between Auxin and Cytokinin Complex interactions between auxin and cytokinin in the expression of the habituation phenotype have been observed. There are several reports that the requirement for cytokinin can be affected by externally supplied auxin, and vice versa (e.g., Witham, 1968; Sogeke and Butcher, 1976; Einset, 1977; Palni et al., 1988). Relatively high concentrations (1 mg/ liter) of cytokinins permitted continuous growth of auxin-requiring and cytokinin-nonrequiring tobacco callus T22 on a medium without auxin. This effect of cytokinin was not due to perpetuation of change in the character of tissue, because the tissue required auxin when it was returned to a cytokinin-free medium (Sybno and Furuya, 1972a)(Fig. 1). This effect was observed only in T22-type cell lines, including cell line G89, from N. glauca and not in XD6SZtype lines. 4. Inoculum Size

Growth of tissues in liquid cultures is known to be influenced by the amount of the initial inoculum (Nitsch, 1963; Sybno and Furuya, 1968). The different growth responses were dependent on the ratio of inoculum to culture medium and the character of the tissues used (Sybno and Furuya, 1968). Viability of cells to form colonies was found to increase with increasing cell density on the plates (Blakely and Steward, 1964; Nagata and Takebe, 1971), to decrease upon washing the cells prior to plating (Street et al., 1965),and to increase when cells were plated on conditioned medium (Jones et al., 1960). Similarly, nurse cells or tissues supported the division of cells with low density, even single cells (Muir et al., 1958). The Oc line of rice cells (Baba et al., 1986) is used frequently as effective nurse cells supporting protoplast cultures (Kyozuka et al., 1987). All these phenomena show that diffusible factor(s) are involved in the interaction of cells to support cell division. Inoculum size is also another factor that influences early aspects of the induction of habituation. After a change in concentration of auxins, habituated cells were induced from the treated auxin-requiring callus segments (T22) when the fresh weight of the inoculum was more than 60 mg, but not when it was less than 30 mg. Even though an exogenous supply of auxin supported the growth of a small piece (30 mg) of auxin-treated callus, the resultant growing calluses were auxin-requiring and not habituated (Fig. 1) (K. Sybno, unpublished data). Probably supply of auxin reversed the partially habituated state so that cells became auxin-requiring again. Fully habituated calluses, after prolonged cultivation after induction of habituation, showed no such inoculum effect and only gradual, progressive changes were evident during subculture. A similar result was also reported in cytokinin-habituated tissues (Meins et al., 1980a).

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As described earlier, the optimal concentration of auxin for the growth of cultured tissues falls during serial subcultures and finally tissues lose their requirement for an exogenous source of auxin. After prolonged subculture for more than 10 years, habituated cells of line XD6S2 can be induced by treating XD6S2 cells with 1 mg/liter of NAA for 3 days at 26°C or for only 1 day at 36°C. Compared with the results obtained with the same line subcultured for shorter periods, a lower concentration of auxin and a shorter period of treatment are sufficient for induction. This observation may also reflect the gradual, progressive process of habituation (K. SyCmo, unpublished data). Using slightly cytokinin-habituated clones of tobacco, changes in the degree of habituation were monitored in subcultures over a long time. The distribution of subclones gradually increased in degree of habituation with time in cultures. It was concluded that changes in the requirement of cytokinin-habituated tissues for exogenous cytokinin result from gradual, progressive changes at the cellular level, and not from the accumulation (as a result of faster growth than less- or nonhabituated cells) of a few fully habituated or variant cells that arise in the tissue during subculturing (Meins and Binns, 1977).

5. Temperature The importance of the concentration of auxin and of temperature for the appearance of habituated tissues during 2 years in subculture has been reported (Gautheret, 1957). The heritable conversion of nonhabituated cells to the habituated cells is a process that, unlike mutation, is strongly influenced by the physiological state of cells. Cytokinin habituation occurred at high rates, higher than 1 in lo3,in response to low concentrations of cytokinin at 35°C treatment (Meins and Lutz, 1980). Tobacco cell lines from N. tabacum cv. Bright Yellow (Sy6no and Furuya, 1971) and the cultivar Havana 425 (Binns and Meins, 1979)required an exogenous source of cytokinin for growth at 16"C, but not at 26"C, the standard culture temperature (Fig. 2). On prolonged culture, cold-sensitive cells gave rise to cold-resistant variants that exhibited the habituated phenotype at both low and normal temperatures (Binns and Meins, 1979).

6. Genetic Background Intra- and interspecific differences in the cytokinin requirements of calluses of Phaseolus vulgaris L. and P. lunatus have been examined. Among the ten genotypes of P. vulgaris tested, one was cytokinin-dependent for callus growth, three grew uniformly in the absence of cytokinin, and the remaining six grew moderately well on cytokinin-free medium. By contrast, six genotypes of P. lunatus were strictly cytokinin-dependent,

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KUNlHlKO SYONO AND TOMOMICHI FUJITA

FIG. 2 Effects of temperature on growth of cytokinin-requiring(T2) and -nonrequiring (T22) calluses. Callus T22 grew rapidly on the medium without added kinetin (0.01 mg/liter) at 26°C but required kinetin at 16°C. (Sydno and Furuya, 1971.)

while four displayed irregular rates of callus growth on cytokinin-free medium. The genotype-specific behavior was observed irrespective of the origin of tissues from which the callus tissues had been isolated and also of the time in culture. Results from crosses between a strictly cytokinindependent genotype and two independent genotypes of P. uulgaris showed that cytokinin autonomy in cultured P. uulgaris tissue is a genetic trait under nuclear control and may be regulated by a single set of alleles (Mok et al., 1980). As described earlier, the extent of habituation generally increases during subculture. There are, however, some exceptions to this rule. For example, in a study of tobacco species, N. bigelouii was the most unstable in terms of hormone requirements among the species examined. The tissues were efficiently habituated after a very short hormone treatment (Buiatti and Bennici, 1970). Another example can be found in Lilium longi$orum, which becomes autotrophic after a short period of growth on IAA-containing medium (Sheridan, 1968). Since these phenomena were observed only in specific species, they seem to be genetically determined.

habituated state

Q Q induction growth

+ transfer

-

basal medium

a

a

&+a a &+a /

////I IAA(1 mg/l) medium

rn

clone

high concentration of auzin (NAA 10-100 mg/l)

recovery

/

FIG. 3 Schematic illustration of the induction of habituation and its reversal with a newly isolated single-cell clone. Habituated calluses were induced in an auxin-requiring cell line, XD6S2, by high concentrations of synthetic auxins. The habituated cells reversed to the auxin-requiring phenotype when they were transferred to a medium that contained the regular concentration ( I mglliter) of auxin.

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KUNlHlKO SYONO AND TOMOMlCHl FUJITA

6. Reversibility In spite of many reports of habituated cells in culture, there are fewer well-documented cases of epigenetic changes. The habituation of cells described above (T22 and XD6S2) was fully reversible by treatment with 1 mghter IAA (the regular concentration used for subculture) at an early stage of subculturing on basal medium, but not after prolonged subculturing (SyOno and Furuya, 1974). Once established, the habituated state is extremely stable. The interconversion between the normal (nonhabituated) state and the tumorous (habituated) state has been demonstrated repeatedly merely by changing the concentration of auxin in the growth medium. Even in newly isolated clones of single cells, similar results were obtained (K. SyOno, unpublished data) (Fig. 3). Thus, the phenomenon involves epigenetic changes and not the selection of mutants. Another example has been reported for cytokinin habituation in tobacco. The habituated state was very stable, as in the case of full habituation, and reversion only occurred at high rates when cloned lines were induced to form plants. Cultivated tissues derived from the cortex of tobacco stem exhibited a cytokinin-autotrophic (C' ) phenotype. By contrast, tissues cultured from the leaf lamina had a cytokinin-requiring (C- ) phenotype. When cells from the two types of tissue were cloned, each clone persisted in its original phenotype, which depended on the origin of the tissue from which the clone was derived. Thus, leaf- and stemspecific requirements for cytokinin are inherited by individual cells during cultivation. These inheritable traits were not, however, accompanied by genetic mutation because the two types of cytokinin requirement were

cortex segment

single cell clones

leaf segment

single cell clones

FIG. 4 Developmental regulation of states of habituation. The cytokinin-habituated phenotype ( C * )of cortex reverts to a nonhabituated phenotype (C-) of leaf during regeneration and vice versa. (Data from Meins et al., 1983.)

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275

shown to be interchangeableduring the regeneration of plants. Regardless of whether C- leaf clones or C+ cortex clones were used to regenerate plants, the leaf and cortex tissues of the regenerated plants exhibited the cytokinin requirement of the analogous tissue taken from seed-grown plants. Namely, cells in a particular state undergo transdetermination during the plant regeneration process (Meins et al., 1983) (Fig. 4). In the case of tobacco pith cells, at least two types of cytokinin-requiring cells have been observed. The first type habituates rapidly under inductive conditions. The second type continues to express the cytokinin-requiring phenotype for many cell generations in culture but retains the capacity for habituation. These findings suggest that pith cells differ in their competence to habituate and that different states of competence are inherited by individual cells (Meins et al., 1980a).

111. Properties of Habituated Cells

A. Hormonal Aspects

Autonomy with respect to phytohormones may be due to (1) increased rates of biosynthesis of the phytohormones, (2) interactions between phytohormones or other substances, (3) decreased degradation of phytohormones, and/or (4) altered sensitivity of cells to phytohormones (Jackson and Lyndon, 1990). In the case of crown gall, the hormonal autonomy of the cells is derived from the overproduction of phytohormones that is caused by the expression of genes that encode enzymes for phytohormone biosynthesis and are transferred from the bacteria into the plant genome (Akiyoshi et al., 1983), as described above. Also, it has often been claimed that the autonomous growth of habituated plant cells results from the enhanced production of growth-promoting factor(s) (Mousdale e? al., 1985). Positive feedback promotion of phytohormone biosynthesis was postulated as the mechanism of habituation (Meins and Lutz, 1980). It has been also reported that crown gall and fully habituated cells of tobacco can form tumors when grafted onto host plants of the same species, whereas normal and cytokinin-habituatedcells cannot (Bennici et a / ., 1972; Hansen et al., 1985). Only tissues capable of hormone-autonomous growth contained readily detectable amounts of cytokinin (Hansen et al., 1985). By contrast, in some lines of fully habituated tobacco cells, no such elevated levels of auxin and cytokinin can be detected (Nishio et al., 1976; Nakajima et al., 1979). In cytokinin-habituated calluses, no

276

KUNlHlKO SYONO AND TOMOMlCHl FUJITA

enhanced levels of cytokinin have been observed (Wyndaele

et

al.,

1988).

It is difficult to identify a direct correlation between hormone autonomy and levels of endogenous hormone. Interactions between phytohormones apparently influence both levels of endogenous hormone and hormoneautonomous growth. In the case of cytokinin-habituated cells of the soybean cultivar Mandarin, no enhanced levels of endogenous cytokinin seemed to be required for growth on cytokinin-free medium (Wyndaele et al., 1988). A similar result was also obtained with tobacco callus (Hansen et al., 1987). Incubation of fully habituated tissues on auxin-containing medium was found to dramatically inhibit the accumulation of cytokinin in some cell lines, suggesting that auxins may be important in the regulation of cytokinin metabolism (Hansen et al., 1985; Wyndaele et al., 1988).

The inhibitory effects of exogenous auxin on the accumulation of cytokinins may explain the absence of a relation between cytokinin content and cytokinin-independent growth. In tobacco callus, it was reported that as more exogenous cytokinin was supplied, less auxin was needed to obtain the same level of growth (Einset, 1977). Auxin-habituated tobacco and soybean lines cultured on medium containing only cytokinin as a plant hormone had enhanced endogenous level of IAA (Sybno and Furuya, 1972a; Wyndaele et al., 1988). Habituated cultures of vinca and sunflower had a specific requirement for auxin in low-salt medium, but not in highsalt medium in the presence of kinetin (Sogeke and Butcher, 1976). In the case of crown gall, auxin and other organic growth factor(s) required by normal and partially transformed crown gall cultures of vinca were reduced by adding extra inorganic salts. It was postulated that certain inorganic salts are able to activate the biosynthesis of auxin and other growth factor(s) (Wood and Braun, 1961; Braun and Wood, 1962). Alternatively, the differences in the hormone requirement of normal tissues and tumorous cultures, including crown gall tissues, may result from differences in membrane permeability which auxin may affect. This result may explain the growth of the calluses observed on auxin-free medium supplemented with a high concentration of cytokinins. Endogenous levels of phytohormones may also be affected by the rates of their degradation or metabolism. IAA-degradative activities have been repeatedly investigated in this connection. A dramatic rise in the level of an auxin protector, an inhibitor of IAA oxidase, was reported during formation of crown gall induced by A. turnefaciens (Stonier, 1969). By contrast, higher activities of the inhibitor in normal callus tissue than in crown gall or habituated tissues were also reported (Kevers et al., 1981). The results are complicated and it is difficult to define a direct relationship between such activities and hormone autonomy.

HABITUATION AS AN INTERCHANGEABLE TUMOROUS STATE

277

In tobacco callus lines (T22 and XD6S2), decreased IAA-degradation activities, via increases in levels of auxin protector, were observed only when calluses on a medium were favorable for induction of auxinnonrequiring callus, despite marked differences in the conditions for induction of auxin-nonrequiring callus between the two lines. Thus, it is not unreasonable to suppose that an increase in level of auxin protector might be one of the factors involved in the induction of habituation (Sydno, 1979). In addition to increases during induction of crown gall, increases in levels of auxin protector were reported in meristematic tissues, and temporary increases were found at wounded sites in mature tissues, while only low activities were found in unwounded mature tissues. The accumulation of the protectors described above seems to be associated with active cell division. Reduced glutathione (Pilet, 1958), ascorbic acid, NADH, cysteine, and thioglycolic acid are all effective inhibitors of the peroxidasecatalyzed oxidation of IAA (Betz, 1963). The possible involvement of o-dihydroxyphenolics and other protectors in relation to redox potential and cell division has been discussed in detail (Stonier, 1970). It has been suggested that the more important function of auxin protector(s) is to keep the cell in a reduced state or to mediate other effects, as discussed later, and it is not present merely to prevent the destruction of IAA (Stonier, 1970). Judging from the different treatments favorable for inducing habituation among the cultured cell lines, there may be several (at least two) mechanisms that induce habituation (Sydno and Furuya, 1974). In spite of the more sensitive responses of habituated cells to plant growth substances (Sydno and Furuya, 1971), the direct correlation between hormone autonomy and endogenous levels of hormone cannot be found as described earlier. The altered sensitivity to the hormone was hypothesized as the other mechanism of the cause of habituation (Nakajima et al., 1979), although there does not appear to be any firm evidence for the hypothesis. B. Molecular Genetic Aspects

As described above, leaf tissues of Nicotiana tabacum L. cv. Havana 425 normally require an exogenous source of cytokinin for rapid growth in culture. Among the plants regenerated from cloned cultured cells, two types of tobacco plant in particular were isolated which differed in leaf tissue phenotype with respect to their cytokinin requirement in culture (Fig. 5). Leaf tissues of these plants were cytokinin-habituated. Studies on the inheritance of the habituated-leaf trait of HI-1, one of the two above-mentioned types of plants, showed that F, hybrids of Hl1 were intermediate between the parental types in terms of the extent of

278

KUNlHlKO SYONO AND TOMOMICHI FUJITA

leaf segment

cortex segment normal

I

\

@

I

L

leaf segment

/"' I

...........

..:.:...................

-,\

pegeneration single cell clones

single cell clones

FIG. 5 A mutant plant in which leaf tissue exhibits a cytokinin-habituated phenotype (C'). At least two genes (HI-1, HI-2) regulate the cytokinin requirement in culture. (Data from Meins and Foster, 1986.)

habituation. No differences were found between reciprocal hybrids. In addition to these results, the frequency of habituated-leaf progeny in the F, and backcross populations showed that the habituated-leaf trait of HI1 is an incompletely dominant, nuclear trait and is regulated by a single genetic locus. Another plant with the habituated-leaf trait was named HI2. In this case, the trait segregated in breeding tests as expected for a dominant, monogenic trait and was inherited at a different locus from the HI-1 trait. Cytokinin mutants can arise in cell culture and at least two genes regulate the cytokinin requirement of cultured tobacco tissues (Meins and Foster, 1986). Neoplastic growth of crown gall requires the expression of gene 4, which encodes a key enzyme for the biosynthesis of cytokinin, as well as genes 1 and 2 in the tms region of the T-DNA. The habituated-leaf trait was shown to compensate for a defective gene 4 in the tmr region of the Ti plasmid in expression of the tumorous phenotype. The results provide evidence that a specific plant cell gene (HI) has an oncogenic function similar to gene 4 in T-DNA. There is no evidence, however, for sequences homologous to gene 4 in the DNA of normal tobacco cells, as judged by Southern hybridization. Moreover, the function of HI seems to differ from that of gene 4 in tumor development. HI can replace the requirement for tmr in expression of cells with a tumorous phenotype, but it cannot replace the requirement for gene 4 in the induction of tumor (Hansen and Meins, 1986). Mutagenically produced tumors may be some of the most useful experimental materials for investigating the control of plant growth and development. Tumors have been induced by treatment of Arubidopsis seeds with y-irradiation, and they arise on the hypocotyls and shoot tips of the plants

HABITUATION AS AN INTERCHANGEABLE TUMOROUS STATE

279

that develop from irradiai.ed seeds. Such tumors have been excised and grown on hormone-free medium (Campell and Town, 1991). cDNAs complementary to transcripts that are expressed at higher levels in a hormone-autonomous tumor line than in normal, hormone-dependent cultured tissues have been isolated and characterized. They include interesting cDNAs with strong sequence similarities to cDNAs that encode a lipid-transfer protein, membrane-channel proteins, and glycine-rich proteins (GRPs) (Campell and Town, 1992). Overexpression of membranechannel proteins could result in increased rates of nutrient uptake or transport, allowing growth without hormonal stimuli. Crown gall cells have been reported to be associated with increased efficiency of substrate utilization (Braun and Wood, 1962; Wood and Braun, 1961). Recently, it has been proposed that habituation involves perturbation of intracellular metabolic pathways (Hervagault et al., 1991). With respect to GRPs, it has been shown that GRPl of petunia is an indicator of promeristematic cells and that overexpression in transgenic plants causes formation of supernumerary organs. Therefore, it seems likely that overexpression of GRPs might be a causative factor in tumorous growth (Campell and Town, 1992). A newly developed T-DNA tagging vector has been used to isolate hormone-autotrophic cells. The vector consists of multiple transcriptional enhancers derived from the 35s RNA promoter of cauliflower mosaic virus (CaMV), located near the right border sequence of T-DNA. After insertion ofthe T-DNA into the plant genome, mediated by Agrobacterium infection, genes present in the plant DNA that flanks the T-DNA insert are overexpressed. This tagging vector has allowed isolation of plant genes that, upon overexpression, promote the growth and division of protoplasts in hormone-free medium. Northern blot analysis showed that transcripts of the cDNA were present in larger amounts in protoplasts and leaves of transgenic plants than in control tobacco plant cells (Hayashi et al., 1992).

IV. Genetic Tumor as an Example of Exaggerated Habituation A. Introduction

1. Genetic Tumors

Genetic tumors are neoplastic growths that arise, without any apparent external cause, in organisms of certain particular genotypes. The genetic constitution seemingly determines the potential of cells to undergo a spon-

280

KUNlHlKO SYONO AND TOMOMlCHl FUJITA

taneous change from normal to tumorous growth. In a number of different plant species, tumors have been reported to occur with no obvious external cause, such as attack by viruses, bacteria, fungi, or insects, although no stringent genetic tests were carried out on these plants and their progeny. Thus, these tumors may also loosely be termed genetic tumors'. The involvement of genetic constitution in the formation of spontaneous tumors is much more apparent in interspecies hybrids, as well as in hybrids within species. Root tumors on interspecies hybrids of the genus Brassica, namely on the results of turnip-rutabaga crosses, appear to be the earliest recorded abnormal outgrowths of probable genetic origin observed in plants (Caspary, 1873; Kajanus, 1917). Similar abnormal proliferation or development has been reported in hybrids within species, such as sweet clover (Melilotus alba) (Littau and Black, 1950), white spruce (Picea glauca) (White and Millington, 19541, pea (Pisum satiuum) (Dodds and Mathews, 1966), and Japanese morning glory (Pharbitis nil) (Takenaka and Yoneda, 1963, 1965), as well as in several interspecies hybrids, for example, root tumors in Brassica rapa L. x B . pekinensis (Kehr, 1965),ovular tumors incrosses between Datura stramonium and other species of Datura (Rappaport et al., 1950), aberrant growth of apical meristems and the cortical parenchyma of stems and leaves in crosses of Bryophyllum calycinum x B . daigremontianum (Resende, 19571, tumors on germinating seeds of Lilium speciosum Album x L . auratum (Emsweller et al., 1962), eruptions on the lower sides of leaves in crosses of Licopersicon esculentum x L . chilense (Martin, 19661, and tumors in interspecies hybrids of Nicotiana species (Kehr, 1951). As described earlier, tumors arise at a specific developmental stage and in specific parts of plants, depending on the species. Formation of genetic tumor is usually accompanied by active proliferation of cells. In the case of Lycopersicon, however, leaf outgrowth was the result of the elongation of cells. The causes of these differences remain to be determined. 2. Genetic Tumors in Nicotianu

Among tumor-prone hybrids reported in a number of species of plants, genetic tumors within the genus Nicotiana have been studied most extensively. Since the earliest report of spontaneous tumors in Nicotiana ( N . 1angsdorfJii x N . glauca) (Kostoff, 1930), more than 300 interspecies hybrids among the 64 species in the genus have been examined. About 30 hybrids from combinations among these species produce tumors regularly and at least 22 other hybrids develop similar but restricted or irregular growth abnormalities (Kehr and Smith, 1954). The species involved in crosses that result in development of genetic

281

HABITUATION AS AN INTERCHANGEABLE TUMOROUS STATE

tumors have been classified into two separate groups, arbitrarily designated the plus group and the minus group. Hybrids between a member of each group produce tumors, whereas hybrids within each group do not. The plus group consists of species that belong mainly to the section Alatae, with a chromosome number ( n ) of 9 or 10. The minus group consists of a variety of other species derived from various sections. Species such as N. glauca, N. tabacum, and N . debneyi are found in this group. In most of the minus group, n equals 12 or 14 (Naf, 1958) (Fig. 6). The same results in terms of tumor formation are obtained in interspecific hybrids regardless of which species is used as the male or female parent. Thus, it appears that tumor formation is determined not by cytoplasmic elements but by nuclear elements contributed equally by the male and the female parent (Kehr, 1951; Smith and Stevenson, 1961). A single extra chromosome of N. longiyora (plus group) on the nontumorous amphidiploid N. debneyi-tabacum (minus group) background is sufficient for the development of spontaneous tumors after repeated backcrossing of N. debneyi-tabacum with N . LongiJIora (Ahuja, 1965). Simi-

Minus group

Plus group

N. glauca N. tabacum N. suaveolens N. debneyi N. rustica N. paniculata N. miersii N. bigelovii

N. langsdorffii N. alata N. longiflora N. plumbaginifolia N. sanderae N. forgetiana N. bonariensis N. nectiflora

n = 9 or 10 factor I

1, I

n=12or14 factor ee

1, ee

ee, ee

I

I

I

normal hybrids

tumor-prone hybrids

normal hybrids

FIG. 6 Tumor formation of interspecies hybrids in Nicotinna. Tobacco species have been classified into two groups. Hybrids between a member from each group produce tumors. I and ee are hypothesized genetic factor(s) involved in tumor formation. (Data from N&f, 1958; Ahuja, 1965.)

282

KUNlHlKO SYONO AND TOMOMlCHl FUJITA

larly, a tumorous hybrid with a single chromosome of N. glauca (minus group) on the N. langsdorfii (plus group) background has also been obtained (Smith, 1988). These results suggest that in tobacco the gene(s) responsible for formation of genetic tumors are located on a particular chromosome in both the plus and the minus group, respectively. 6. Induction of Genetic Tumors and Their Reversion

1. Phenomena in Vivo In general, genetic tumors of Nicotiana hybrids appear spontaneously at the maturation stage, after the plant has gone from vegetative to reproductive growth, Tumors also develop when young plants are exposed to stress conditions. Irradiation of various parts of tumor-prone hybrid plants accelerates tumor formation. Tumors appear earlier than they do in unirradiated control plants (Smith, 1965). Elevated temperatures and crowding also induce tumors prematurely. Several chemicals, such as derivatives of phenol and benzene, mercaptoethanol, and even paint mixtures, also accelerate tumor formation in young plants (Ames and Smith, 1969). Among the phytohormones, cytokinin alone or in the presence of auxin promotes the induction of tumors in Nicotiana seedlings, whereas auxin alone has no effects (Schaeffer, 1962). Since decapitation promotes tumor formation and this effect is inhibited by a supply of auxin to the decapitated shoot apex, it was suggested that the decline in levels of auxin caused by decapitation promotes tumor formation in young plants. Auxin may thus have somewhat inhibitory effects (Ames, 1974; Ames and Mistretta, 1975). Shoot-like protuberances that emerge and extend from partially differentiated (teratomatous) regions of hybrid plants (Nicotiana suaueolens X N. langsdorfii) sometimes elongate into abnormally thick stems. These aberrant branches, which apparently originate from tumor tissues, occasionally yield flowers that produce seeds. The progeny germinated from the seeds on aberrant stems do not show any differences, even in the tumor-forming trait, from the progeny derived from the seeds produced on normal stems on the same hybrid (Smith, 1965).

2. Phenomena in Vitro a. Fusion of Protoplusts

Ultimate proof of the reversal of genetic plant tumors would require the regeneration of a plant similar to the progeny of tumor-prone hybrids from a single cell with a genetically defined tumorous nature. Such experimental proof has not yet been reported, but similar

HABITUATION AS AN INTERCHANGEABLE TUMOROUS STATE

283

evidence has been presented from experiments with protoplasts (Smith et al., 1976).

Protoplasts of N. gfauca and N . langsdorjjfii were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol. After cell division in hormone-containing medium for a short period, the mixture of fusion products of the protoplasts was transferred to a medium that lacked phytohormones for selection against the parental protoplasts which required hormones in culture. Among the 130 tumorous clones with the hormone-nonrequiring phenotype, 70 differentiated into leaf-like teratomatous structures and some of these occasionally formed roots. Mature plants obtained by either grafting onto parental stock or from teratomotous structures that had rooted, developed into independent plantlets. All of the 23 regenerated plants that reached sexual maturity formed tumors spontaneously, usually about a month after initiation of flowering, confirming their hybrid nature. The parasexual hybrids were somewhat different from but similar to true amphidiploids (GGLL) produced by cross-pollination and were different from either parent (Smith et al., 1976).

b. Interconversion System in Vitro In the greenhouse, genetic tumors appear most frequently on mature hybrid plants during and after the flowering period, when growth of the shoot apex is reduced (Smith, 1965). Since the tumors develop spontaneously on the mature plant, it takes a very long time and is hard to regulate the timing of tumor formation. Involvement of microorganisms in tumor formation cannot be excluded. In in vitro aseptically germinated seedlings of N. gfauca x N . lungsdorffii, however, tumorous outgrowths developed more easily on the hormone-free medium, most frequently at the upper axillary nodes and leaf scars; on areas subjected to stress, such as tissues that touched the surface of the glass culture flask; directly on seeds; and around the border between the stem and agar medium (Fig. 7). Cutting induced tumor tissues synchronously and rapidly on every segment excised from morphologically normal plantlets, without exception (Ichikawa and Sybno, 1988). This system might be appropriate for studying genetic tumor induction at the molecular level. In this in uitro system, tumorous tissues induced by cutting grew rapidly on the basal medium, with a 10-fold increase in fresh weight every 6 days for 15 days. The tumorous state was maintained during repeated subculturing under continuous light (ca. 10 W/mZ)at 27°C. When this tumor was cultured in the dark or under a light intensity less than 0.1 W/mZ, normal etiolated shoots developed frequently (Fig. 8). After the regenerated shoots which were more than 3 cm in height were stuck into fresh basal medium, they were cultivated under light conditions of more than 0.24 W/mz.The shoots gradually turned green, and developed

KUNlHlKO SYONO AND TOMOMlCHl FUJITA

FIG. 7 Spontaneous tumor formation in uitro. Tumors developed at the transitional region between shoot and root on a hormone-free medium. (Ichikawa and Sybno, 1988.)

normal leaves and adventitious roots. Finally, whole plants with a normal appearance regenerated. The state of the tissues can be regarded as the same one as that of plants derived from seeds, since the regenerated plants could develop tumor tissues in the same manner as the plants grown from seeds. Although both the germinated seedlings and the regenerated plantlet

FIG. 8 Two different states of genetic tumor cells. (Right) Tumorous state under continuous light. (Leff)Morphologically normal shoots regenerated from tumors in darkness. Ichikawa and Sybno, 1988.)

HABITUATION AS AN INTERCHANGEABLE TUMOROUS STATE

285

Teratoma

f

Light

Y 0 Seeds

seedlings Normal

\$

Regenerated shoots A

g

h

t

Normal regenerated shoots FIG. 9 I n uirro system of interconversion between a normal and a tumorous state in a genetic tumor.

appeared to be normal, they had the potential ability to form tumorous tissues (Ichikawa and Sybno, 1988).Consequently, we were able to switch the morphology between the normal and the tumorous states rapidly and with ease in this system (Fig. 9). C. Hormonal Aspects of lnterconversion between Genetic Tumors and Normal States

1. Auxin In general, as observed in the formation of callus that is induced by exogenously supplied phytohormones or the formation of crown gall that is induced by overproduction of endogenous phytohormones (Akiyoshi

286

KUNlHlKO SYONO AND TOMOMlCHl FUJITA

et al., 1983), phytohormones are known to be involved in the induction of tumorous growths from normal tissues. Thus, in genetic tumors also, increases in levels of phytohormones were expected to be involved in the interconversion from a normal to a tumorous state. When stem segments of normal tobacco F, ( N . glauca X N . langsdotfJii) seedlings were cultured on hormone-free medium in the light, the first cell division was observed within 12 hr and tumorous tissues were observed on the cut surfaces of segments within 5 days after cutting. Longitudinal sections of the segments revealed active cell division even in the original cut stem region which, externally, still appeared normal. Moreover, primordium-like structures corresponding to teratomas were observed in the peripheral regions of proliferating tissues that originated from cut surfaces. Subsequently, they developed into a primordium of teratomatous buds 1 1 days after cutting. The teratomatous tumors developed very rapidly, with a 10-fold increase in weight every 6 days (Ichikawa et al., 1989). The level of endogenous IAA in normal internodes of F, seedlings grown in the light was a few nanograms per gram fresh weight. The level increased dramatically after cutting and reached about 100 ng/g fresh weight within 5 days. The level rapidly decreased close to the initial level, remaining a little bit higher than that in normal F, tissues even after transfer to fresh medium for subculture. Similarly, low levels of auxin in vigorously growing tumors cultured in uitro in darkness have also been reported (Palni and Summons, 1987; Fujita et a)., 1991). Temporary increases and subsequent reductions in levels of auxin might be important for the induction of tumors, but it remains unclear whether variations in levels of auxin are a cause of, coincide with, or are a result of tumor induction. A close correlation has, however, been demonstrated between a decrease in levels of endogenous auxin and the induction of tumor formation in hybrids of greenhouse-grown plants (Ames and Mistretta, 1975). In another related study of auxin production, it was reported that the rates of synthesis of water-soluble, bound auxin were higher in tumorous hybrids than in the parents (Liu et af., 1978). Release of free auxin from bound auxin that accumulated in the hybrids may explain the increase in levels of free auxin during tumor-inducing treatment in uitro (Ichikawa et al., 1989). Metabolic interconversion between water-soluble, bound auxins and the auxins in their free, active forms should be examined in relation to the morphological interconversion between the tumorous and the normal state (Ichikawa and Syono, 1991). 2. Cytokinins

The level of cytokinin nucleotides is about five times higher in tumorprone hybrid tobacco plants than in the parental species (Nandi ef al.,

HABITUATION AS AN INTERCHANGEABLE TUMOROUS STATE

287

1990a,b). However, interconversion of nucleotides and free cytokinin has not been examined in relation to the development of the tumorous state. The morphological observations described earlier demonstrated that tumors developing on tobacco hybrids proliferate vigorously and differentiate into the primordia of many aberrant buds or leaves to form teratomas. These observations suggest that a cytokinin-like agent is involved in the induction of genetic tumors since similar effects of cytokinin are observed in tobacco pith tissue in culture (Skoog and Miller, 1957). Tumor induction was enhanced by treatment with cytokinin as described above (Schaeffer, 1962. The introduction of a gene required for cytokinin biosynthesis in the T-DNA of A. tumefaciens into a nontumorous mutant resulted in recovery of the tumorous character (Feng et al., 1990). The level of endogenous cytokinin in cultured tumorous tissues was, however, not as high as expected (Palni et al., 1988). D. Molecular Basis for lnterconversion 1, cT-DNA Genes

Agrobacterium rhizogenes causes the plant disease known as hairy root syndrome in a large number of dicotyledonous plants. As in the case of crown gall tumors, a large plasmid called root-inducing (Ri) plasmid is essential for development of the disease. Two small portions (TL-DNA and T,-DNA) of the plasmid are transferred to the plant genome and are responsible for the formation of hairy roots. Four loci, designated rolA, rolB, rolC, rolD (rol-root locus), have been identified as being involved in the formation of hairy roots (Zambryski et al., 1989). Even if neoplastic growth results, tissue transformed by A. rhizogenes tissue can regenerate to yield fertile normal plants that give rise to transformed progeny, an example of interconversion between the neoplastic state and normal state.

a. Homology with T-DNA of Ri Plasmid. The genome of untransformed Nicotiana glauca contains a DNA sequence homologous to the TL region of the Ri plasmid that has been designated cT-DNA (cellular T-DNA). cT-DNA is organized as an inverted repeat which is imperfect at its center, such that the left arm is longer than the right (White et al., 1983; Furner et al., 1986) (Fig. 10). On the left arm of cT-DNA, there is an open reading frame (ORF), named the Ng rolB gene, that corresponds to ORFl 1 (rolB)on the T,-DNA of the Ri plasmid and another ORF, named Ng rolC gene, corresponds to ORF12 (rolC) on the Ri plasmid. The Ng rolB can potentially encode a similar polypeptide, although a 1-bp substitution at a site 633 bp away

TL-DNA mL4 mIB

A. hizOgenes Ri plasmid

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FIG. 10 Relationship between cT-DNA in Nicotiana glauca and TL-DNA in Agrobacterium rhizogenes. N . glauca contains a DNA sequence (cT-DNA) homologous to the bacterial gene. cT-DNA is organized as an inverted repeat which is imperfect at its center, such that the left arm is longer than the right. The sequences of four ORFs corresponding to those in Ri plasmid are highly conserved. (Data from Furner er al., 1986; Aoki et al., 1994.)

HABITUATION AS AN INTERCHANGEABLE TUMOROUS STATE

289

from an initiation codon generates a termination codon and results in polypeptides that are 48 amino acids shorter, namely 80% of the length of those encoded by Ri roll?. The left arm ORF of Ng rofC is intact and codons for the initiation and termination of translation are present at positions that correspond to those of the Ri rofC gene. The Ng rolC gene on the right arm may, however, be a pseudogene. The ORF is interrupted by a frameshift-generated stop codon near the start codon of the gene (Furner et al., 1986) (Fig. 10). Recently, the sequence of the remainder of the left arm of the cT-DNA was determined and two other intact ORFs, corresponding to ORFs 13 and 14 of the T,-DNA of the Ri plasmid, were found (Aoki et al., 1994). These genes were designated Ng ORF13 and Ng ORF14, respectively. The sequence of these genes is highly conserved. The homology within reading frames between A. rhizogenes and N . glauca in the rol B-C region is 83%, whereas the homology in the intergenic regions averages 75% at the nucleotide level. The amino acid sequences of predicted polypeptides are 75% homologous when those encoded by Ng rolC and Ri rolC are compared (Furner et al., 1986). The reading frames ofORFs13 and 14 in N. glauca are 87.2% and 83.9% homologous to DNA sequences of corresponding ORFs in Ri plasmid, respectively, The amino acid sequences of the predicted peptides are 80% and 74% homologous in ORFs 13 and 14, respectively (Aoki et a f . , 1994) (Fig. 10).

b. Expression of cT-DNA. These genes in the cT-DNA were believed to be pseudogenes until transcripts of the Ng rolB and Ng rolC genes were detected in the teratomatous tissues of F, hybrids of N . glauca x N . h g s d o r f i i , but not in the seedlings of N. gluuca, one of the parental species, which contained cT-DNA (Ichikawa et al., 1990). Subsequently, similar results were obtained for ORFs 13 and 14 (Aoki et a f . , 1994). Ng rolB and Ng rolC are transcribed most extensively in tumorous tissues cultivated in virro. The level of expression of these genes is reduced in teratomatous tissues grown in the dark and no expression can be detected in normal shoots regenerated from the tumorous tissues. These results indicate that the extent of expression of the Ng rofE and Ng rofC genes is qualitatively correlated with the extent of the tumorous state in F, hybrids (SyBno et al., 1992). Recently, all the cT-DNA genes were found to be expressed in the habituated tissues of N.glauca cultivated in the light (line GHL) and in darkness (line GHD), as well as in the auxin-requiringcallus tissues (G89) of N. glauca from which the habituated lines were derived. Since these genes are expressed in the auxin-requiring parental line, G89, no direct correlation between habituation and expression of these cT-DNA genes

290

KUNlHlKO SYONO AND TOMOMlCHl FUJITA

is apparent. In the case of intact plants, at flowering, the F, hybrid of N. glauca x N . langsdorffii and N . glauca itself contained detectable transcripts in their stem tissues but not in their leaf tissues. This observation may reflect a much easier transition of stem tissues than leaf tissues to the tumorous state (S. Aoki and K. Sy6no, unpublished data). 2. Tumor-Specific Genes

One of the most useful approaches to understanding the mechanism of interconversion between the normal and the tumorous state involves identification of the genes expressed specifically in tissues in each state. With this aim, cDNA probes specific for the tumorous state in interspecies hybrids from the cross of N. glauca x N . 1angsdotfFi were prepared by subtractive hybridization procedures and used to screen a genetic tumor cDNA library, As a result, 17 distinct cDNA clones were isolated for genes that are specifically or preferentially expressed in tissues in a tumorous state but are not expressed at all or are scarcely expressed in F, hybrids in the normal state (Fujita et al., 1994). About half of these cDNA clones exhibit significant homology to known genes. These clones encoded proteins homologous to glucan endo- 1,3-P-gIucosidase, wound-induced proteinase inhibitor I of tomato, pathogenesis-related (PR) protein la, osmotin, and the basic form of PR1 protein, respectively. All of them are induced by viral infection or wounding and are so-called stress proteins. Three other clones also exhibited significant homology to the 14-kDa protein of carrots, miraculin in miracle fruit (Richadella dulcijca), and a major allergenic protein of rye grass (Lo1PI). Transcripts that corresponded to these eight clones accumulated at high levels but only transiently after F, hybrid stems in the normal state were cut (Fujita et al., 1993; 1994). Using the kinetics of accumulation of the transcripts that correspond to the eight novel cDNA clones, these clones could be divided into three classes (Fig. 11). The mRNAs that correspond to members of the first group of clones (four clones) were expressed transiently at the early stages of tumorigenesis after cutting. This transient increase after wounding is similar to that in the expression of wound-inducible genes. Some kind of stress is a prerequisite for the formation of genetic tumors. These genes, in addition to those corresponding to clones that exhibit significant homology to known genes, as described earlier, may be involved in the transition from the normal to the tumorous state although the expression may be merely a defense response to wounding or to stress that is independent of the transition. The expression of genes that correspond to the second group of clones

HABITUATION AS AN INTERCHANGEABLE TUMOROUS STATE

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the first- group - - - -of- -clones

--------

I

the third group of clones

-------

'

dividing

0-n-mcells

0

2

5

I

teratoma tissues

------11

subculture

days after cutting

FIG. 11 Schematic representation of three types of gene expression patterns during the development of geneitc tumors. The appearance and decay of the expression of the genes corresponding to each group of genetic tumor-specific cDNA clones are shown.

(two clones) was induced immediately after F, stems were cut and was maintained in subcultured genetic tumor tissues (Fig. 11). Expressed strongly in callus tissues derived from the parent plants, these genes are suggested to be involved in the proliferation of genetic tumor cells and in the maintenance of a tumorous state. Genes corresponding to the third group of clones (two clones) were first expressed at the late stage of genetic tumor formation (Fig. 11). This stage coincides with the initiation of teratomatous differentiation of the bud primordia (Fujita et al., 1994). Expression of these genes cannot be detected in unorganized variants of genetic tumor tissues, which grow without differentiation of leaf buds. These results support the idea that these genes are involved in the process by which unorganized tissues are converted to organized teratomas (T. Fujita, unpublished data). Almost all genes were expressed in callus tissues derived from the parent plants. However, genes corresponding to the specific 2 in 17 clones were not expressed at all in the callus tissues or in stems and leaves of parent and hybrid plants. These two genes are noteworthy because their expression is specifically restricted to F, hybrids in a tumorous state (Fujita et al., 1994).

292

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V. Concluding Remarks

After treatment to induce habituation, growth of treated tissues on hormone-free medium is greatly affected by the size of the inoculum. Failure of growth with a small inoculum can be overcome by a supply of auxin to the culture medium. The resultant growing tissues are, however, auxinrequiring and not habituated (Fig. 1). A supply of auxin may inhibit transition to the habituated state. This result suggests that, in the transition stage that leads to habituation, auxin itself or some substance, the transport of which is affected by auxin, is easily released from the tissue. In fully habituated tissue, such transport might be inhibited. This working hypothesis is supported by the fact that treatment with specific inhibitor of auxin polar transport, triiodobenzoic acid, induces habituation in both soybeans (Christou, 1988) and tobacco (K. SyMo, unpublished data). Levels of flavonoid, such as quercetin, reported as natural inhibitors of auxin transport (Jacobs and Rubery, 1988), increase when the tissues are treated with cytokinins (Miller, 1969) that maintain growth in place of auxin in some lines of calluses (Syono and Furuya, 1972a; Sogeke and Butcher, 1976; Einset, 1977). Levels of phenolics, including flavonoids, some of which show auxin-protector activity, are remarkably increased when calluses are treated with concentrations of auxins favorable for induction of habituation (Syono, 1979). Furthermore, in pea shoots, decapitation of growing shoots or internodal segments excised from these shoots resulted in the loss of polar transport of 14C-labeledIAA, a natural auxin, and a supply of auxin is required to maintain the activity of polar transport. It seems likely that the reversible loss of polar transport of auxin is the result of a gradual randomization of the distribution of efflux carriers in the plasma membrane after withdrawal of an apical supply of auxin, and that the recovery of polar transport involves reestablishment of effluxcarrier asymmetry under the influence of gradients in the concentration of auxin (Morris and Johnson, 1990). The relationship between the inhibition of induction of habituation and application of physiological concentrations of auxin is very similar to the relationship between the maintenance of polar transport of auxin and application of physiological concentrations of auxin in pea stem segments. Further studies on the transport of auxin and other substances in relation to induction of habituation (Hervagault et al., 1991) can be expected to clarify some aspects of the reversible transition of a requirement for hormones at the molecular level. In this connection, it is very interesting that cDNAs complementary to transcripts that are expressed at a higher level in a hormone-autonomous tumor line than in a normal hormone-

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dependent line include membrane-channel proteins and a lipid transfer protein (Campell and Town, 1992). Recent progress in the molecular genetics of higher plants may assist in an analysis of the reversible transition between the normal and tumorous state at the molecular level. A cloned gene that supports the division of protoplasts in hormone-free medium is undoubtedly useful. Northern hybridization showed that the transcripts correspondingto the clone accumulated not only in protoplasts but also in leaves (Hayashi et al., 1992). Thus, the function of the correspondinggene in inducing tumorous growth is repressed in leaves. Proteins and mRNAs that specifically accumulate in a normal state have been detected and isolated in genetic tumors (Fujita et al., 1991, 1994). The analysis of such macromolecules may provide useful information about the transition between normal and tumorous states. The possible involvement of cT-DNA genes cannot be excluded even if the expression of these genes is not restricted to hybrid plants in the tumorous state. The sensitivity to auxin of cells transformed by A. rhizogenes was reported to be 100-1000 times higher than that of normal untransformed cells. Since DNA sequences homologous to cT-DNA are absent from some of tumor-prone hybrids, such as those from crosses of N. suaveolens x N . plumbaginifalia and N . gossie x N . longiyora (Kung, 1989), cT-DNA may not be essential for tumor formation. However, if the function of cT-DNA genes, including Ng rol genes, is similar to that of Ri genes, it is probable that cT-DNA is a factor in promotion of tumor formation, The function and regulation of expression of cT-DNA genes requires detailed analysis. With respect to reversion from a tumorous to a normal state, there are a few reports in the case of crown gall (Amasino et al., 1984) and hairy root (Sinkar et al., 1988) induced by Agrobacterium infection. In these cases, evidence for methylation and subsequent inactivation of genes in the T-DNA have been presented. Similar modifications of genes may occur in genetic tumors, but the modification reactions must be much more rapidly reversible than methylation. In the case of habituation, reversibility to a normal, hormone-requiring state is reduced during subcultures. This is the case in decreases of organforming capacity (Syijno, 1965) and also in increases in the variability of regenerated plants (Sybno and Furuya, 1972b). Chromosomal variation and movable elements might be other candidates for the cause of these phenomena. Recently, it was reported that retrotransposons are activated in regenerated plants from cultured cells and also in cultured cells (Hirochika, 1993), especially in established cell lines such as the tobacco BY2 line (Nagata et al., 1992) and rice Oc line (Baba et al., 1986) subcultured

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for a prolonged period (Hirochika, 1993). One possible mechanism suggested for somaclonal variation is the activation of transposable elements (Scowcroft, 1985). Although the molecular mechanisms of interconversion between a normal and a tumorous state still remain to be elucidated, recent advances in plant molecular genetics enable us to analyze these curious but developmentally important phenomena at the molecular level. Acknowledgments I thank all the persons who worked with me on these subjects. This work was supported by a grant-in-aid for scientific research and a grant-in-aid for scientific research on a priority area to K.S. from the Ministry of Education, Science, and Culture in Japan.

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Index

A ACC synthase, see 1-Aminocyclopropane-1-carboxylate synthase Agrobacterium rhizogenes, genetic tumors caused by DNA sequence analysis, 288-289 interconversion mechanism, 287 Agrobacterium tumefaciens, tumorous outgrowth induction by, 265-266 1-Aminocyclopropane-I-carboxylate synthase, auxin-induced expression, 132-133 Antiauxins, habituation in tumorous plant cells, induction, 269 Antigens CDllKD18, role in inflammation, 75-76 in monocyte/macrophage procoagulant activity, 60 Arabidopsis, auxin-regulated gene expression, 133 Ascaris univalens, holokinetic chromosomes, 33 Auxins affected plant cells elongation tissues, 111-1 17 growth, 109 molecular mechanisms, 137 suspension culture, 126, 128-132 tobacco mesophyll protoplasts, 117-127 habituation in tumorous plant cells, induction concentration changes, 267-269 cytokinin interactions with, 270 inoculum size, 270-271

mechanisms, 265-266.276-277, 285-286,292 Auxin-regulated genes, see Genes, auxin-regulated

B Basement membrane, effects on retinal pigment epithelium, 253 Blood, coagulation, cellular mechanisms activating in pathological conditions, 89 phospholipid-dependent activity, 54-55 process, 49 procoagulants adhesion receptor regulation of, 75-77 expression suppression, 86-89 and malignancy, 82-86 properties, 77-82 tissue factor characteristics, 50-51 as cytokine growth factor superfamily member, 51-53 function, 53-55 localization, 55 cytokine-mediated induction, 59-64 endothelial cell regulation, 64-73 on fibroblasts, 73-74 monocyte-macrophage, 57-59 Bombyx mori, male eupyrene meiosis, 19 Bovine serum albumin, advanced glycosylation end products, endothelial cell tissue factor induction by, 70 Brassica, genetic tumors, 280

301

302

INDEX

C Calcium in tissue factor expression on fibroblasts, 74 Calcium signal mammalian egg-activating analysis of effects, 200-201 calcium-induced calcium release, 188-189, 191, 196-200 decoding, 210-213 fertilization, 183-189, 213 mechanism, 183-184, 189,206-210 monotonic calcium increase, 202-206 oscillation mechanisms, 196- 199,206-2 10 models, 199-200 requirements, 201-202 signal generation hypotheses calcium bomb, 189-191 G protein-inositol phosphate production, 191- I94 soluble sperm factor, 194-196 Calmodulin-dependent protein kinase 11, activation of mammalian eggs, 210-211 Cancer, and procoagulants, 82-86 Centromeres, lepidopteran apyrene spindle organization, 35 DNA analysis, 33-34 kinetic organization, 11 Centrosomes, lepidopteran, in male eupyrene meiosis, 20 Chromatin elimination, lepidopteran, in female meiosis, 14-15 lepidopteran, in male apyrene meiosis, 27-28 Chromosomes, lepidopteran spindle structure experimental systems, 32-35 holokinetic, 33 kinetic organization, 8-12 meiosis female, 13-15 male apyrene, 24-30,35-36 male eupyrene, 16-24,34-36 microtubules, post-translational modifications, 30-32 mitosis, 12-13 structure, 33-34 technical aspects, 3-8

Clones auxin-regulated genes arcA, cDNA, 129 mechanism, 11 1 complementary DNA, tumor-specific genes in habituated plant cells, 290-291 Coagulation, blood, cellular mechanisms activating, see Blood, coagulation, cellular mechanisms activating disseminated intravascular, see Disseminated intravascular coagulation Crown gall disease, and habituation of plant tissue, 266 Cytokine growth factors, tissue factor as superfamily member, 51-53 Cytokines, see also specific cytokines monocytehacrophage procoagulant activity, modulation by, 61, 63 Cytokinins, habituation in tumorous plant cells, induction of auxin interactions with, 270 concentration changes, 268-269 genetic aspects, 271-272 inoculum size, 271 mechanisms, 265-266,276,286-287,292 molecular genetic aspects, 277-278 temperature effects, 271 Cytoskeleton, retinal pigment epithelium affected by changes in, 255 Cytostatic factor, in calcium activation of mammalian eggs, 210

D 2,4-Dichlorophenoxyaceticacid, messenger RNA response to, 116 Disseminated intravascular coagulation leukemia associated with, 83-84 tissue factor role, 57 DNA cellular transfer, genes, in habituated cells expression, 289-290 homology with transfer DNA of Ri plasmid, 287-289 role, 287,293 complementary arcA, clone, 129

INDEX

303

in habituated plant cells expression, 279 tumor-specific genes, clones, 290-291 lepidopteran, 33-34 transfer, tumorous outgrowth induction by, 266, 278-279

E Effector cell protease receptor I , procoagulant activity in prothrombinase complex, 80-8 I Eggs, mammalian, calcium signal activating, see Calcium signal Endothelial cell tissue factor, regulation, 64-73 Ephestia kuehniella kinetic chromosome organization, 9-1 1 meiosis female, 14 male apyrene, 24. 26-27 male eupyrene, 16-19,21 microtubules, post-translational modifications, 30-32 structural features, 3-8 Epithelium, retinal pigment, see Retinal pigment epithelium Extracellular matrix, retinal pigment epithelium regeneration affected by, 253-254

F Fertilization, and mammalian egg activation by calcium signal, see Calcium signal Fibrin and cytokine-mediated tissue factor induction, 59 and endothelial cell tissue factor induction, 71-72 in tumor cells, 81 Fibroblasts, tissue factor on, 73-74

G Genes auxin-regulated ACC synthase, 132-133

arcA, cDNA clone, 129 biochemical analysis, 110 cloning, 11 1 dbp in Arabidopsis, 133 downregulation, 135 function, 136 GH3, expression. 115 GUS, activity, 126, 128, 134 parA characterization, 121 responsiveness, 126 parB, in tobacco mesophyll protoplasts, 123- 124 prokaryotic, 133- I34 rolB, activation, 134 SAURs, characterization, 115 in strawberry fruits, 133 targeting, 136 cellular transfer DNA, in habituated cells expression, 289-290 homology with transfer DNA of Ri plasmid, 287-289 role, 287, 293 mitochondrial, expression in Saccharomyces cereuisiae, see Saccharomyces cerevisiae tumor-specific, and habituation, 290-291 Genetic factors, in habituated tumorous plant cells induction, 271-272 molecular aspects, 277-279 Genetic tumors, and habituation auxin effects, 265-266,285-286,292 cDNA clones, specificity, 291 cellular transfer DNA genes, 287-290, 293 characterization, 279-280 cytokinin effects, 265-266, 286-287, 292 development, 266-267 hormonal aspects, 265-266, 285-287, 292 induction in vitro, 282-285 in viuo, 282 interconversion molecular basis, 287-291, 293 in vitro system, 283-285 in Nicotiana, 280-282 protoplast fusion, 282-283 stress-induced formation, 290 tumor-specific genes, 290-291

INDEX

Glutathione S-transferase, and auxin-regulated genes expression, 124 functions, 128 neoplastic transformation, 126 parB homology to, 123 Glycine-rich proteins, overexpression as cause of tumorous growth in habituated plant cells, 279 Glycosylation, advanced, endproducts, induced endothelial cell procoagulants, 70 Growth factors cytokine, tissue factor as superfamily member, 51-53 retinal pigment epithelium regeneration affected by, 253-254

H Habituation, in tumorous plant cells, see Plant cells, tumorous, habituation in Hormones, plant, see Phytohormones Hyperpolarizing responses, in calcium-activated mammalian eggs, I85 Hypersensitivity, delayed-type, and fibrin deposition, 59

I Immunity, cell-mediated, and fibrin deposition, 59 Inachis io, male eupyrene meiosis, 20 Indole-3-acetic acid, induced habituation in tumorous plant cells, 267,276-277 Inflammation CDlIKD18 antigen role, 75-76 monoc ytehacrophage procoagulant activity stimulated by, 59 procoagulant expression suppression, 89 Inositol(1,4,5)-trisphosphate,role in calcium oscillation in mammalian egg, 191-194 Inositol(1,4,5)-trisphosphatereceptor, in calcium oscillation mechanism, 197-199 Interferon-y endothelial cell tissue factor induction by, 69-70

as procoagulant response regulator, 60-63 Interleukin-1, endothelial cell tissue factor induction by, 68-69, 71-72 Interleukin-4, procoagulant expression suppression by, 88-89

K Kinetochore microtubules, lepidopteran meiosis female, 14 male apyrene, 25 male eupyrene, 22-23 organization, 12 post-translational modifications, 3 1 structural features, 33

L Lepidoptera, spindle structure characterization, 1-3 chromosomes kinetic organization, 8-12 structure, 33-34 experimental systems, 32-35 meiosis female, 13-15 male apyrene, 24-30,35-36 male eupyrene, 16-24,34-36 microtubules, post-translational modifications, 30-32 mitosis, 12-13 technical aspects of analysis, 3-8 infectious particles, 4 tissue preparation, 6 Leukemia, and procoagulants, 83-85 Lilium longiflorum, cytokinin-induced habituation, 272 Lipopolysaccharide, coagulation effects endothelial cell tissue factor induction, 66-69,71-12 monocyte-macrophage tissue factor induction, 57-58 procoagulant s activity, 79, 81, 85 expresssion modulation, 86-88 tissue factor induction, 61-63 Lipoprotein, low-density, endothelial cell tissue factor induction, 70-71

INDEX

305 M

Macrophage procoagulant-inducing factor production, 60 tissue factor expression, stimulation of, 63 Macrophages, and coagulation cellular activators, 78-79 cytokine-mediated induction of tissue factor, 59-64 pathway protein expression by, 77 procoagulant activity, 85 suppression of procoagulant expression, 86,88-89 tissue factor induction, 81-82 Macrophage tissue factor, 57-59 Malignancy, and procoagulants, 82-86 Maturation-promoting factor, in calcium activation of mammalian eggs, 210 Meiosis, and lepidopteran spindle structure female, 13-15 male apyrene, 24-30, 35-36 male eupyrene, 16-24,34-36 Microtubules, lepidopteran in experimental systems, 33-35 kinetic organization, 8, 12 kinetochore meiosis, 14,22-23,25 organization, 12 post-translational modifications, 3 I structure, 33 in meiosis female, 14-15 male apyrene, 24-25,27-28 male eupyrene, 16, 18-24 post-translational modifications, 30-32 structure, 1 Mitochondria1 genes, expression in Saccharomyces cerevisiae, see Saccharomyces cereuisiae Mitosis, and lepidopteran spindle structure, 12-13 Monocyte-macrophage procoagulant activity, see Procoagulants, monoc yte-macrophage Monocyte-macrophage tissue factor adhesion receptors, induction by, 75-76 properties, 57-59 Monocytes, and coagulation cellular activators, 78-79 cytokine-mediated induction of tissue factor, 59-64

pathway protein expression by, 77 prothrombinase activity, 80 tissue factor induction, 81

N Nicotiana. habituation cellular transfer DNA expression, 289-290 genetic tumors, 266, 280-282 tumor-specific genes, 290 Nicotiana bigelovii, cytokinin-induced habituation, 272 Nicotiana debneyi, genetic tumors, 281 Nicotiana glauca, habituation cellular transfer DNA genes, 287, 289 DNA sequence analysis, 288-289 genetic tumors, 281-282 hormone concentration, 267 induction by triiodobenzoic acid, 269 in vitro, 283 Nicotiana longijorum, genetic tumors, 281-283 Nicotiana tabacum auxin-regulated genes in, 128 habituation cytokinin-induced, 277, 281 hormone concentration, 267, 269 temperature effects, 271 Nuclear division, in eupyrene meiosis of Lepidoptera, 21

0 Orgyia antiqua, male eupyrene meiosis, 20 Orgyia thyellina, male apyrene meiosis, 24-25

P Pericentriolar material, lepidopteran male eupyrene meiosis, 19, 22 structural features, 1 Phaseolus lunatus, cytokinin-induced habituation, 271 Phaseolus vulgaris, cytokinin-induced habituation, 271-272 Phenotype, habituated, reversal in plant cells, 273-275

306 Phorbol myristate acetate, tissue factor induction by, 67-68 Phospholipids, coagulation affected by, 54-55 Phragmatpbia fuliginosa,male eupyrene meiosis in, 21, 34 Phytohemagglutinin, tissue factor induction by, 68 Ph ytohormones in habituated plant cells autonomy, 275-277 concentration changes, 267-269 Plant cells, tumorous, habituation in auxin-cytokinin interactions, 270 cell properties hormonal, 275-277 molecular genetic, 277-279 genetic tumor as example auxin effects, 285-286 cellular transfer DNA genes, 287-290, 293 characterization, 279-280 cytokinin effects, 286-287 hormonal aspects, 265-266 induction by auxin, 265-266, 292 by cytokinins, 265-266, 292 by hormones, 265-266,292 in vitro. 282-285 in vivo, 282 interconversion molecular basis, 287-291, 293 system, 283-285 in Nicotiana, 280-282 protoplast fusion, 282-283 tumor-specific genes, 290-291 induction by antiauxins, 269 genetic background, 271-272 hormone concentration changes, 267-269 inoculum size, 270-271 temperature, 271-272 mechanism, 265-267 reversibility, 273-275, 293-294 Plant hormones, see Phytohormones Plants, see specific plants Plasma membrane, retinal pigment epithelium alterations, effect of, 252-253 location, 230-231 properties, 227,229

INDEX

Platelet activating factor, as procoagulant response regulator, 64 Polarizing responses, see Hyperpolarizing responses Polyphosphoinositides, messenger system, calcium oscillation in mammalian eggs by, 191 Procoagulants adhesion receptor regulation, 75-77 expression modulation, 86-89 and malignancy, 82-86 monocyte-macrophage cytokine modulation, 61,63 mechanism, 57-59 protein antigen induction, 60 properties, 77-82 Prokaryotic genes, auxin-regulated, 133- 134 Prostaglandins, procoagulant expression modulation by, 88 Protein kinase, calmodulin-dependent , activation of mammalian eggs, 210-211 Protein kinase C, in calcium activation of mammalian eggs, 21 1-212 Proteins ABF2, yeast mitochondria1 gene expression affected by, 149 advanced glycosylation end products, endothelial cell tissue factor induction by, 70 auxin-binding, analysis, 110-1 1 1 in calcium activation of mammalian eggs, 212 in genetic tumors of habituated plant cells, 290 glycine-rich, overexpression as cause of tumorous growth in habituated plant cells, 279 GTP-binding, role in calcium oscillation in mammalian eggs, 191-194 in monocytehnacrophage procoagulant activity, 60 parA, in tobacco mesophyll protoplasts, 122-124 parB, in tobacco mesophyll protoplasts, 123-124 yeast mitochondria1 gene expression effects categorization, 154-155 function, 156

307

INDEX

post-translational activity, 163-164 regulation, 166-167 reverse transcriptase activity, 153 Prothrombinase complex assembly, 79-80 viral infection effects, 81 Protoplasts, fusion, in genetic tumors,

282-283

R Retinal pigment epithelium, mammalian, regeneration basement membrane effects, 253 cytologic changes, 226-227 cytoskeletal changes, effects, 255 extracellular matrix effects, 253-254 growth factor effects, 253-254 interactions, 223-224,254 laser lesions, 224-225 mechanisms, 255 models, 225-226 plasma membrane alterations, 252-

253

polarity, 227-232 in rabbits, 251 sodium iodate retinopathy cytology, 234,236-251 as model, 232-235 RNA messenger in auxin-regulated genes, 112-114,

116

expression in mouse tumors, 57 SAUR induction, 115 response to 2,4-dichlorophenoxyacetic acid, 116 in Saccharomyces cerevisiae gene expression enzyme-dependent processing, 168 processing, 150- 158,168-169 regulation, 165-166 translation, 159-162 turnover, 158-159 RNA polymerase, in Saccharomyces cerevisiae gene expression, 149 Ryanodine receptor, in calcium activation of mammalian eggs, 197-198

S Saccharomyces cerevisiae, mitochondria1 gene expression mechanism, 145,167 post-translational factors, 162-164,169 regulation, 164-167 RNA messenger, 15 1- 162,165-166,168-169 modification, I5 1, 168 processing, 151-158,168 regulation, 165 ribosomal, 150-151, 168 splicing, 153 transfer, 150-151, 165,168 translation, 159-162 turnover, 158-159 RNA polymerase role, 149 transcription, 146-150, 167-169 transcription factors, 149-150 transcription promoters, 146-149 translation, 159-162 Schizosaccharomyces pombe, chromosome structure, 34 Serum albumin, advanced glycosylation end products of, endothelial cell tissue factor induction by, 70 Sodium iodate retinopathy cytology of, 234,236,238-251 as model of retinal pigment epithelium regeneration, 232-235 Spermatogenesis, double definition, 2 development in Lepidoptera, 36 Spindle structure, lepidopteran, see Lepidoptera, spindle structure Synaptonemal complexes, lepidopteran, in meiosis female, 14 male eupyrene, 20

T Tissue factor characteristics, 50-5 1 as cytokine growth factor superfamily member, 51-53 cytokine-mediated induction, 59-64 endothelial cell, regulation of, 64-73 on fibroblasts, 73-74

308 function, 53-55 localization, 55-57 monocyte-macrophage, 57-59 Transcription, mitochondrial, in Saccharomyces cerevisiae mechanism, 167- 168 promoters, 146-149 RNA polymerase role, 149 transcription factors, 149-150 Translation, mitochondrial, in Saccharomyces cerevisiae, 159-162 Translation factors, mitochondrial, in Saccharornyces cerevisiae, 166-167, I69 Trichoplusia ni, kinetic chromosome organization, 9

INDEX

Triiodobenzoic acid, as inducer of habituation in plant cells, 269 Tumor necrosis factor, endothelial cell tissue factor induction by, 69-70,72 Tumors coagulation effects, 81, 83 genetic, see Genetic tumors

Y Yeast, see also Saccharomyces cerevisiae Schizosaccharomyces pombe, chromosome structure, 34

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