MICROSCOPY RESEARCH AND TECHNIQUE 2223-48 (1992)

Role of the Cytoskeleton During Early Development WILLIAM M. BEMENT, G. IAN GALLICANO, AND DAVID G. CAPCO Molecular and Cellular Biology Program, Department of Zoology, Arizona State University, Tempe, Arizona 85287-1501

KEY WORDS

Signal transduction, Localization, Detergent extraction, Embryogenesis

ABSTRACT Oocytes, eggs, and embryos from a diverse array of species have evolved cytoskeleta1 specializations which allow them to meet the needs of early embryogenesis. While each species studied possesses one or more specializations which are unique, several cytoskeletal features are widely conserved across different animal phyla. These features include highly-developed cortical cytoskeletal domains associated with developmental information, microtubule-mediated pronuclear transport, and rapid intracellular signal-regulated control of cytoskeletal organization. 0 1992 Wiley-Liss, Inc.

INTRODUCTION The cytoskeleton is a ubiquitous component of eukaryotic cells that exhibits significant variation in composition and organization. The ubiquity of the cytoskeleton suggests that it fulfills needs possessed by all cells, whereas its variation indicates that it is also responsible for numerous specialized cell functions. Intensive study of somatic cell systems has helped define many of the conserved roles of the cytoskeleton and provides a useful foundation for framing questions about the role of the cytoskeleton in gametes and developing embryos. Conversely, study of the cytoskeleton in early development yields insight into the many specializations exhibited by cytoskeletal structures. In this review, we sketch a framework of data and hypotheses derived from research on somatic cell cytoskeletons. We also discuss problems faced by eggs and embryos which are not normally encountered by somatic cells. Because the information obtained concerning the role of the cytoskeleton in different systems is dependent on the approach employed, we briefly describe pertinent techniques for studying cytoskeletons. We then examine specific developmental systems in turn, applying to each the framework developed from analysis of somatic cell systems, and describing cytoskeletal specializations which may have evolved to manage the distinct challenges encountered by that system. We conclude by discussing universal features of embryonic cytoskeletal systems, and their relevance to general understanding of cytoskeletal structure, function, and dynamics.

The Cytoskeleton in Somatic Cells The cytoskeleton has four general functions in somatic cells: (1)control of cell shape, (2) translocation of cellular components, (3) binding of RNA and protein, and (4) control of cell motility. (1) Control of Cell Shape. Individual cells have three-dimensional organizations which reflect the particular role they perform in the organism. The three main filament systems of the cytoskeleton (actin filaments, microtubules, and intermediate filaments) establish and maintain not only the organelle distribution characteristic of the cell interior, but also the

0 1992 WILEY-LISS, INC.

three-dimensional configuration of the cell exterior (for reviews of experimental evidence for the role of the cytoskeleton in maintaining cell structure, see Brinkley et al., 1980; Fey et al., 1984). This role is complicated by the fact that cells undergo extensive internal and external reorganizations during cell cycle progression, locomotion, and other processes characteristic of cell life. Thus, the cytoskeleton acts both to support the cell infrastructure, and to effect dynamic cell rearrangements. (2)Translocation of Cellular Components. Cells frequently exhibit nonrandom patterns of organelle biogenesis and distribution; to maintain this polarity, organelles must be actively transported to and from different regions of the cell. The cytoskeleton attaches to, and a t least in some cases moves, organelles and other components of the cell using both microtubuleand actin filament-based systems of transport. Microtubules in squid axons transport organelles between the cell axon and the cell body (Vale et al., 1986; see reviews by Schroer and Kelley, 1985; Vale et al., 1986). Movement away from the cell body is associated with the molecule kinesin, and movement toward the cell body appears to be associated with cytoplasmic dynein (Vale et al., 1986). Evidence suggests that microtubules play a similar role in other cell types as well, such as the amoeba (Koonce and Schliwa, 1986), and other more widely-used model systems. For example, in cultured mammalian cells, microtubules appear to move endosomes toward the cell center where they contact lysosomes, which are also associated with microtubules (Matteoni and Kreis, 1987). Actin-based organelle transport has been demonstrated in the plant, Nitella, where it was shown that beads coated with heavy meromyosin move along the length of actin cables in the cytoplasm of ruptured Nitella cells (Sheetz and Spudich, 1983). Myosin-coated beads also move along isolated actin bundles in vitro,

Received July 15, 1990; accepted in revised form October 15, 1990. Address reprint requests to Dr. David G. Capco, Department of Zoology, Arizona State University, Tempe, AZ 85287-1501.

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and organelles move in register with actin filaments in permeabilized amoebae (Koonce and Schliwa, 19861, providing further support for the existence of actinbased organelle movement. (3)Binding of mRNA and Protein. As in the case of organelles, cells also exhibit nonrandom patterns of mRNA and protein distribution, which may allow spatial coordination of protein synthesis and utilization. For example, specific cytoskeletal mRNAs are localized near the site where their cognate protein is needed: actin mRNAs can be found a t the cell periphery, a region enriched in actin filaments, and tubulin mRNA is concentrated near the nucleus, a region enriched in microtubules (Lawrence and Singer, 1986). At least part of the heterogeneous protein distribution is controlled by the cytoskeleton, which is associated with a number of different mRNAs and proteins (Lenk et al., 1977; Fulton et al., 1980; Ben-Ze’ev et al., 1981; Van Venrooij et al., 1981; Bonneau et al., 1985; Singer et al., 1989). Evidence also suggests that the protein synthetic machinery is associated with the cytoskeleton (Dang et al., 1983; Howe and Hershey, 1984) and protein synthesis is known to occur on cytoskeletonassociated mRNA (Lenk and Penman, 1979; Van Venrooij et al., 1981; Fulton and Wan, 1983). A variety of enzymes are also thought to associate with the cytoskeleton, including tRNA-synthase (Dang et al., 1983), creatine phosphokinase (Eckert et al., 19801, phosphofructokinase (Liou and Anderson, 1980; Hand and Somero, 19841, and aldolase (Pagliaro and Taylor, 1988). The functional significance of these interactions is far from clear, but in some cases it appears that association with the cytoskeleton modifies the properties of the protein or mRNA, potentially resulting in up- or down-regulation. Recently, it has also become evident that the cytoskeleton may modulate intracellular signalling activities by controlling signal transduction across the plasma membrane. For example, tryrosyl kinase (Cinton and Finley-Whelan, 1984), growth factor receptor kinase (Landreth et al., 1985), and GTP-binding proteins (Jesaitis et al., 1988) associate with the cytoskeleton, and in some cases the association is correlated with changes in the activity of these proteins (Jesaitis et al., 1988). (4) Control of Cell Motility. Many cell types are required to undergo some form of movement. In somatic cells, movement is usually of the amoeboid type, which is mediated by contracting stress fibers (bundles of actin filaments) coordinated with adhesion plaque formation and membrane flow (for reviews see Schliwa, 1986; Singer and Kupfer, 1986). In some single-celled organisms, movement is accomplished by the beating of numerous cilia, actin filament-containing structures which extend from the cell surface or by the rotation of one or several flagella, microtubule-containing structures which also extend from the cell surface. Problems Faced by Eggs and Embryos In addition to performing the functions described above, gamete and embryonic cytoskeletons also help these cells deal with constraints imposed upon them by early development. Eggs and embryos face several

problems which are either not encountered by other cells or are greatly exacerbated in eggs and embryos. First, size: most eggs and embryos are much larger than other cell types; therefore, specializations must compensate for this large size. Second, totipotency: a single cell (the fertilized egg) must give rise to a whole organism instead of merely another cell, thus, the egg must possess and organize the appropriate developmental information. Third, fertilization: sperm-egg fusion is required to restore diploidy and to initiate early development; thus, the egg must have specializations which allow it to fuse with the sperm and which drive pronuclear fusion. Further, since polyspermy is usually lethal, the egg must have specializations which prevent it from fusing with more than one sperm. Fourth, cleavage: many embryos undergo several fast (30 minutes or less) cell divisions in which G1 and G2 are greatly reduced or omitted, hence the cell must be able to execute transit through the cell cycle and all of the accompanying processes (e.g., mitosis, cytokinesis) with extreme rapidity. As we shall see, different organisms have evolved ingenious mechanisms for coping with these problems, many of which are based on cytoskeletal specializations. Often, the specializations are elaborations of routine cytoskeletal functions, or integration of more than one function. At least three specializations appear to be widely conserved across the different phyla: (1)all systems examined have a highly developed cortical cytoskeletal domain in the eggs which undergoes reorganization after fertilization. In many cases, the domain is associated with components containing developmental information, such as mRNA or proteins. The reorganizations following fertilization position these components in the appropriate location to direct early development; (2) all systems examined employ microtubule networks to move pronuclei. This ensures that even when the sperm fuses with the egg at a region distant from the female pronucleus, syngamy can nevertheless be achieved; and (3) in those systems where control of cytoskeletal organization has been examined, complex cytoskeletal structures are under the regulation of intracellular signalling pathways. This allows developing embryos to rapidly assemble and disassemble structures necessary for specific developmental events without having to rely on the comparatively slow process of regulated protein expression. Methods for Analysis of the Cytoskeleton There are many different methods for analysis of the cytoskeleton. We will discuss only methods which are used for microscopic examination, and particular emphasis will be given to detergent extraction. Detergent extraction uses a nonionic detergent together with other cytoskeletal stabilizing components to produce a medium which removes lipids from the plasma membrane and the internal organelles, thereby allowing release of soluble components. The material remaining after such a treatment, by definition, represents the detergent-resistant cytoskeleton. A number of different nonionic detergents have been employed for this purpose, although the detergent used most frequently is Triton X-100. In cases where the integrity of the cy-

ROLE OF CYTOSKELETON DURING EARLY DEVELOPMENT

toskeleton is disrupted by Triton X-100, as has been reported for some embryos (Jeffery, 1985b; Mutchler et al., 1988; Gallicano et al., 1991), replacement of Triton X-100 with either NP-40 or Tween 20 has circumvented this problem. The detergent extraction method requires rapid uniform extraction of the cell to generate a cytoskeleton with reproducible characteristics. This requires that the detergent be present a t a concentration above its critical micelle concentration in order for individual components to intercalate into the lipid bilayer and displace the lipid and, conversely, for the lipid to intercalate into the micelle. The critical micelle concentration for various detergents can be determined from information provided by the supplier or in a reference list supplied by CalBiochemicals. To allow rapid and uniform penetration of detergent, the specimen cannot be more than a few cell layers thick, otherwise the external cells become completely extracted while internal cells either remain intact or slowly release soluble components. Either condition prevents obtaining a reproducible cytoskeleton. Size is also a problem in studies of early development, as eggs of some species are very large (i.e., over a millimeter in diameter). Although some investigators have attempted to detergent-extract such cells, there is no guarantee that the structure they isolate is equivalent to the cytoskeleton of somatic cells. Large amounts of extracellular matrix also prevent rapid and uniform extraction and lead to unreproducible results. Recently, a freeze-sectioning procedure has been developed which permits detergent extraction of larger multicellular aggregates containing extracellular matrices (Capco et al., 1987). This approach has been shown to produce a cytoskeleton equivalent to those yielded by more standard procedures applied to somatic cells, and has been successfully applied to the large eggs of the amphibian (Hauptman et al., 1989). In addition to the detergent, other extraction medium components are also important, and should mirror, as closely as possible, ionic conditions of the intracellular milieu. Such conditions vary, depending on whether the organism is a vertebrate or invertebrate, and whether the organism is terrestrial, aquatic, or marine. A protease inhibitorb) is also required to prevent autolysis. Phenylmethylsulfonyl fluoride is often used but other inhibitors may be required (Issacs et al., 1989). Once detergent extract is complete, the sample can be processed for structural or biochemical analysis. For structural analysis, the fixative (2% glutaraldehyde andlor 2% paraformaldehyde) is made in detergent extraction medium. The osmolarity of this medium is less important, as the membranes have already been permeabilized. It is important, however, not to alter the composition of the medium the cytoskeleton is in until after fixation, as this can destabilize the cytoskeleton. An exception to this is fixation for immunofluorescence, in which case if the PMSF is present with the fixative (usually paraformaldehyde), the samples will have a greater level of autofluorescence. After the initial fixation, the detergent-resistant cytoskeleton can be processed as any other tissue for examination in the light or electron microscope. Cy-

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toskeletal preparations can be viewed as a wholemount after critical point drying or they can be embedded and sectioned. Such cytoskeletal preparations are particularly well suited for examination as embedment-free sections (Capco et al., 1984). This is because, in the absence of soluble components and embedding resin, the cytoskeleton can be sharply imaged. The image is created by scattering of electrons by the biological material in a vacuum, rather than the biological material in a resin. In the latter case, the biological material and resin scatter electrons nearly equally, and heavy metal stains are required to provide enough contrast to view the image. Moreover, in the absence of resin, relatively thick specimens can be observed, and thus, more spatial information obtained. One disadvantage of embedment-free preparation is that it produces a lower yield of specimens than obtained with plasticembedded sections. Specimen loss comes during removal of the embedding medium and during critical point drying, procedures which apparently cause enough turbulence to dislodge the specimen from the grid. Also, special precautions must be taken to reduce beam damage to the specimen. The principal change is to reduce the gun bias to decrease the number of electrons which contact the specimen. In addition, a precooled cold trap and low beam intensity are also helpful. Images of such specimens are high in contrast and to obtain an easily printable image, we have found it useful to dilute the developer (D-19) 1:8 and develop for eight minutes. Such a developer can be used only once. While embedment-free sections have several advantages, they are not suitable for all types of questions. When it is desired to resolve details in filament substructure, either negative stained preparations or replicas are more appropriate. A particularly elegant method is to quick freeze and deep-etch detergent-resistant cytoskeletons after fixation. Such views provide exquisite substructural detail and a wealth of fine spatial information (Hirokawa and Heuser, 1981). The identity of specific cytoskeletal components can be determined in samples where a primary antibody is directed against a specific cytoskeletal protein and that antibody, or a secondary antibody, is coupled to gold beads. At the level of resolution of the light microscope, the cytoskeleton has also been examined by fluorescent probes. Usually this takes the form of antibodies to known cytoskeletal proteins being imaged after binding of a fluorescently tagged second antibody. The biological material for such treatments can be processed by detergent extraction. Some investigators apply living cells with detergent and fixative at the same time; other investigators apply the detergent after fixation. In these latter cases, a detergent-resistant cytoskeleton is not produced, but rather, the detergent is used to permeabilize the cells, and while some soluble components are removed, others remain immobilized in the specimen. Some investigators fix and permeabilize the cells by treatment of living cells with cold methanol (-20°C) and others also include DMSO in such a treatment. Such specimens demonstrate excellent fluorescence (Lehtonen and Badley, 1980; Houliston et al.,

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1987) but clearly would not be suited for ultrastructural examination. Each approach has advantages and disadvantages and provides complementary types of information about the cytoskeleton. Electron microscopic analysis of detergent extracted specimens provides high resolution images of the cytoskeleton and allows examination of changes in organization of all cytoskeletal proteins. However, this approach is time consuming (preparation can occupy several days), whereas the immunofluorescent approach allows more rapid acquisition of results. Since immunofluorescence utilizes antibodies to specific cytoskeletal proteins, one is immediately aware whether the protein of interest has altered its organization. However, this type of analysis is limited to the cytoskeletal proteins for which antibodies are available. Finally, the limited resolution of this approach does not make it possible to detect interactions between two cytoskeletal elements and is restricted to the observation that the elements co-localize.

The Cytoskeleton in Nonchordate Development Annelids. Oocytes, eggs, and embryos of Chaetopterus, a marine annelid, provide an excellent example of how cytoskeletal specializations are employed to deal with problems entailed by early development. This system has been extensively studied by Jeffery and colleagues who have shown that two aspects of normal (i.e., somatic) cytoskeleton function have been highly developed to organize and partition developmental information. Specifically, the cytoskeleton of Chaetopterus oocytes, eggs, and embryos not only bind mRNA, they also appear to translocate these components to the region of the embryo where they will be needed to direct development. The cytoplasm of Chaetopterus oocytes is organized into regions that possess different staining properties, allowing the fate of different egg regions to be followed in subsequent development. Three regions of the egg cytoplasm are recognized: the endoplasm, which consists of the cell center and has blue-staining yolk and smaller, red-staining granules; the ectoplasm, which lines approximately two-thirds of the cell cortex and has red-staining granules; and the germinal vesicle (nucleus), which is near the cell apex and is transparent. During oocyte maturation, fertilization, and embryogenesis, these plasms move in a stylized pattern and are distributed into different blastomeres. Jeffery and Wilson (1983) and Jeffery (1985b) used in situ hybridization to show that poly(A) RNA, histone mRNA, and actin mRNA are concentrated in the oocyte ectoplasm and move with the ectoplasm during maturation and cleavage. During cleavage, animal hemisphere ectoplasm is equally distributed to the first two blastomeres, while vegetal hemisphere ectoplasm is concentrated in a blastomere evagination known as the polar lobe (Jeffery and Wilson, 1983). Demonstration that these mRNAs are associated with the egg and embryo cytoskeleton was achieved by showing that the mRNA patterns were not altered by detergent extraction, a procedure which removes soluble cell components but leaves the cytoskeleton behind (see Methods for Analysis of the Cytoskeleton; Jeffery, 1985b). Jef+

fery further strengthened this notion by stratifying (via centrifugation) the living egg and then detergentextracting the stratified egg (Jeffery, 198513). In situ hybridization of eggs prepared in this manner revealed that poly(A)+RNA, histone mRNA, and actin mRNA stratify to the centrifugal pole, along with the detergent-resistant cytoskeleton. Given that the mRNAs not only colocalize with the cytoskeleton, but also partition with it following centrifugation, it is likely that the mRNAs are by some means securely connected to the cytoskeleton. Further work by Jeffery and coworkers has demonstrated that the Chaetopterus egg cytoskeleton is required for normal cleavage (Swalla et al., 1985). Eggs subjected to protracted low speed centrifugation separate into two fragments, only one of which (that a t the centrifugal pole) contains the cytoskeleton. Either the centrifugal or the centripetal fragment may contain the female pronucleus, and, when fertilized, the fragment with the egg nucleus emits polar bodies. However, only the fragment which contains both the nucleus and the cytoskeleton will undergo cleavage when fertilized, whereupon such fragments develop to the blastula stage. In contrast, very few fragments containing the nucleus but lacking the cytoskeleton are capable of cleavage. It thus appears that factors intrinsic to the cytoskeletal domain are required for cleavage. In conclusion, oocytes of Chaetopterus possess a cytoskeletal domain in their cortex which: (1) localizes mRNA in the ectoplasm; (2) undergoes profound rearrangement during early development, taking the attached mRNA with it; and (3) is essential for normal development. Oligochaetes. Fertilized eggs of the oligochaete, Tubifex, also contains a highly developed cortical cytoskeleton (Shimizu, 19841, which, like the domain of Chaetopterus, contains developmental information and undergoes characteristic rearrangements during early embryogenesis (Shimizu, 1988). Actin filaments are the predominant component of this network, as revealed by labeling with heavy meromyosin and rhodamine phalloidin (Shimizu, 1984, 1988). In both the animal and the vegetal hemispheres of the egg, actin filaments form polar caps, which, upon fertilization, are partitioned into the “CD” blastomere. The CD blastomere subsequently gives rise t o the C and D blastomeres. The caps are distributed to the D blastomere, and it is this blastomere that contains information required for development of specific body parts. Like the granules in the ectoplasm of Chaetopterus which follow the redistribution of the cytoskeletal domain, organelles in Tubifex partition with the actin filament caps, suggesting an association between the organelles and the caps, as well as a role for actin filaments in organelle translocation. Ultimately, the actin filament caps fuse and associate with the nuclei of some of the D blastomere daughter cells (Shimizu, 1988). While it is not known how the polar caps are partitioned into the correct blastomeres, colchicine-treated zygotes fail to translocate the caps, indicating that microtubules may play a role in directing cap movement. Thus, oligochaete eggs possess a highly organized cortical cytoskeleton which undergoes extensive reorganization

ROLE OF CYTOSKELETON DURING EARLY DEVELOPMENT

upon fertilization, and this reorganization appears to be important for proper partitioning of developmental information. Nematodes. Another important developmental system, the nematode Caenorhabditis elegans, exhibits dynamic reorganizations of the both actin and microtubule networks following fertilization. Prior to fertilization, the cortical actin filament network, myosin, P granules (determinants of the germ line) and yolk are distributed equally in both the posterior and the anterior of the egg. Only the location of the nucleus in the egg anterior reveals egg polarity. Upon fertilization, however, the sperm pronucleus is targeted to the posterior end of the zygote, and the array of cortical actin becomes more apparent and filamentous (Strome, 1986; Strome and Hill, 1988). Later, a t 70-85 minutes following fertilization, a transient cleavage furrow forms as a result of contraction of the zygote anterior. Simultaneously, the P granules segregate to the posterior cortex and the female pronucleus moves to the posterior pole, while actin and myosin filaments become concentrated in the anterior cortex. Moving toward the zygote anterior, the male pronucleus centrosome begins to emanate microtubules which apparently mediate pronuclear migration, and upon meeting the female pronucleus, both move to the zygote center. The whole complex rotates, and then translocates to the zygote posterior. Cleavage separates the zygote into a large anterior blastomere, which has a disproportionately high concentration of actin filaments, and a small posterior blastomere, which contains the P granules. Formation of the transient cleavage furrow and P granule segregation are sensitive to actin filament inhibitors, whereas pronuclear migration is sensitive to microtubule poisons (Strome and Wood, 1983; Hill and Strome, 1988, 1990). Thus, as is the case in Chaetopterus and Tubifex, localization and translocation of zygote organelles in C. elegans appears to be mediated by the cytoskeleton. Because P granules direct germ line development, C. elegans provides a particularly straightforward example of how an embryonic system has utilized a known function of the cytoskeleton (i.e., binding and translocation of cellular components) to cope with requirement imposed by embryogenesis (i.e., specification of cell fate). Insects. Study of Drosophila development has demonstrated not only a dynamic cortical cytoskeleton, but has also resulted in the surprising discovery that centrosomes, rather than nuclei, regulate aspects of the process of cellularization, a major developmental transition in Drosophila which entails dramatic changes in cell cycle regulation as well as cell structure. In the fertilized Drosophila egg, immunofluorescence and phalloidin staining have demonstrated that microtubules, actin filaments, and myosin are distributed throughout the entire cortex in a layer approximately 3 FM deep (Warn et al., 1980, 1984; Warn and Warn, 1986; Karr and Alberts, 1986). Early development is syncytial, with nuclei dividing in the zygote center, in the absence of accompanying cytokinesis. The dividing nuclei are surrounded by yolk, and each nucleus is associated with an aster-like array of microtubules. After the 8-9th nuclear division, the nuclei

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migrate to the zygote cortex. In the cortex, the nuclei develop individual cytoskeletal arrays, such that the entire zygote cortex is subdivided into numerous units, each containing one nucleus surrounded by actin filaments, myosin, and microtubules. Microtubules that were formerly dispersed throughout the cortex are localized in the areas between the nuclei and the zygote plasma membrane. These microtubules run adjacent to the nuclei and extend inward into the zygote (Fullilove and Jacobson, 1971; Karr and Alberts, 1986; Warn and Warn, 1986). The actin filaments which were formerly located throughout the cortex form cap-like arrangements at the region between the apical microtubules of each nucleus and the plasma membrane (Karr and Alberts, 1986). As nuclear divisions continue, the cytoskeleton of the cortex is further partitioned until the time of cellularization, when cytokinesis occurs and individual cortical cytoskeletal domains are divided by the plasma membrane into blastomeres. The above progression of cytoskeletal reorganizations were intially identified using fixed specimens. These results have been confirmed in living zygotes by immunofluorescent microscopy of specimens injected with fluorescently-labeled anti-tubulin antibodies (Warn et al., 1987)or fluorescently-labeled tubulin or actin (Kellogg et al., 1988). The process whereby cells form in the zygote cortex is referred to as “cellularization”, and occurs coincident with major changes in cell cycle control (Edgar et al., 1986; Edgar and Schubiger, 1986; OFarrell et al., 1989). Recently, Raff and Glover have demonstrated that a t least some aspects of Drosophila cellularization are controlled by the centrosomes rather than the nuclei themselves (Raff and Glover, 1989). These researchers uncoupled nuclear replication from centrosome replication by treating zygotes with the DNA synthesis inhibitor, aphidicolin, a treatment which blocks nuclear replication but allows the centrosomes to continue dividing. Under these conditions, the centrosomes migrate to the cortex at the appropriate time and multiple individual actin caps form, as normally occurs during cellularization. Moreover, the pole cells, which form around nuclei that migrate to the posterior pole of the embryo prior to migration of the other nuclei, actually form in the absence of nuclei (Raff and Glover, 1989). These results have several important implications: first, it appears that the centrosomes may possess their own endogenous cell cycle clock which can function even when essential functions of the cell cycle (i.e., DNA synthesis) are arrested. Second, since migration occurs without the nuclei, it is likely that the forces which translocate the nuclei to the cortex do so by virtue of the interaction between the nucleus and the centrosome, rather than the nucleus itself. This may also be the case during pronuclear migration, although this point has yet to be established. Third, the centrosome rather than the nucleus must carry the requisite information for organization of the actin cap and pole cell formation. Thus, like other nonchordates, insect embryos exhibit cytoskeletal structures which undergo dynamic changes and appear to mediate essential developmental transitions. The similarity between insects and

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other systems is even more evident when the relationship between the insect cytoskeleton and RNA localizations are examined. It is now well known that insect oocytes, eggs, and embryos exhibit dynamic, nonrandom distributions of RNA (Capco and Jeffery, 1978, 1979; Edgar et al., 1987). For example, at cellularization, distribution of mRNA for the segmentation gene fushi tarazu changes from a uniform distribution in the syncytial blastoderm to 7-banded pattern which grows increasing defined as cellularization proceeds (Edgar et al., 1986, 1987). Using in situ hybridization to follow the distribution of fushi tarazu mRNA in embryos treated with cytoskeletal inhibitors, or mutant embryos which fail to cellularize, Edgar et al. (1987) demonstrated that the cytoskeletal rearrangements of cellularizations enable the embryo to partition fushi tarazu mRNA in it’s characteristic 7-banded pattern. Disruption of the embryonic cytoskeleton led to “drifting” of fushi tarazu mRNA and failure to resolve tight mRNA bands. They proposed that these mRNAs are sequestered between the invaginating plasma membrane and the growing microtubule lattice a t the apex of cortical nuclei, perhaps by virtue of an interaction with intermediate filaments (Edgar et al., 1987). Echinoderms. Echinoderms, particularly sea urchins, have been popular model systems for investigation of cytoskeletal dynamics. Studies performed on sea urchin eggs have provided considerable insight into problems posed by fertilization. For at least one of these problems, preventing polyspermy, the organism has turned to the cytoskeleton. To prevent more than one sperm from penetrating, the sea urchin egg, like eggs of many other species, undergoes a massive exocytosis reaction, releasing the contents of cortical granules into the extracellular matrix. Granule contents, in conjunction with the matrix, form a barrier impenetrable to sperm, the fertilization envelope. As discussed below, not only does the cytoskeleton anchor the cortical granule, it also mitigates potentially unfavorable consequences of exocytosis. Unfertilized starfish eggs (Otto and Schroeder, 1984) and sea urchin eggs possess a cortical cytoskeletal domain which contains actin filaments (Spudich and Spudich, 1979; Kidd and Mazia, 1980; Schatten et al., 198613; Bonder et al., 1989). In the sea urchin egg, filamentous actin in the cortex is concentrated predominantly in the numerous microvilli which stud the surface of the egg, as demonstrated by phalloidin staining, which reveals a punctate pattern on the egg surface (Bonder et al., 1989).Fodrin (also known as spectrin), a peripheral membrane protein which associates with actin, also exhibits a punctate pattern of staining in the unfertilized egg (Schatten et al., 1986b; Henson and Begg, 1988; Bonder et al., 1989). Beside the microvilli, ultrastructural analysis of the egg cortex reveals that short, actin-sized filaments connect the cortical granules to the plasma membrane (Fig. 1)(Chandler, 1984; Henson and Begg, 1988). Actin filaments also run adjacent to the plasma membrane (Fig. 2) (Henson and Begg, 1988) and the cortical granules are underlain by a layer of filamentous actin, as demonstrated by immunofluorescence (Bonder et al., 1989). Detailed biochemical, ultrastructural, and immuno-

Fig. 1. Quick-freeze, deep-etch image of sea urchin egg cortex, cytoplasmic face. Actin-sized filaments (arrows)run along the plasma membrane and anchor cortical granules (CG) to the plasma membrane. (Reproduced from Henson and Begg, 1988, with permission of Academic Press.)

fluorescent studies by several laboratories have revealed that, in addition to the filamentous actin described above, the sea urchin egg cortex contains a second nonfilamentous pool (Spudich and Spudich, 1979; Henson and Begg, 1988; Spudich et al., 1988; Bonder et al., 1989). Spudich and Spudich (1979) initially noted that although isolated sea urchin egg cortices contained a large amount of actin, very few actin filaments could be seen by electron microscopy, leading them to propose that much of the cortical actin was in a nonfilamentous form. Using immunof luorescence and immunoelectron microscopy, Spudich et al. (1988) demonstrated that although actin immunolocalizes in the microvilli, the subplasma membrane region, and around the cortical granules (Fig. 31, the actin surrounding the cortical granules is nonfilamentous. Bonder et al. (1989) confirmed and extended these findings, and have proposed a precise spatial map of filamentous and nonfilamentous actin pools in the cortex, wherein filamentous actin is located in the microvilli and in a layer beneath the cortical granules, whereas nonfilamentous actin is localized around the cortical granules (Fig. 4). What is the purpose of the unpolymerized pool of actin? Apparently, it is needed immediately upon fertilization: fertilization triggers a wave of exocytosis, and hence the insertion of massive amounts of membrane into the plasma membrane. As a result, the membrane surface area of the egg increases dramat-

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Fig. 2. Quick-freeze, deep-etch image of sea urchin egg cortex, cytoplasmic face, after S-1labelling. Actin filaments run along the plasma membrane (arrows). (Reproduced from Henson and Begg, 1988,with permission of Academic Press.)

ically (Schroeder, 1979). The egg copes with this increase by following the wave of exocytosis with a wave of microvillar elongation (Begg and Rebhun, 1979; Schroeder, 1979). As exocytosis proceeds, the formerly unpolymerized pool of actin is rapidly recruited into the microvilli, which lengthen nearly 3fold (Schroeder, 1979). The microvilli remain elongate until endocytosis retrieves sufficient membrane to return the plasma membrane surface area to normal (Schroeder, 19791, thereby mitigating the potentially unfavorable consequences of massive insertion of cortical granule membranes into the egg plasma membrane. In addition to an increase in microvillar length, fertilization also triggers an increase in the amount of filamentous actin in the cortex below the plasma membrane (Spudich and Spudich, 1979). Detergent extraction of sea urchin eggs and embryos has demonstrated that an increase in the binding of poly(A)+RNA to the cytoskeletal fraction is associated with an increase in the number of filaments in the cortex, and an increase in the amount of protein synthesis in the embryo (Moon et al., 1983). Taking a cue from work done on somatic cells, Moon et al. (1983) suggested that association of embryonic mRNA with the cytoskeleton promotes protein synthesis. Cortical microtubules are found in both sea urchin and starfish eggs (Schroeder and Otto, 1984; Boyle and Ernst, 1989). In sea urchin eggs, microtubule reorganizations play an important role following fertilization. Microtubules elongate from the sperm centrosome and

extend to the female pronucleus (Bestor and Schatten, 1981) and the male and female pronuclei appear to migrate along the microtubules until pronuclear fusion. Moreover, pronuclear migration is blocked by agents which disrupt microtubules (Schatten et al., 19891, further supporting the idea that a microtubulebased system of motility mediates pronuclear migration. Because of intense interest in the intracellular signalling events of sea urchin fertilization, insights have been gained into the regulatory mechanisms which govern some of the cytoskeletal transitions which accompany fertilization. The key signalling event in sea urchin fertilization, as in fertilization of other organisms (Jaffe, 1983), is a transient increase in intracellular free calcium ([Ca2+Ii)(Steinhardt et al., 1977). The [Ca2+li rise travels from the sperm entry point across the egg in a wave-like manner, acting as the initial stimulus for a multitude of morphological and biochemical events. For example, the [Ca2+I,wave is followed rapidly by exocytosis, microvillar elongation, and an increase in intracellular pH, and each of these events can be triggered in the absence of fertilization by artificially raising [Ca2+Ii.The precise relationship between the [Ca2+Iiwave, the rise in pH, and microvillar elongation has been assessed by studies wherein calcium was increased under conditions which prevented the pH increase, or pH was increased under conditions which did not result in a [Ca2 Ii rise (Begg and Rebhun, 1979; Begg et al., 1982; Carron and Longo, 1982). It appears that the [Ca2+lirise and the pH increase each play a separate role in microvillar elongation: the [Ca2 Ii rise triggers actin filament extension, thereby resulting in microvillar elongation, whereas the pH increase triggers bundling of the actin filaments, thereby stabilizing the newly elongated microvilli (Fig. 5) (Carron and Longo, 1982).Thus, the sea urchin embryo utilizes specific intracellular signals to execute particular cytoskeletal transformation. +

+

The Cytoskeleton in Chordate Development Ascidians. Specializations of the cortical cytoskeleton seen in lower organisms are also apparent in chordates. Indeed, one of the most extensively studied cortical cytoskeletal domains is that of the ascidian, Styela. This organism, which was adopted for investigation of cytoskeletal rearrangement during early development principally by Jeffery and coworkers, has several features that make it ideal for such studies. For example, the eggs are transparent and have differentially colored components associated with different egg regions. Thus, it is possible to follow the fate of the different egg regions in living embryos by observing the redistribution of the colored components during embryogenesis. Styela has three obvious egg cytoplasmic regions, each of which has a specific developmental fate. Ectoplasm, a clear region originating from the oocyte germinal vesicle, accumulates in ectodermal cells. Myoplasm, a region with yellow pigment granules, accumulates in muscle and mesenchyme cells. Endoplasm, a white region containing yolk, accumulates in endodermal cells. As might be expected given their different develop-

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W.M. BEMENT ET AL.

Fig. 3. Immunogold labelling of actin in sea urchin egg cortex. Gold particles are concentrated in the microvilli and around the cortical granules. Inset: Linear array of gold particles demonstrating the presence of filamentous actin in the microvilli. (Reproduced from Spudich et al., 1988, with permission of Alan R. Liss, Inc.)

mental fates, each region of Styela oocytes and embryos exhibit characteristic patterns of mRNA distribution. This was demonstrated by in situ hybridization of nucleic acid probes to histological sections. In the full grown oocyte, poly(A)+RNA is enriched in the germinal vesicle (Jeffery and Capco, 1978). In contrast, actin mRNA is found in the yellow cytoplasm of the egg cortex and the germinal vesicle, whereas histone mRNA is apportioned equally to all regions of the cytoplasm (Jeffery et al., 1983). Fertilization triggers a dramatic redistribution of these regions of the cytoplasm and their constituent RNAs. The myoplasm and its associated actin mRNA become concentrated in the vegetal hemisphere cortex. Both then move to the subequator of the zygote and form the yellow crescent, which later gives rise to the posterior pole. The ectoplasm becomes enriched in poly(A) +RNA and, later, the poly(A) +RNA (like the ectoplasm) is predominantly distributed to ectodermal cells (Jeffery et al., 1983). Histone mRNA, however, remains uniformly distributed and is equally partitioned into the blastomeres. How does the Styela embryo accomplish localization and subsequent segregation of the different regions of the cytoplasm and their associated RNAs? Several lines of evidence indicate that the cytoskeleton controls both localization and redistribution of Styela cytoplasm and RNAs. First, detergent-extracted Styela eggs, which lack soluble components but possess intact cy-

toskeletons, retain the bulk of their RNA (Jeffery, 1984). Second, the spatial distribution of RNA in detergent-extracted eggs is essentially the same as that in unextracted eggs (Jeffery, 1984). Third, eggs stratified by centrifugation have distinct bands corresponding to the ectoplasm, endoplasm and myoplasm, and in situ hybridization reveals that RNA stays in the same region it was located in before stratification (Jeffery, 1984). Fourth, electron microscopy and biochemical analysis of detergent-extracted eggs and embryos has revealed the presence of a filamentous network in the cortex which contains actin and intermediate filament proteins and which colocalizes with the yellow pigment and mRNA of the myoplasm (Jeffery and Meier, 1983, 1984;Jeffery, 1985a). Fifth, upon fertilization, this cortical cytoskeleton domain undergoes contraction and migration to the vegetal cortex, and then to the region of the yellow crescent, precisely reflecting pattern of reorganization undergone by the myoplasm in normal development (Jeffery and Meier, 1983, 1984). The above studies show that (1) RNA protein components of the different plasms are very tightly associated with elements in the cell; (2) these elements remain behind after detergent extraction; and (3) RNA and other plasm components move in temporal and spatial concert with embryonic cytoskeletal domains. Taken together, these investigations clearly demonstrate the role of the cytoskeleton in localization and segregation of cytoplasmic components during Styela

ROLE OF CYTOSKELETON DURING EARLY DEVELOPMENT

Q

A

......

..

B )

@@LO

4

Fig. 4. Diagram depicting the distribution of nonfilamentous actin (A; closed circles), filamentous actin (A; cross-hatch),and spectrin (B; thin strands) in the sea urchin egg cortex. CG denotes cortical Danules; AV denotes acidic vesicles. (Reproducedfrom Bonder et al., 1989, with permission of Academic Press.)

development. Moreover, the fact that the different domains maintained and transported by the cytoskeleton have distinct developmental fates further illustrates how embryonic patterning events are dependent on the cytoskeleton. In addition to their cortical actin filament network, ascidians possess an elaborate network of microtubules. Using immunofluorescence, Sawada and Schatten (1988) demonstrated that the unfertilized egg con-

31

tains microtubules which extend throughout the cytoplasm. Fertilization triggers a reorganization of this network, resulting in microtubules projecting from a centrosome located in the myoplasm a t the vegetal pole. The precise role of this network is unknown, but it is thought to be required for migration and syngamy of the male and female pronuclei. The means by which ascidians regulate these cytoskeletal transitions are not clear; however, preliminary evidence suggests that a rise in [Ca2+Ii,triggered by fertilization, may be an important signal. For example, Jeffery (1982) demonstrated that translocation of myoplasm to the vegetal pole of Styela could be perturbed by placing eggs next to a rod treated with calcium ionophore. Under these conditions, the myoplasm always migrated toward the side of the egg closest to the rod, without respect to the animal vegetal axis (Jeffery, 1982). Subsequently, Bates and Jeffery (1988) showed that treatment of egg fragments with calcium ionophore disrupts normal patterns of myoplasm migration, and suggested that the normal trigger for myoplasm movement was a rise in [Ca2+Ii,and, in un er turbed development, an endogenous gradient of [Ca% +Iiregulates migration of the myoplasm to the vegetal pole. Amphibians. Amphibian oocytes, eggs, and zygotes, particularly those of the frog Xenopus laevis, have been extremely popular systems for study of many developmental and cellular phenomena, including cytoskeletal dynamics. Actin filaments, intermediate filaments, and microtubules all undergo dramatic reorganizations a t key transitions of Xenopus oogenesis and early development (Bement and Capco, 1990a,c). Some of these cytoskeletal reorganizations are crucial for the processes of fertilization, cytokinesis, and axis determination; others reflect specializations to allow the egg to cope with its tremendous size. Further, at least some of these reorganizations are controlled by intracellular signalling pathways, allowing rapid transient regulation of complex cytoskeletal events (Bement and Capco, 1990~). Again, as seen in other organisms, amphibian oocytes and eggs have a cortical domain which undergoes profound changes in structure and composition during oogenesis and early development. The cortex of amphibian oocytes and eggs contain actin filaments as demonstrated by electron microscopy (Franke et al., 1976), immunofluorescence et 1981), and immunoelectron microscoPY (Gall et al., 1983). While both the oocyte and the egg have cortical actin filamerits, in the oocyte, many of the filaments appear to be contained in the microvilli while the actin filament network underlying the plasma membrane is relatively sparse, whereas in the egg, the microvilli are greatly reduced in size (Charbonneau and Grey, 1984; Bement and Capco, 1989a) but the filament network beneath the plasma membrane is much more dense (Ryabova, 1982). Fertilization initiates a wave of cortical granule exocytosis (Grey et al., 1974), microvillar enlargement (Ezzell et al., 1985), and contraction of the egg cortex, such that the animal hemisphere surface appears to shrink with respect to the vegetal hemisphere surface

32

W.M. BEMENT E T AL.

Fig. 5. Electron micrographs of sea urchin egg cortices depicting microvillar elongation. A The plasma membrane of the unfertilized egg is studded with short microvilli (MV). EJ denotes egg jelly, CG denotes cortical granule. B The fertilized egg possesses elongate microvilli with a bundled actin filament core (arrowheads). F M denotes

fertilization membrane. C: An egg treated with calcium ionophore in Na-free medium to induce a rise in calcium while preventing an increase in pH has elongate microvilli (MV) which lack bundled actin filaments. Arrowheads denote fertilization membrane. (Reproduced from Carron and Longo, 1982, with permission of Academic Press.)

(Wolf, 1974). Microvillar elongation allows the egg plasma membrane to cope with the insertion of cortical granule membrane, as it does in sea urchins. Cortical contraction is followed by relaxation of the cortex, and then later, in synchrony with each cleavage cycle, the

egg cortex contracts and relaxes again (Hara e t al., 1980). The initial contraction may be necessary for emission of the second polar body, and evidence suggests that it helps position the sperm near to the site where the female pronucleus will form (Elinson, 1977).

ROLE OF CYTOSKELETON DURING EARLY DEVELOPMENT

The subsequent contractions are thought to be required for cytokinesis (Hara et al., 19801, a presumption borne out by our observation that eggs which fail to contract also fail to form cleavage furrows (Bement and Capco, 1990b). Microvillar enlargement and cortical contraction are apparently mediated by interaction of the cortical actin filament network with myosin since they are sensitive to N-ethylmelamide-modified heavy meromyosin (Ezzell et al., 1985), and depletion of myosin from eggs sliced in half prevents contraction, whereas readdition of myosin restores contractility (Christensen et al., 1984). Intermediate filaments are also found in amphibian oocytes, and, like actin filaments, undergo reorganization as the oocyte becomes the egg, and again as the egg becomes the zygote. Cytokeratin filaments were first identified in Xenopus oocytes by immunofluorescence and immunoelectron microscopy (Gall et al., 1983; Franz et al., 1983; Godsave et al., 1984a). The clearest picture of the oocyte cytokeratin network has been provided by Klymkowsky et al. (1987),who, using wholemount immunofluorescence, demonstrated the presence of a highly organized “geodesic” cytokeratin network which extends throughout the vegetal hemisphere cortex (Fig. 6). Another intermediate filament, vimentin, is also present in the Xenopus oocyte. In the oocyte animal hemisphere, immunofluorescence reveals that vimentin is present in mitochondria-filled corridors of cytoplasm, while in the vegetal hemisphere it exhibits a punctate pattern (Godsave et al., 1984b). During meiotic resumption, as the oocyte is converted into the egg, virtually all of the cytokeratin network disappears (Godsave et al., 1984b; Gall and Karsenti, 1987; Klymkowsky et al., 1987), leaving a few residual cytokeratin filaments in the egg cortex (Klymkowsky et al., 1987). Vimentin distribution also changes, becoming dispersed uniformly throughout the egg (Godsave et al., 1984b). Fertilization initiates the reformation of the cytokeratin network in the animal hemisphere, and also triggers formation of a somewhat more sparse cytokeratin filament network in the animal hemisphere. The cytokeratin networks maintain this pattern until the early blastula stage. Unlike cytokeratin, vimentin remains randomly distributed after fertilization (Godsave et al., 1984b; Tang et al., 1988). Given that the animal and vegetal hemispheres differ both in the form (i.e., filamentous versus nonfilamentous) and amount of the two intermediate filament proteins they contain, it is reasonable to assume that the different blastomeres derived from each hemisphere will likewise differ in their intermediate filament composition. This difference may well contribute to the different developmental fates of the blastomeres derived from the animal and vegetal hemispheres. Study of microtubules in amphibian oocytes, eggs, and zygotes has revealed similarities to somatic microtubules, as well as fascinating specializations of this cytoskeletal element which are entirely unanticipated by somatic cell studies. In the Xenopus oocyte, immunofluorescence and electron microscopy have shown that microtubules are found both in the cortex (Dumont and Wallace, 1972; Huchon et al., 1988) and in

33

Fig. 6. Wholemount immunofluorescence micrograph of cytokeratin in the Xenopus oocyte. Cytokeratin filaments from a “geodesic” array in the oocyte vegetal hemisphere cortex. (Reproduced from Klymkowsky et al., 1987, with permission of The Company of Biologists, Ltd.)

association with the germinal vesicle (Jessus et al., 1986), a distribution similar to that seen in other cell types (Brinkley, 1985). Oocyte microtubules may help position the germinal vesicle within the cytoplasm, since treatment of oocytes with microtubule poisons induces displacement of the germinal vesicle toward the oocyte cortex (Lessman, 1987). Meiotic maturation entails extensive reorganizations of oocyte microtubules. When the germinal vesicle of the oocyte breaks down, a prominent microtubule network becomes visible (by light microscopy and immunofluorescence) a t the vegetal side of the disintegrating germinal vesicle (Huchon et al., 1981;Jessus et al., 1986). As meiotic maturation progresses, the network moves to the animal pole, and upon assembly of

34

W.M. BEMENT ET AL.

the first meiotic spindle, the cytoplasmic network disappears (Huchon et al., 1981). Curiously, in the meiotically mature egg, which is arrested at metaphase 11, microtubules are present both in the cytoplasm (Elinson, 1983; Huchon et al., 1988) and in the meiotic spindle, a situation unheard of in somatic cells (Brinkley, 1985). Fertilization results in an initial decrease in the level of polymerized tubulin (Elinson, 1985) followed by an explosive increase (Stewart-Savage and Grey, 1982; Gard and Kirschner, 1987a,b; Elinson and Rowning, 1988). Two distinct microtubule networks form a t this time, each accomplishing a different task. First, microtubules from the sperm aster elongate dramatically, ultimately contacting the female pronucleus and accomplishing pronuclear migration (Stewart-Savage and Grey, 1982). The sperm aster microtubules elongate at 12 pm per minute (Stewart-Savage and Grey, 1982) a rate 10-fold faster than somatic cell microtubules (Gard and Kirschner, 1987a). This rapid elongation is required to overcome the long distances traveled by the male and female pronuclei through the relatively vast cytoplasm of the egg. If the microtubules elongated at the rate of somatic cell microtubules, pronuclear migration would take 5 hours instead of 30 minutes. As the pronuclei migrate, a second microtubule network arises in the egg. This network forms in the vegetal cortex and is composed of numerous parallel microtubules (Fig. 7) (Elinson and Rowning, 1988). The formation of this network occurs at the same time the zygote cortex rotates with respect t o the zygote interior. This rotation establishes the dorsallventral axis of the embryo (Gerhart et al., 19861, and several lines of evidence indicate that it is mediated by the parallel microtubule network: (1) the network is in a “shear zone” between the egg cortex and the interior cytoplasm, which is the appropriate position to rotate or facilitate rotation of the cortical cytoplasm (Elinson and Rowning, 1988); (2) microtubule poisons, which disrupt the network, also prevent rotation and axis formation (Manes et al., 1978; Elinson, 1983; Ubbels et al., 1983); and (3) irradiation of the zygote at the time the network is forming (but not before or after) disrupts the microtubule network and prevents rotation (Elinson and Rowning, 1988). Like the organisms discussed above, Xenopus oocytes, eggs, and zygotes exhibit dynamic, nonrandom RNA distributions. In the periphery of the oocyte vegetal hemisphere, in situ hybridization has revealed a striking localization of poly(A) RNA (Fig. 8) (Capco and Jeffery, 1982; Larabell and Capco, 1988). Moreover, the vegetal periphery is also enriched in specific mRNAs, including Vgl (which encodes a protein related to transforming growth factor beta; Weeks and Melton, 1987; Meoton, 1987) and tubulin mRNA (Larabell and Capco, 1988). Interestingly, the pattern of poly(A)+RNA localization throughout oogenesis (Fig. 9) (Capco and Jeffery, 1982) precisely parallels that exhibited by the Vgl mRNA (see Melton, 1987). In addition to the vegetal periphery, biochemical analysis has revealed that other regions of the oocyte contain localizations of specific mRNAs. By separating oocytes into animal and vegetal hemispheres, and each +

Fig. 7. Electron micrograph of the vegetal, subcortical region of a fertilized Xenopus egg. Microtubules are apparent in the cytoplasm, forming a parallel array. (Reproduced from Elinson and Rowning, 1988, with permission of Academic Press.)

hemisphere into peripheral and central regions (Fig. 10) (“spatial fractionation”; Capco and Mecca, 19881, we were able to purify and analyze (by blotting and probing with radiolabeled nucleic acid probes) mRNA from each of these different regions (Perry and Capco, 1988). Using this approach, we showed that actin and tubulin mRNA are concentrated in the oocyte periphery, whereas histone mRNA is distributed equally throughout the egg (Perry and Capco, 1988). Spatial fractionation further revealed that meiotic maturation is accompanied by RNA redistribution. In the egg, tubulin mRNA is concentrated in the egg center rather than the periphery, and a slight increase in the concentration of actin mRNA in the egg center is also apparent (Fig. 11). The distribution of histone mRNA, on the other hand, does not change. In situ hybridization provides further confirmation that meiotic maturation triggers mRNA redistribution. For example, in the egg, poly(A)+RNA is no longer concentrated in the vegetal periphery (Capco and Jeffery, 1982) nor is mRNA for Vgl or tubulin (Melton, 1987; Larabell and Capco, 1988). Using timecourse in situ analysis of the poly(A)+RNA distribution, we have determined that the localization in the vegetal periphery disappears at about the time the germinal vesicle breaks down (Larabell and Capco, 1988). Fertilization reestablishes the concentration of tubu-

ROLE OF CYTOSKELETON DURING EARLY DEVELOPMENT

35

Fig. 8. Darkfield micrograph of full grown Xenopus oocyte vegetal cortex after in situ hybridization with [3H]poly(U). Concentrations of silver grains (which appear as white spots) demonstrate that Poly(A)+RNA is localized in the oocyte vegetal subcortex. (Reproduced from Capco and Jeffery, 1982, with permission of Academic Press.)

lin and actin mRNAs in the zygote periphery, whereas histone mRNA is concentrated in the animal hemisphere (Fig. 11) (Perry and Capco, 1988). While it is unclear whether these RNA redistributions reflect active translocation or compartmentalized synthesis and degradation, two reports suggest the translocation can account for at least part of the observed patterns of RNA distribution. In the first, labeled poly(A) +RNA isolated from the vegetal hemisphere and then injected into the animal pole of the activated egg was found to redistribute back to the vegetal pole (Capco and Jeffery, 1981). In the second, labeled Vgl mRNA injected into oocytes was found to translocate to the vegetal periphery, the normal location for endogenous Vgl mRNA (Yisraeli and Melton, 1988). In both of these studies, the kinetics of translocation and the relative stability of the RNA employed suggest that translocation, rather than compartmentalized synthesis and degradation, was responsible for the observed RNA redistribution. Since poly(A)+RNA (Capco and Jeffery, 1982), Vgl mRNA (Melton, 1987) and tubulin mRNA (Larabell and Capco, 1988) colocalize in the oocyte vegetal periphery with the cytokeratin network (Klymkowsky e t al., 19871, while actin mRNA (Perry and Capco, 1988) colocalizes in the periphery with the actin network (Franke et al., 1976; Gall et al., 1983), and in view of the association of RNA and cytoskeletal elements in other systems, it seems likely that the Xenopus oocyte

and egg cytoskeleton might be associated with localized populations of RNA. Indeed, although much work remains to be done, this seems to be the case. For example, it has been shown that Vgl mRNA is associated with the detergent-resistant cytoskeleton of the oocyte, but in the egg, it is associated with soluble components (Pondel and King, 1988). It has also been demonstrated that the normal patterns of Vgl mRNA localization are disrupted by treatment of oocytes with cytoskeletal poisons (Yisraeli et al., 1990). Using a detergent-extraction protocol originally designed for large cells or blocks of tissue (Capco et al., 1987), we have recently examined associations between actin and tubulin mRNA and the cytoskeleton of Xenopus oocytes and eggs (Hauptman et al., 1989). In both the oocyte and the egg, 80% of the actin and tubulin mRNA are associated with the detergent-resistant cytoskeleton (Hauptman et al., 1989). Since actin and tubulin mRNAs have characteristic patterns of localization in both the oocyte and the egg (Perry and Capco, 19881, a simple interpretation of the above reports is that actin and tubulin mRNA retain their characteristic patterns of localization by binding to the cytoskeleton in the region where they are localized. In contrast, Vgl mRNA and other RNAs localized in the vegetal periphery bind to a detergent-resistant network only in the oocyte, and upon meiotic maturation, either detaches from the network, or the network itself, perhaps the geodesic cytokeratin filaments, becomes soluble.

36

W.M. BEMENT ET AL.

Stage 1-2

Stage 3

0 ...... ,.. ..,,

,,

@:;j

.:-,...... .....

Stage 4

Stage 5

pq

Stage 6

Fig. 9. Diagram demonstrating the distribution of poly(A)+RNA throughout Xenopus oogenesis. Stippling denotes concentrations of poly(A)+RNA. (Reproduced from Capco and Jeffery, 1982, with permission of Academic Press.)

In no other developmental system, except perhaps sea urchins, has the relationship between intracellular signalling events and observed patterns of cellular reorganizations been as closely scrutinized as Xenopus (Bement and Capco, 1989a, 1990~). As with sea urchins and ascidians, evidence demonstrates that amphibian eggs and embryos employ intracellular signalling

pathways to control cytoskeletal transitions (Bement and Capco, 1990b,c). Once again, a rise in [Ca2+liacts as the initial stimulus for many of the observed changes, including cortical granule exocytosis and cortical contraction (Schroeder and Strickland, 1974; Busa and Nuccitelli, 1985; Kubota et al., 1987). The rise in [Ca2+litriggered by fertilization is coupled to cortical granule exocytosis, microvillar enlargement, and cortical contraction by protein kinase C (PKC; Fig. 12) (Bement and Capco, 1989b; 1990b; Bement and Capco, unpublished results). Evidence also indicates that PKC regulates formation and closing of the contractile actomyosin ring which mediates Xenopus embryo cytokinesis (Fig. 13) (Bement and Capco, 1989b, 1990a,c; Brice and Capco, unpublished results). PKC is a calcium- and phospholipid-dependent kinase, and presumably therefore exerts its effects by way of phosphorylation. What are the relevant PKC substrates underlying cortical contraction and cytokinesis? All of the available evidence suggests that phosphorylation of myosin light chain may represent the key trigger for PKC-mediated contraction and cytokinesis: first, myosin is required for cortical contraction both in vitro and in vivo (see above). Second, phosphorylation of myosin light chain is correlated with actin gel contraction in Xenopus egg extracts (Ezzell et al., 1983). Third, phosphorylation of myosin light chain in vivo is correlated with cortical contraction, and this phosphorylation is mediated by PKC (Bement and Capco, unpublished data). Fourth, phosphorylation of embryonic myosin light chain is known to increase the actin-activated ATPase activity of myosin (de Lanerolle and Nishikawa, 1988). These data, and the fact that PKC antagonists prevent cytokinesis in Xenopus embryos (Brice and Capco, unpublished results), lead us to suggest the following hypothesis for amphibian embryonic cytokinesis: the rise in [Ca2+liwhich accompanies each meiotic and mitotic division (Whitaker and Patel, 1990) transiently activates PKC. Upon activation, PKC phosphorylates myosin light chain, thereby imparting an increase in the actin-binding affinity and actin-activated ATPase activity of myosin (de Lanerolle and Nishikawa, 1988). This results in an increase in actomyosin-based force production and consequently, cell contraction and cytokinesis. Intracellular signals also appear to regulate microtubule dynamics during meiotic resumption and egg activation. Maturation promoting factor (MPF), a complex of phosphoproteins which acts at the top of a cascade of kinaselphosphatase reactions, converts the cytoplasmic microtubule array of the oocyte into the spindle array of the egg in vitro (Lohka and Maller, 1985) and in vivo (Karsenti et al., 1984). Similarly, phosphorylation of microtubule associated proteins (MAPS)may regulate sperm aster formation and possibly formation of the vegetal microtubule array. For example, Gard and Kirschner (1987b) identified "XMAP", a 215 kD MAP found in the cytoplasm of Xenopus eggs. XMAP promotes extremely rapid microtubule polymerization (Fig. 141, and high XMAP activity is correlated with dephosphorylation of XMAP. Since formation of the sperm aster and the subcortical microtubule array coincide with high XMAP activity,

ROLE OF CYTOSKELETON DURING EARLY DEVELOPMENT

37

Fig. 10. The “spatial fractionation” technique. Xenopus oocytes, eggs, or zygotes are placed in 100% ethanol (a), divided into animal and vegetal hemispheres with a microscalpel (b), further divided into periphery and central regions by scooping with a microspoon (1.9,and then the vitelline envelope and follicle cells are removed (d). (Reproduced from Capco and Mecca, 1988, with permission of Elsevier Scientific Publishers.) FULL GROWN

MEIOTICALLY

UNICELLULAR

OOCYTE

MATURE EGG

ZYGOTE

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ALPHA-TUBULIN

.

.

.

BETA-TUBULIN

.

.

.

.

. . . .. . . . .

ACTIN

. .

.

.

.

.

.

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.

.

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.

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Fig. 11. Diagram representing the distribution of actin, tubulin, and histone mRNA in the full grown Xenopus oocyte, the metaphase I1 egg, and the fertilized egg. Stippling represents the distribution of the different mRNA. (Based on the results of Perry and Capco, 1988.)

XMAP likely contributes to the regulation of these tain cytoskeletal domains which reorganize during early development. Interestingly, however, mammastructures. Mammals. Mammalian eggs and embryos also con- lian eggs and embryos, unlike other systems described

38

W.M. BEMENT ET AL.

+