A yellow crescent cytoskeletal domain in ascidian eggs and its role in early development

DEVELOPMENTAL BIOLOGY g&125-143 (1983)

A Yellow Crescent Cytoskeletal Domain in Ascidian Eggs and Its Role in Early Development

Department of Zoology, Ckiversily qf Te~us, R~slin., Texns 78712

In this investigation, Triton X-100 extraction was utilized to examine the cytuskeleton of ascidian eggs and embryos.

The eytoskeleton contained little carbohydrate or lipid and only about 20-2596 of the total cellular protein and RNA.

It was enriched in polypeptides of molecular weight (H,) S&48, and 43 X l@. The 43 X IO3 ~&&polypeptide was identified

as a&in based on it.8 &, isoelectric point, and affinity for DNase I. Electron microscopy of the detergent-extracted

eggs showed that they contained cytoskeletal domains corresponding to colored cytaplasmic regions of specific mor-

phogenetic fate in the living egg. A yellow crescent cyioskeletal domain in the mgoplasm was examined and shown

to consist af a plasma membrane lamina (PML) and a deeper lattice of filaments which appeared to connect the yellnw

crescent pigment granules to the PML. The PML prahably consists of integral membrane proteins stabilized by an

underlying network of actin Biaments since NBD-phallacidin stained this area of the egg cortex and the PML was

extracted from the cytoskeleton by DNase I treatment. The yellow crescent cytoskeletal domain was found throughout

the cortex of the unfertilized egg. During ooplasmic segregation it progressively receded into the vegetal hemisphere

and xv-as subsequently partitioned to the presumptive muscle and mescnchyme cells of the 3%cell embryo. It is suggested

that contraction of the actin network in the yellow crescent cytoskeletal domain is the motive force for ouplasmic

segregation. This structure may also serve as a framework for the positioning of morphogenetic determinants involved

in muscle cell development.

The developmental fate of embryonic cells is thought to be governed by morphogenetic determinants Iocal- ized in the egg cytoplasm (see Davidson, 1976, for re- view). Although compelling evidence argues for the ex- istence of these substances {Wilson, 1925), their molecular identity, mode of action, and manner of localization are unknown. It has often been assumed that morphogenetic determinants are associated with membranes or the cortical cytoplasm of the egg. This assumption is based on the fact that many of these substances are resistant to displacement by low centrifugal forces. In certain molluscan embryos, for example, the displacement of cytoplasm from the polar lobe by centrifugation does not prevent the development of lobe-dependent larval structures (Morgan, 1935; Clement, 1968; Verdonk, 1968). Likewise, t,he displacement of cytoplasmic organelles from the yellow crescent region of Stycla eggs

by centrifugation does not prevent the development of the larval muscle cells (Conklin, 1931). The inability to alter normal developmental patterns by removing part of the fluid cytoplasm from eggs (van den Biggelaar, cited in Dohmen and Verdonk, 1979; Laufer and von Ehrenstein, 1980), further suggests that morphogenetic determinants may be bound to rigid cytoplasmic structures.

’ To whom all correspondence should be addressed.

Our understanding of the eytoplasmic organization of eukaryotic cells has changed markedly in recent years. It is now known that the cytoplasm contains elaborate networks of mierotubules, actin filaments and intermediate filaments, collectively known as the cytoskel- et,on (Wolosewick and Porter, 1979; Heuser and Kir- schner, 1980; Batten et al., 1980). The distribution and organization of these filaments has been established by immunocytochemistry (&born et al., 1978) and electron microscopy of detergent-extracted cells (Brown et al., 1976; Small and Celis, 1978; Schliwa et al,, 1981). Inte- gral membrane proteins (Ben Ze’ev, 19’79; Sheetz, 1979f, enzymes (Eckcrt et al., 1980), centrioles (Lenk et at., 1977), and polyribosomes (Lenk et al., 19’77; Cervern CL al., 1980; van Venrooij et ccl., 1981) represent some of the cytopl~smic components reported to be associated with the cytoskeleton. It is possible that substances such BY the morphogenetic determinants of eggs, which act in a spatially distinct fashion, may also be associated with the cytoskeieton. Such an anchorage would explain the resistance of determinants to low-speed centrifugation and their failure to be removed from the egg with the fiuid cytoplasm.

As an initial step in investigating the role of cytoskeletal elements in the spatial aspects of early development, we have characterized the cytoskeleton of ascidian eggs by a combination of biochemical and mi- croscopic methods. We used the eggs of S@& and

126 DEVELOPMENTAL BIOLOGY VOLUME 96, 1983

Bolt&a, because they contain colored cytoplasmic regions which are extensively rearranged after fertilization and partitioned into specific cell lineages during early development ~~onklin, 1905). One of these regions, the myoplasm or yellow crescent cytoplasm (in Styela), has been shown to contain determinants which specify the development of acetylcholinesterase in the muscle cell lineage (Whittaker, 1980). In this report we show that the yellow crescent region contains a cytoskeletal domain consisting of pigment granules embedded in a lattice of filaments that are connected to a submembrane network of F actin. This yellow crescent cytoskeletal domain participates in ooplasmic segregation and eventually enters the presumptive muscle and mesenchyme cells during embryogenesis.

MATERIALS AND METHODS

~~olo~i~a~ ~~e~a~. The ascidian species utilized in this investigation were Styela clava (collected at Woods Hole, Mass.), S. plicata (supplied by Pacific Biomarine Inc., Venice, Calif.), and B&e&a villosa (collected at Friday Harbor, San Juan Island, Wash.). The ascidians were maintained in Instant Ocean culture systems under constant light for 2 weeks in order to promote rip- ening of gametes and to prevent premature spawning. Eggs and sperm were obtained from surgically removed gonads of S. clava and Boltenia or obtained by natural spawning of S. plicata (West and Lambert, 1975). Fer- tilization was elicited by the mixture of gamete sus- pensions obtained from different individuals as described previously (Jeffery and Capco, 1978). Fertilized eggs were successively washed in ten 5-ml volumes of artificial sea water and cultured in Millipore-filtered artificial sea water (MFASW) at 18°C.

Separation and sorting of ernbrgmic cells. Fertilized eggs were dechrionated by treatment with 2 mg/ml protease (Type IV, Sigma, St. Louis, MO.) for 20 min (Bol- tenia) or 2 hr (StyeZa), washed 10 times with 20 vol of MFASW, and when they reached the 32cell stage were suspended in MFASW containing 3 mM EGTA. Cells were separated by agitating the embryos for lo-15 min. The separated embryonic cells were washed in 5 ml of MFASW three times and divided into three groups based on color. Yellow cells represent the myoplasmic blastomeres, clear cells represent mainly the eetodermal blastomeres, and white cells represent endodermal blastomeres. Each group of balstomeres was treated with Triton X-100 to form eytoskeletons and attached to grids for electron microscopy as described below.

Cytoskeletm preparation. It was necessary to remove the chorion and accessory cells prior to the preparation and examination of the egg cytoskeleton. Usually this was accomplished by protease digestion, as described

above. Alternately, the chorion and accessory cells were removed by manual dissection. Eggs were incubated in 0.5% Triton X-100 extraction buffer (see below) for about 10-15 min. During this period the follicle cells become loosened from the chorion and float free. The chorion is hardened so that it can be fractured by gentle compression of the egg between the bottom of a glass petri dish and an electron microscope grid. The compression procedure releases test cells and denuded eggs into the medium. Eggs are further incubated in the extraction buffer until cytoskeieton formation is complete.

Denuded eggs were extracted with a number of different buffers supplemented with Triton X-100, including the extraction media developed by Cervera et al. (1981) and by Heuser and Kirschner (1980). Although the structures we describe in this paper were seen in cytoskeletons prepared in all the extraction buffers, a modification of the buffer developed by Cervera et al. (1981) produced cytoskeletons of high quality and was used in this investigation. This cytoskeletal extraction buffer (CEB) consisted of 10 mM piperazine-N&V-bis[2- ethanesulfoni~ acid], 300 mM sucrose, 100 mM KCl, 5 mM Mg acetate, 1 mM EGTA, 10 PM leupeptin (PH. 6.8). Eggs and zygotes were washed in CEB without detergent and then extracted in CEB containing 0.5% Triton X-100 for 30-60 min at 4°C. Cytoskeleton formation was judged as complete when the opaque eggs became completely transparent. Isolated cytoskeletons were prepared for biochemical analyses or electron microscopy as described below.

Bioch,ernical analyses. Cytoskeletons were collected by eentrifugation at 30009 for 1 min, the supernatant fraction was decanted, and the pellet was washed twice in 5 ml of CEB (4°C). The pooled supernatants were designated as the detergent-soluble fraction (SOL). The pellet was resuspended in a small volume of CEB and designated the cytoskeletal fraction (CSK).

The protein content of the SOL and CSK was esti- mated by the method of Lowry et al. (1951). The carbohydrate content of the SOL and CSK was determined by the method of Van Handel (1965). The SOL and CSK were extracted with a mixture of 1:l chloroform-meth- anol for lipid analysis. The organic phase was evapo- rated to dryness and its lipid content was determined by the method of Zollner and Kirsch (1962). RNA content was determined by absorbance at 260 nm after the SOL and CSK was extracted with phenol. Poly(A) content of phenol-extracted RNA was measured by [3H]poly(U) hybridization as described by Jeffery and Brawerman (1974).

DNase I-Sepharose chromatography. The CSK was di- luted to 5-10 ml with IO mM Tris-HCl, 1 mM CaClz, 1 mM dithiothreitol, 5 mg/ml leupeptin, pH 7.5 (appli-

JEFPERY AND MEIER Yellow Crescent Cytoskeletm 127

cation buffer), and mixed with a slurry of DNase I (Wor- thington, Freehold, N. J.) covalently linked to Sepharose 4B (Pharmacia) (Lazarides and Lindberg, 1974) for 2 hr at 4°C. The DNase I-Sepharose was pelleted by low- speed centrifugation, the supernatant containing the unbound material was removed, and the pellet was washed several times in the application buffer. The washed DNase I-Sepharose slurry was poured into a glass column and eluted with 8 M urea. The column fractions with absorbance at 280 nm were pooled and analyzed by gel electrophoresis.

Gel e~~c~ro~~~es~s. SOL and CSK polypeptides were analyzed by two-dimensional (2D) gel electrophoresis as described by O’Farrell (1975). Prior to electrophoresis the samples were treated with DNase and RNase (50 and 5 pg/ml, respectively, for 5 min at 4”C), brought to 9 M urea and 50 mM lysine, and mixed with an equal volume of 5% &mercaptoethanol, 2% Nonidet P40,2% ampholines (1.6% pH range 5-7 and 0.4% pH range 3.5- 10), and 9.5 M urea (lysis buffer). The sample was applied to 4% isoelectric focusing gels and, after electrophoresis at 400 V for 15 hr, the tube gels were equil- ibrated in 0.063 M Tris-HC1, 2.5% SDS, 5% 2-mercaptoethanol, and 10% (w/v) glycerol (pH 6.8) for 1 hr and loaded onto 10% polyacrylamide slab gels containing 0.1% SDS for electrophoresis in the second dimension. Electrophoresis in the second dimension was for 4.5 hr at 80 V through the stacking gel, then at 100 V through the separating gel. The gels were stained with silver nitrate (Wray et al., 1981) or Coomassie blue.

Pre~~a,rat~~T~ of cytosk~~etal whole mmmts and spreads. Eggs or embryonic cells which had been detergent extracted were washed several times in CEB lacking Tri- ton X-100 and then suspended in CEB in a small glass petri dish. In order to prepare whole mounts, a 400- mesh, poly-L-lysine-coated electron microscope grid was placed under the cytoskeletons with a forceps and they were carefully lifted out of the solution. Some cytoskeletons usually attached to the grid during this process. In order to prepare a cytoskeletal spread the grid containing attached cytoskeletons was inverted in the solution and gently pulled across the glass substrate with a forceps. The whole mounts and spreads were immediately fixed and processed for electron microscopy as described below.

~lec~~~ microscopy. Intact eggs, cytoskeletons, or grids containing cytoskeletal whole mounts or spreads were fixed for 30 min at 20°C with 2% glutaraldehyde in 0.1 Mphosphate buffer (pH 7.2), washed three times in 0.1 M phosphate buffer, and postfixed in 1% 0~0, for 5 min (whole mounts and spreads) or 1 hr (all other specimens) at 20°C. For transmission electron microscopy (TEM), specimens were embedded in Spurrs, thin sectioned, stained with lead citrate and uranyl acetate,

TABLE 1 BIOCHEMICAL COMPOSITION OF TRITON X-100 EXTRACTED

Styeia EGGS AND ZYGOTES

Percentage of unextracted counterpart

Component J&g Zygote’

Carbohydrate 3.4 6.2 Lipid 7.7 9.8 Protein 22.0 19.8 RNA 27.8 25.7 Poly(A) 55.7 65.2

a Zygotes were extracted with Triton X-100 10 min after insemi- nation. The values shown in this table represent the average of three separate determinations.

and examined with a Hitachi H-300 transmission electron microscope. Specimens attached to grids were crit- ical point dried using COZ as the exchange fluid. For TEM, grids could be examined immediately. For scanning electron microscopy (SEM), dried grids were sput- ter-coated with 70 nm of gold-palladium alloy and observed in an IS1 Super IIIA scanning electron microscope.

Flzcoremmee microscopy. Eggs or embryos were rinsed in phosphate-buffered saline (PBS), fixed in 3% form- aldehyde for 5 min at 2O”C, rinsed twice with PBS, placed on coverslips, and extracted with acetone for 2 min at -20°C. Cells were incubated with 3.3 X lo-? &f ‘I-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin (Molecular Probes Inc., Plano, Tex.) for 60 min at 2O”C, rinsed twice with PBS, mounted, and viewed with a fluorescence mi- eroscope at a wavelength of 460 nm.

RESULTS

Detergent Extraction of Egcqs and Zy.gotes

We chose to study the eytoskeleton of eggs and zygotes extracted with the nonionic detergent Triton X- 100 (Buckley and Raju, 1976; Brown et al., 1976; Lenk et al., 1977; Small and Celis, 1978). As shown in Table 1, Triton X-100 extraction of Styela eggs or zygotes produced cytoskeletons nearly depleted of carbohydrate and lipid. Most of the residual lipid probably represents lipid pigment granules which, as shown below, are preserved in the eytoskeleton. Cytoskeletons also contain about 20-25% of the total protein and RNA of unextracted eggs and zygotes. Despite the extraction of most of the RNA, the cytoskeleton was relatively rich in poly(A). This is probably due to the presence of poly(A)-containing RNA in the cytoskeleton of Styela eggs and zygotes, as has been previously reported for detergent- extraeted mammalian cells (Lenk et al., 1977; Cervera

DEVELOPMENTAL BIOLOGY VOLUME 96,1983

FIG. 1. Light micrographs of living and Triton X-loo-extracted eggs and embryos of St&a. (A) A living zygote at the yellow crescent stage. Opaque white yolk obscures the position of the yellow crescent (YC). (B) A detergent-extracted zygote at the yellow crescent stage illustrating the clarification and retention of the yellow pigmentation in the cytoskeleton. (C) A detergent-extracted, unfertilized egg adjacent to a zygote showing a thin layer of yellow pigmentation localized in the cortex. (D) A detergent-extracted zygote involved in ooplasmic segregation showing the ring of intense yellow pigmentation progressing into the vegetal hemisphere. (E) A detergent-extracted zygote at the maximal crescent stage showing three eytoskeletal regions, endoplasm (pale uppermost region), ectoplasm (clear middle region), and myoplasm (bright yellow, lowermost region). (F) A detergent-extracted, two-cell embryo showing yellow pigmentation localized in the vegetal regions of the left and right blastomeres. The chorion (bright line surrounding the cytoskeletons) was not removed from these specimens prior to extraction. All micrographs Xl.24

et at., 1981; Van Venrooij et ad., 1981). No significant differences were detected in the macromolecular composition of cytoskeletons prepared from eggs and one- eel1 zygotes (TabIe I).

Light micrographs of detergent extracted eggs and zygotes are shown in Fig. 1. Unfertilized Styela eggs contain three colored eytoplasmic regions; a clear ee- toplasm in the animal hemisphere, a white, yolk-filled endoplasm primarily localized in the vegetal hemisphere, and a yellow cortical region, the myoplasm. These cytoplasmic regions are extensively rearranged during a period of ooplasmie segregation which is ini- tiated after fertilization (Conklin, 1905). The myoplasm moves into the vegetal hemisphere focusing around the

point of sperm entry and is followed in its wake by the ectoplasm. At the height of ooplasmic segregation, the myoplasm and ectoplasm form a cap within a transient cytoplasmie lobe in the vegeta1 hemisphere of the egg (Zalokar, 1974; Sawada and Osanai, 1981; Jeffery, 1982). At the same time the endoplasm streams in the opposite direction filling the animal hemisphere. After the initial movements of ooplasmic segregation are complete the ectoplasm eventually moves back into the animal hemisphere with the male pronucleus and the myoplasm mi- grates to the future posterior pole of the embryo ex- tending itself into a yellow crescent. Despite its coloration, the myoplasm of St~eZa eggs is often difficult to detect by microscopy due to the opacity of the en-

doplasmic yolk platelets and substantial variations in the intensity of yellow pigmentation between clutches of eggs (Conklin, 1905) (Fig. 1A). Detergent-extracted zygotes, however, became transparent and the crescent could be clearly distin~ished by light microscopy (Fig. 1B). A similar preservation of myoplasmic crescent material, in this case due to orange pigment granules, was seen in Bdtenia eggs (data not shown).

These observations suggest that the myoplasmic pigment granules are associated with cytoskeletal elements and that these interactions may be responsible for their characteristic movement during ooplasmic segregation. However, if this is so, the pigment granules should show cytoskeletal affinities during the process of ooplasmic segregation, as well as after its completion. Therefore, ~ytoskeletons were prepared from unfertilized eggs and zygotes undergoing ooplasmic segregation. Yellow pigment granules were present throughout the cortical region of the cytoskeleton of unfertilized eggs (Fig. lC), whereas cortical rings of concentrated pigment granules were detected in cytoskeletons made from zygotes in which myoplasmic migration was in progress (Fig. 1D). Cytoskeletons from eggs with veg- eta1 cytoplasmic lobes also exhibited pigment granules within a yellow vegetal cap (Fig. 1E). In addition, domains corresponding to the positions normally occupied by ectoplasm and endoplasm were also evident in these cytoskeletons (Fig. 1E). The results suggest that myoplasmic pigment granules show cytoskeletal affinities throughout ooplasmic segregation.

Developing embryos were extracted with Triton X- 100 to determine whether the interaction between the myoplasmic pigment granules and the cytoskeleton per- sisted during later development. The pigment granules were preserved in cells containing the myoplasm when detergent extracted embryos were prepared between the early cleavage stages (Fig. 1F) and the tail bud stage (data not shown). Thus, interactions between the yellow pigment granules and the cytoskeleton are maintained during embryogenesis. Moreover, as in the living embryo, the pigment granules are preferentially localized in cytoskeletons of the cells of the muscle and mesen- thyme lineages.

Protein. Composition of the Cgtoskeletm

We examined the protein composition of the eytoskeleton by 2D gel electrophoresis of the SOL and CSK. Silver-stained gels of the SOL and CSK from unfertilized eggs are shown in Fig. 2. Three major spots, each with some microheterogeneity, could be seen in the egg CSK. In contrast, many spots were visible in the SOL. Some of the proteins in these gels can be identified. The family of abundant SOL proteins which exhibit molec-

ular weights (M,.) between 23 and 56 X lo3 and isoelectric points (pl) between 5.9 and 6.2 (arrows, Fig. 2A) are identical in electrophoretic mobility to proteins extracted from purified yolk platelets and are thus likely to be yolk proteins. The putative yolk proteins are ef- ficiently extracted by Triton X-100 and consequently are virtually absent from the CSK (Fig. 2B). One of the spots present in both SOL and CSK was identified as actin, based on its M, (43 X 103), its pI (5.7) and its affinity for DNase I (Lazarides and Lindberg, 1974) (Fig. 3A). This conclusion was confirmed by treating the detergent-extracted eggs with DNase I, a procedure which is known to depolymerize F actin filaments and extract them from the cytoskeleton (Hitchcock et (II., 1976; Raju et ccl., 1978). The CSK from DNase I-treated cytoskeletons was selectively depleted in the 43 X lo3 M, protein (Fig. 3B). The two other major spots seen in gels of the CSK, representing polypeptides of 48 X 10” M, (PI 5.5) and 54 x l@ IV, (PI 5.5), cannot be identified at this time, although their electrophoretic properties are similar to the vertebrate intermediate filament proteins desmin and vimentin (Jackson et al., 1980). The 54 X lo3 nia, polypeptide was also found to coelute with actin from DNase I-Sepharose columns (Fig. 3A), suggesting that it might be an a&in-associated protein.

The cytoskeletal extraction procedure is carried out at 4°C. Hence, if microtubules comprise part of the cytoskeleton they may not have been detected by our methods since they would be unstable at this temper- ature. Therefore, in order to determine whether the cytoskeleton also contains microtubules or other cold-sen- sitive structures we extracted unfertilized eggs with detergent at 18°C and checked for the presence of the tubulins in 2D gels of the CSK. No new polypeptides were detected in these gels (Fig. 3C). These observations suggest that tubulin is not a major constituent of the CSK and confirm earlier ultrastructural studies which indicated a paucity of microtubules in the Styela egg (Berg and Humphreys, 1960).

We utilized Triton X-100 extracted zygotes at the height of ooplasmic segregation for fine structural analysis of the cytoskeleton since the characteristic cytoplasmic regions were distinct and easily identified at this stage at the light level of microscopy (Fig. 1E). Figure 4 shows TEMs of sections taken through normal and detergent-extracted zygotes. As first shown by Berg and Humphreys (1960) the coloration in the various regions of the Styela egg is due to different types of inclusions embedded in a cytoplasmic ground substance. These include yolk granules of varous sizes in the endoplasm (Fig. 4A) and pigment granules and associated

DEVELOPMENTAL BIOLOGY VOLUME96,1983

FIG. 2. 2D polyacrylamide gel eleetrophoresis of proteins present in the SOL (A) and the CSK (B) of Triton X-lo-extracted S&e&, eggs. Upward pointing arrows indicate positions of the yolk proteins. Downward pointing arrows indicate the position of actin. Equivalent volumes of SOL and CSK were applied to each gel.

JEFFERY AND MEIEB Yellow Crescent Cytoskeleton 131

r IEF

SDS

mitochondria in the myoplasm (Fig. 43). The trans- parency of the ectoplasm, on the other hand, is due to a paucity of large inclusions (Fig. 4B). Despite the removal of 75% of the protein (including most of the major protein species; Fig. 2B) and nearly all the lipid, the detergent-extracted zygotes showed a surprising degree of structural detail so that the cytoskeletal frameworks underlying the three cytoplasmic regions could be readily identified (Figs. 4C-D). As viewed with the TEM, two features of these cytoskeletons are most conspic- uous; large vacant spaces, sites where endoplasmic yolk granules have been extracted leaving fibrous periph- eries (Fig. 4C), and, in agreement with our light mi- croscopic studies, pigment granules preserved in the myoplasmic cytoskeleton (Fig. 4D). The pigment granules are surrounded by well-outlined spaces, once occupied by mitochondria, and a line network of filaments associated with electron-dense spheres which may be ribosomes (Fig. 4E). A thin, densely stained line was also evident along the other boundary of the myoplasmic region of the cytoskeleton (Fig. 4F). This line was specific to the myoplasm; it could not. be found at the boundary of other parts of the zygote cytoskeleton (Fig. 4G). A similar structure, the plasma membrane lamina (PML), has been described at the outer boundary of detergent-extracted mammalian cells (Ben Ze’ev et al., 1979; Sheetz, 1979). It apparently consists of integral membrane proteins stabilized by an underlying network of cytoskeletal elements.

These ultrastructural results suggest that Styela eggs contain characteristic cytoskeletal domains which cor- respond to their colored cytoplasmic regions visible with the light microscope. In the remainder of this report we describe the structure and development of the yellow crescent eytoskeletal domain, a part of the myoplasmic cytoskeleton.

SEM was found to be a suitable means to investigate the yellow crescent PML and its underlying cytoskeletal features since these structures are present at the surface of the egg during early development, As shown in Fig. 5, the PML gradually became visible in the yellow crescent region of zygotes during the course of detergent extra&ion. In completely extracted Styela or Rol-

FIG. 3. The major cytoskeletal protein region of 2D polyacrylamide gels of Triton X-loo-extracted Stgetn eggs. (A) Cytoskeletal proteins which bind to DNase I-Sepharose columns. (B) Cytoskeletal proteins from eggs extracted in the presence of 2 mg/ml DNase 1. (C) Cyto- skeletal proteins from eggs extracted at 18°C. A is stained with Coom- assie blue. B and C are stained with silver nitrate. Upward-pointing arrows designate position of actin. In A, 20 pg of protein was applied to the gel. In B and C, 10 pg of protein was applied to each gel.

132 DEVELOPMENTAL BIOLOGY VOLUME 96,1983

JEFFERY AND MEIER Yellozo Crescent Cytoskeletcm 133

tenia zygotes it consisted of an elaborate network of filamentous structures organized parallel to the plane of the plasma membrane. The network is composed of filaments as small as 5-10 nm in diameter with interstices 25-600 nm across (Figs. 5C, D). Most of the interstices at this stage, however, were small; less than 50 nm across (Fig. 13). Submembrane filamentous networks of similar structure have been previously described in mammalian erythrocytes (Hainfield and Steck, 197’7; Sheetz and Sawyer, 19’78; Tsukita et al.,

1980; Nermut, 1981), hepatocytes (Mesland et al., 1981), and leukocytes (Boyles and Bainton, 1979, 1980).

TEM studies suggested that the PML was restricted to the outer boundary of the yellow crescent region of the cytoskeleton (Figs. 4F-G). This relationship was confirmed by SEM. The PML ended abruptly at the edge of the myoplasmic cytoskeleton, as defined by the extent of the underlying pigment granules (Fig. 6A; also see Fig, 12C in which PML localization is shown during ooplasmic segregation of Boltenia eggs). The intimate relationship between the PML and the myoplasmic cytoskeleton is also well-illustrated during the first cleavage. At this time the advancing cleavage furrow bisects the yellow crescent which is now located in the posterior region of the vegetal hemisphere (Conklin, 1905). As shown in Fig. 6B, the PML covers both the myoplasmic eytoskeleton and the cleavage furrow region.

Cytoskeletal Filaments in the Myoplasnz

A more interior system of cytoskeletal filaments, presumably corresponding to those shown to surround the myoplasmic pigment granules by TEM (Fig. 4E), could be detected under the PML by SEM (Fig. ?A). In contrast to the thin filamentous network of the PML, the myoplasmic cytoskeletal system consisted of a three- dimensional filamentous lattice (Fig. 7B) coursed by bundles of filaments. The lattice appeared to be associated with the PML and to contain embedded pigment granules (Fig. 7B).

In order to obtain an estimate of the diameter of the interior system of filaments, whole mounts were prepared and examined with the TEM. A typical specimen is shown in Fig. 8A. The myoplasmic cytoskeleton can

be identified by its embedded pigment granules. The filaments vary widely in size, exhibiting diameters between 6 and 25 nm (Fig. 8B).

In summary, the yellow crescent region of the zygote appears to contain a cytoskeletal domain composed of a superficial reticulum of filaments in the PML and a deeper lattice of filaments which connects the PML to pigment granules and perhaps other myoplasmic organelles.

Actin in the Yellow Crescent Cytoskeletal Domain

Since the cortical regions of eggs are known to contain microfilaments (Spudich and Spudich, 1979; Wang and Taylor, 1979; Franke et al., 1976; Colombo et al., 1981; Lehtonen and Bradley, 1980) and actin is a major component of the cytoskeleton of ascidian eggs (Fig. 2B), we conducted experiments to determine whether actin filaments were part of the yellow crescent cytoskeletal domain. Two types of analyses were performer fluorescence microscopy of eggs treated with NBD-phallacidin, and TEM and SEM of cytoskeletons prepared in the presence of DNase I. NBD-phallacidin, a specific fluorescent indicator of F actin (Barak et at., 19801, stained the cortical regions of unfertilized eggs and the yellow crescent region of zygotes (Figs. 9A-B).

In a second set of experiments fertilized eggs at the height of ooplasmic segregation were extracted with 0.5% Triton X-100 in CEB supplemented with DNase I or, in controls, with bovine serum albumin. The effect of this treatment on the yellow crescent cytoskeletal domain was determined by TEM and SEM. As shown earlier (Fig. 3B) DNase I selectively extracts actin from the egg cytoskeleton. The myoplasmic pigment granules as well as most of the deeper filaments were present in the DNase I-treated cytoskeletons but the entire filamentous network of the PML was missing (Figs. lOA, C). Control eggs extracted in the presence of bovine serum albumin, did not show this defect (Figs. lOB, D). The NBD-phallacidin staining and DNase I extraction studies suggest that actin is a component of the yellow crescent cytoskeletal domain and that the filamentous network of the PML appears to be the major actin- containing structure.

FIG. 4. TEMs of normal and Triton X-IOO-extracted Styelu zygotes at the height of ooplasmic segregation (A) The endoplasm of a normal zygote, X2700. (B) The borders between endoplasm (EN), ectoplasm (EC), and yellow crescent (YC) of a normal zygote, X2700. (C) Endoplasmic cytoskeleton of an extracted zygote, X2700. (II) The borders between the endoplasmic (EN) and yellow crescent (YC) cytoskeletons of an extracted zygote, ~2700. PG, pigment granule. (E) Higher ma~ni~~ation of cytoskeietal filaments in the yellow crescent region of an extracted zygote show a finely fitamentous network associated with particles that may be ribosomes (arrows), X39,500. (F) Edge of the cytoskeleton in the yellow crescent region of an extracted zygote indicates the presence of the PML (arrows), X3560. (G) Edge of the cytoskeleton (between arrowheads), in the endoplasmic region of an extracted zygote shows the PML is absent, X3560.


FIG. 5. SEMs of the formation and structure of the PML of Triton X-loo-extracted ascidian zygotes. (A) The surface of an unextracted StgeZo zygote. The PML is not visible. (B) The surface of a Styela zygote extracted for 30 min. The PML becomes apparent. (C) The surface of a St~ela zygote extracted for 1 br showing the PML and underlying pigment granules. (D) The surface of a Bolt&o zygote extracted for 1 hr showing the PML and underlying pigment granules. All micrographs X10,000.

U~lasrn~c ~e~eg~t~~ of the Yellow Crescent eta1 hemisphere with the myoplasm after fertilization. Cytoskeletal Domain In order to resolve this issue we examined the cytoskel-

etons of unfertilized eggs and zygotes involved in oo- Several possibilities exist for the origin of the yellow plasmic segregation by SEM. As shown in Fig. llA, the

crescent cytoskeletal domain. It may be polymerized de PML was present over the entire cytoskeletal surface no~o in the cortex after fertilization, as is true of the of the egg. The actin filament network of the PML, how- cortical actin cytoskeleton in fertilized sea urchin eggs ever, appeared to be more dispersed in egg cytoskeletons (Spudich and Spudich, 1979; Wang and Taylor, 1979). compared to their counterparts from zygotes (compare On the other hand, it may already be present in the Figs. 11B and 5C, D). The width of interstices in the cortex of the unfertilized egg, as suggested by NBD- PML of unfertilized eggs and one-cell zygotes which phallacidin staining (Fig. 9A), and segregate to the veg- have completed ooplasmic segregation is compared in

JEFFERV AND MEIER

FIG. 6. SEMs showing the retationship between the PML and the yellow crescent pigment granules in Triton X-loo-extracted 8&a embryos undergoing the first cleavage. (A) The edge of the yellow crescent (YC) cytoskeletal domain. Note the PML over the area of pigment granules and its abrupt termination where the endoplasmic (EN) cytoskeleton begins, X2000. (B) The posterior pole of an extracted two-cell embryo showing the PML over the yellow crescent region which has been bisected by the cleavage furrow, X9500. Ag- gregates at the upper right are extracted test cells which have re- mained associated with the embryonic cytoskeleton.

FIG. 7. SEMs of cytoskeletal filaments associated with yellow crescent pigment granules of Triton X-loo-extracted Styela zygotes. (A) A lesion in the PML in the yellow crescent region reveals an underlying filamentous lattice, X9500. (B) A tear in the PML shows it contains embedded pigment granules and is associated with the underlying filamentous lattice, X10,000.

136 DEVELOPMENTAL BIOLOGY VOI~~ME 96, 1983

FIG. 8. TEM of whole mount spreads of Triton-X-100~extracted Styelu zygotes. (A) A pigment granule embedded in eytoskeletal filaments, X26,400. (B) A portion of the filamentous lattice, Xl60,O~.

Fig. 12. The average size of interstices was reduced two to three times between fertilization and the height of ooplasmic segregation. As ooplasmic segregation pro- ceeds the PML appears to receed, presumably toward the vegetal hemisphere, along with the myoplasmic pigment granules (Fig. 11C). This behavior would be ex- pected if the yellow crescent cytoskeletal domain was originally present throughout the egg cortex and con- tracted to a localized region during ooplasmic segregation. Our results suggest that the yellow crescent cytoskeletal domain is present in the unfertilized egg and probably originates during oogenesis.

Fate of the Yellow Crescent Cytoskeletd Domain

The retention of pigment granules in cytoskeletons prepared from relatively advanced embryos suggests that the yellow crescent cytoskeletal domain may be present in the presumptive muscle and mesenchyme cells. In order to test this possibility we separated 32- cell embryos into ectodermal, endodermal, and myoplasmic blastomeres and extracted them with Triton X- 100. The identity of the dissociated blastomeres can be determined with the light microscope by their color; myoplasm cells are yellow, endoderm cells are white, and ectoderm cells are colorless. As shown in Figs. 13A- B, cytoskeletons from the yellow cells exhibit a PML, which is similarly organized to that of the yellow crescent region of the zygote. Internal filaments associated with the pigment granules could also be distinguished beneath the PML in the detergent-extracted yellow cells (data not shown). In contrast to the yellow cells, the white and transparent cells did not exhibit an extensive PML after detergent extraction (Figs. 13C-D). Ecto- derm cells occasionally contain a few yellow pigment granules (Fig. 13C; Conklin, 1905). Isolated pigment granules in these cells were accompanied by a small patch of PML (Fig. 13C), suggesting that satellite units may be detached from the yellow crescent cytoskeletal domain during early development and be partitioned to cells other than those of the muscle and mesenchyme lineages. Despite the lack of a PML, the white and transparent cells show distinct cytoskeletal features. The surface of detergent extracted ectoderm cells was traversed by long filaments while detergent-extracted endoderm cells exhibited filamentous shells surrounding the sites of vacant spaces once occupied by yolk granules (Fig. 13D). Thus the three cell types charac-

FIG. 9. bight micrographs of NBD-phailicidin staining of Styela eggs and embryos. (A) An unfertilized egg focused at the edge showing peripheral actin staining. (B) A fertilized egg facused over part of the yellow crescent showing actin staining in the crescent region. Both micrographs, X100.

JEFFERY AND MEIER Yellnw Crescent Cgtoskeleton 137

FIG. 10. Ultrastrueture of ~~~en~ zygotes extracted with Triton X-100. (A) SEM of a cytoskeleton prepared in the presence of 2 mg/ml DNase I. The PML is absent, X2000. (B) SEM of a cytoskeleton prepared in the presence of a 2 mg/mi bovine serum albumin, as a parallel control to A. The PMb is present, X2000. (C) TEM of a cytoskeleton prepared in 2 mg/ml DNase I. The PML is absent and the complexity of less superficial portions of the cytoskeleton ia also reduced, X3400. (D) TEM of a cytoskeleton prepared in the presence of 2 mg/ml bovine serum albumin as a parallel control. to C. The PML (arrows) is present, X3400.

teristic of 32-cell embryos show distinct cytoskeletal terized by distinct cytoskeletal organizations. One of organizations and the yellow crescent cytoskeletal do- these regions, the myoplasm or yellow crescent cyto- main persists at least until the 32-cell stage where it plasm (in ~~~e~), has been examined in detail in the is segregated primarily to the presumptive muscle and present study. The yellow crescent cytoplasm is of par- mesenchyme cells. titular embryological interest since it contains mor-

phogenetic determinants responsible for some of the

DISCUSSION features of muscle cell development (Whittaker, 1980). After soluble proteins, lipids, and other cellular com-

The results suggest that cytoplasmic regions of spe- ponents were extracted by Triton X-100, we were able cific morphogenetic fate in ascidian eggs are charac- to demonstrate that the yellow crescent cytoplasm con-

138 DEVELOPMENTAL BIOLOGY VOLUME 96. 1983

tains a cytoskeletal domain consisting of a PML and a more internal lattice of filaments which connect the PML to yellow crescent organelles.

The yellow crescent PML probably consists of integral membrane proteins stabilized by their interaction with an underlying network of actin filaments. Struc- tures of this kind have previously been reported at the outer boundaries of detergent-extracted HeLa cells (Ben Ze’ev et al., 1979), hepatocytes (Mesland et al., 1981), leukocytes (Boyles and Bainton, 19’79, 1981), and red blood cells (Hainfield and Steck, 1977; Sheetz and Saw- yer, 1978; Tsukita et al., 1980; Nermut, 1981). Evidence that the PML of ascidian eggs, like those of the mammalian cells listed above, contains actin filaments stems from two sources. First, the pattern of NBD-phallacidin staining indicates that actin filaments are present in the cortex of unfertilized eggs and cosegregate with the myoplasm after fertilization. Second, the PML disap- pears from cytoskeletons treated with DNase I in order to selectively extract F actin. It is well known that actin filament networks occur in the cortex of eggs (Spudich and Spudich, 1979; Wang and Taylor, 1979; Franke et al., 1976; Colombo et al., 1981; Lehtonen and BradIey, 1980). To our knowledge, however, this is the first report of the ooplasmic segregation and subsequent localization of an actin filament network.

The PML we observe by SEM at the height of ooplasmic segregation in Styeta and Bo~~~~a eggs is probably identical to the electron-dense, submembrane layer previously shown by TEM to be restricted to the vegetal lobe of Cimn eggs (Sawada and Osanai, 1981). The more internal filaments of the yellow crescent cytoskeletal domain, however, have not been previously observed and differ in organization and composition from those which comprise the PML. Unlike the PML network, which is organized in the plane of the plasma membrane, the internal cytoskeleton is a three-dimensional structure consisting of a filamentous lattice traversed by distinct bundles of filaments. This characteristic per- mits the internal filaments to be discerned in thin sections by TEM whereas the PML can only be detected as a thin dense line. Most of the internal filaments are not extracted by DNase I implying that they are composed of substances other than actin. The diameter of

FIG. 11. SEMs of Triton X-loo-extracted ascidian eggs and zygotes involved in ooplasmic segregation. (A) An unfertilized Sty& egg. The yellow crescent pigment granules and the PML are present around the entire surface of the cytoskeleton, X400. (B) Higher magnification of a region from A showing the structure of the PML, X10,000. (C) A ~~~~~~~ff zygote undergoing ooplasmic segregation. The domain containing the pigment granules and PML is elevated over the surface of the cytoskeleton. The presumed direction of pigment migration is indicated by the arrow, X200.

139

40

A

0 1 2 3 4 5 6 7 8 9 10 11 l2 13 14 15 I6 0 1 2 3 4 5 6 7 8 9 10 11 12 13 VI 15

INTERSTICE DIAMETER (nm x 102)

FIG. 12. A comparison of the distribution of interstice lengths in PML networks of unfertilized eggs (A) and fertilized zygotes (B) at the height of ooplasmic segregation. The histograms are derived from 100 measurements made from SEMs of each stage.

the internal filaments suggests they could be intermediate filaments. The 48 X lo3 and 54 X lo” IV, polypeptides, which remain in the CSK after DNase I treatment and show electrophoretic mobilities similar to those of vertebrate vimentin and desmin (Jackson et al., 1980), may be major constituents of the internal filaments.

SEM observations of detergent-extracted eggs undergoing ooplasmic segregation indicate that the yellow crescent cytoskeletal domain moves into the vegetal hemisphere as a unit. Our interpretation of the movement of the PML is supported by previous reports of the behavior during ooplasmic segregation of substances attached to the surface of the egg. Chalk granules (Ortolani, 1955), carmine particles and supernu- merary spermatozoa (Sawada and Osanai, 19811, lectins (Monroy et al., 1973; O’Dell et al., 1974; Ortolani et al., 1977; Zalokar, 1980), and sometimes even the test cells (Conklin, 1905) move from as far as the animal pole region to cap with the myoplasm in the vegetal hemi-

sphere. Presumably, this behavior occurs by virtue of the binding of these substances to PML proteins or their associated components which are exposed on the egg surface. Capping of underlying actin filaments with cell surface ligands is also known to occur in mammalian cells (Albertini and Anderson, 1977; Gabbiani et al., 1977; Toh and Hard, 1977).

Ooplasmic segregation in ascidian eggs has been at- tributed to the contraction of the cortex (Zalokar, 1980). The inhibition of ooplasmic segregation by cytochal- asins has implicated actin filaments in this process (Za- lokar, 19’74; Reverberi, 19’75; Sawada and Osanai, 1981). We propose that the motive force for these cytoplasmic movements is the contraction of the actin network of the PML. A model for the proposed role of the yellow crescent cytoskeletal domain in ooplasmic segregation is presented in Fig. 14. This model has several key elements for which evidence is presented in this paper or in previous works on ooplasmic segregation in ascidian


FIG. 13. SEMs of Triton X-loo-extracted cells from Styela embryos at the 32-cell stage. (A) A yellow cell of the muscle-mesenchyme lineage. Note the presence of numerous pigment granules, X2500. (B) At higher magnification, the yellow cell cytoskeleton shows a PML, X9500. (C) The cytoskeleton of a transparent cell of the ectoplasmic lineage. The PML is not present over most of the cytoskeleton. Note the presence of a pigment granule (PG) with a small patch of PML (arrow) nearby, X10,000. (D) The cytoskeleton of a white cell of the endoplasmic lineage. The PML is not evident and large vacant spaces indicate places where yolk spheres were extracted by the detergent. Filaments (arrowheads) surround the spaces where yolk spheres were located, X4500.

eggs. First, the internal cytoskeletal filaments connect the PML actin network to the yellow crescent pigment granules and probably to other organelles located in this cytoplasmic region. Our ultrastructural observations of the yellow crescent cytoskeletal domain of detergent extracted eggs provide direct evidence for these connections. Second, the PML appears to shorten and pull the pigment granules along during ooplasmic segregation. Whether the shortening is due to an active, mechanoehemical process requiring metabolic energy or recoil of a previously stretched elastic system is an

unanswered and important question raised by this study. Finally, the PML contracts uniformly toward a single point on the egg surface, presumably the point of sperm entry (Conklin, 1905). As pointed out previously (Sa- wada and Osanai, 1981), this assumption is necessary to account for the movement of exogenous substances bound to the egg surface over great distances during ooplasmic segregation.

Altogether, these three elements of our model offer an explanation for the cytoplasmic movements which occur during ooplasmic segregation (Fig. 14). The initial

JEFFE~Y AND MEIER Yellow Crescext Cytoskeleton

0 c D E

AP

141

I VP

FIG. 14. Diagrammatic representation of the behavior of the yellow crescent cytoskeletal domain during ooplasmic segregation. {A) Un- fertilized egg. Arrow indicates the future focal point of PML contraction. (B-D) Fertilized eggs in the process of ooplasmic segregation. Arrows with continuous lines represent the direction of myoplasmic movement generated by PML contraction. Arrows with broken lines represent the direction of endoplasmic movement. (E) Zygote after completion of ooplasmic segregation. The thin egg boundaries represent membrane without the PML. The thick egg boundaries represent membrane with the PML. Structures at.tached to the inside of the PML represent the interior filamentous lattice and attached myoplasmic organelles in the yellow crescent region, AP, animal pole; VP, vegetal pole.

event of this sequence would be a contraction of the PML toward a single point on the egg surface. The focal point of this contraction may be specified by a sperm- mediated, local increase in Ca+2, a situation recently shown to polarize the formation of myoplasmic caps in ionophore A2318’7-activated ascidian eggs (Jeffery, 1982). The contraction of the PML would provide for the movement of the internal filamentous lattice of the yellow crescent and its associated organelles into the vegetal hemisphere as well as contribute to the force that dis- places the endoplasm into the animal hemisphere. The latter, coupled with a possible weakening of that portion of the plasma membrane which would now lack the PML, could lead to the bulging of the animal hemisphere often seen during the early stages of ooplasmic segregation (Sawada and Osanai, 1981). Continued shortening of the PML and endoplasmic bulging at the opposite side of the cell would ultimately cause a myoplasmic lobe to occur in the vegetal hemisphere of the egg.

Our model does not account for the movement of the ectoplasm which streams into the vegetal hemisphere in the wake of the myoplasm. It is possible that this movement could also be linked to that caused by the PML if physical connections existed between the ectoplasmic and myoplasmic cytoskeletal domains. More de- tailed studies of the structure of the ectoplasmic cytoskeleton will be necessary to establish whether such connections exist.

Our studies indicate that, aside from its role in ooplasmic segregation, the yellow crescent cytoskeletal domain is also likely to be involved in subsequent embryogenesis. This is evidenced by the maintenance of its major constituents, the PML and the internal filaments, in the developing embryo after the conclusion

of ooplasmie segregation and their eventual partition- ing to the presumptive muscle and mesenchyme cells. The yellow crescent cytoskeleton probably serves to an- chor the myoplasmi~ constit,uents in a position which will insure their entry into the correct. blastomeres during development. The muscle cell determinants may be among these constituents. Although there is no evidence for a eytoskeletal association of these determinants at this time, low-speed centrifugation experiments suggest that determinants are not easily dis- placed from the myoplasm (Conklin, 1931). In future experiments it will be important to determine whether a relationship exists between muscle cell development and centrifugal forces which disrupt the integrity of the yellow crescent cytoskeletal domain.

We are grateful to Ms. Lynn Hunter, Ms. Priscilla Kemp, and Mr. Christopher Drake for technical assistance. Mr. Richard Brodeur prepared the DNase I-Sepharose, Mr. Bob Riess performed some of the TEM, Dr. Mary Ann Rankin provided the carbohydrate and lipid analyses, and Ms. Bonnie Brodeur executed the line drawings. We are also grateful to J. Render, J. Henry, and R. Moon who collected Bol- teniu vi&so. This research was supported by grants from the NIH (I’D-~3970 to W.R.J. and DE-05616 to S.M.), the NSF (PCM-791472 to S.M.), and the Muscular Dystrophy Association (W.R.J.). Part of this work was done when W.R.J. was an instructor in the Embryology course at the Marine Biological Laboratory, Woods Hole (NIH Train- ing Grant HD-0’7098).

REFERENCES

ALBERTINI, D. I?., and ANDERSON, E. (1977). Microtubule and micro- filament rearrangements during capping of concanavalin A reeep- tors on cultured ovarian granulosa cells. f. Cell Biol. 73, 111-127.

BARAK, L. S., YOCUM, R. R., NOTHNAGEL, E. A., and WEBB, W. W. (1980). Fluorescence staining of the actin cytoskeleton in living cells with ~-nitrobenz-Z-oxa-1,3-diazole phallaeidin. Proc. Na,t. Acud Sci USA 77, 980-984.

142 DEVELOPMENTAL BIOL~

BATIEN, B. E., AALBF,RG, J. J., and ANDERSON, E. (1980). The eytoplasmic filamentous network in cultured ovarian granulosa cells. Cell 21, 885-895.

BEN-ZE’EV, A., DUERR, A., SOLOMON, F., and PENMAN, S. (1979). The outer boundary of the cytoskeleton: A lamina derived from plasma membrane proteins. Cell 17, 859-865.

BERG, W. E., and HUMPHREYS, W. J. (1960). Electron microscopy of four-cell stages of the ascidians Ciona and Styeb Dev. Biol 2, 4% 60.

BOYLES, J., and BAINTON, D. F. (1979). Changing patterns of plasma membrane-associated filaments during the initial phases of poly- morphonuclear leukocyte adherence. J. Cell Biol. 82, 347-368.

BOYLES, J., and BAINTON, D. F. (1981). Changes in plasma-membrane- associated filaments during endoeytosis and exoeytosis in poly- morphonuclear leukocytes. Cell 24,905-914.

BROWN, S., LEVINSON, W., and SP’IJUICH, J. A. (1976). Cytoskeletal elements of chick embryo fibroblasts revealed by detergent extraction. J. ?suprumol strakct. 5, 119-130.

BUCKLEY, I. K., and RAJU, T. R. (1976). Form and distribution of actin and myosin in non-muscle cells: A study using chick embryo fibroblasts. J. Microsc. 107, 129-149.

CERVERA, M., DREYFUSS, G., and PENMAN, S. (1981). Messenger RNA is translated when associated with the cytoskeletat framework in normal and VSV-infected HeLa cells. &El 23, 113-120.

CLEMENT, A. C. (1968). Development of the vegetal half of the Ily- anassa egg after removal of most of the yolk by centrifugal force, compared with the development of animal halves of similar visible composition. Dw. Biol. 17, 165-186.

COLOMBO, R., BENEDUSI, P., and VALLE, G. (1981). Actin in Xenow development: Indirect immunofluorescence study of actin loealiza- tion. Difl~entiation 20, 45-51.

CONKLIN, E. G. (1905). The organization and cell-lineage of the ascidian egg. J. Aend. Not, Sci. Phila. 13, l-119.

CONKLIN, E. G. (1931). The development of centrifuged eggs of ascidians. J. E;cn. 2001 60, l-119.

DAVIDSON, E. (1976). “Gene Activity in Early Development,.” Aca- demic Press, New York.

DOHMEN, R., and VERDONK, N. H. (1979). The ultrastructure and role of the polar lobe in development of molluscs. In “Determinants of Spatial Organization” (S. Subtelny and I. R. Konigsberg. eds.), pp. 3-17. Academic Press, New York.

ECKERT, B. S., KOONS, S. J., SCHANTZ, A. W., and ZOBEL, C. R. (1980). Association of creatine phosphokinase with the cytoskeleton of cultured mammalian cells. J. Cell Biol. 86, 1-5.

FRANKE, W. W., RATHKE, P. C., SEIH, E., TRENDELENBERG, M. F., Os- BORN, M., and WEBEN, K. (1976). Distribution and mode of arrangement of microfilamentous structures and actin in the cortex of the amphibian oocyte. Cytobiology 14, 111-130.

G~BBIANI, G., CHAPONNIER, G., ZUMBE, A., and VASSALLI, P, (1977). Attin and tubulin co-cap with surface immunoglobulins in mouse B lymphocytes. NatlLre [Londoni 269, 697-698.

HAINFIELD, J. F., and STECK, T. L. (19’77). The sub-membrane reticulum of the human erythrocyte: A scanning electron microscope study. J. Supamol. Stmtct. 6,301-311.

HEUSER, J, E., and KIRSCHNER, M. W. (1980). Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. J. Cell Biol. 86, 212-234.

HITCHCOCK, S. E., CARLSSON, L.. and LINDBERG, U. (1976). Depoly- merization of F-actin by deoxyribonuclease 1. Cell 7, 531-542.

JACKSON, B. W., GRUND, C., SCHMID, E., BURKI, K., FRANKE, W. W., and ILLMENSEE, K. (1980). Formation of cytoskeletal elements during mouse embryogenesis. Intermediate filaments of the cytoker- atin type and desmosomes in preimplantation mouse embryos. oif

~~~~tiat~~7a 7, 161-179.

JEFFERY, W. R., and BRAWERMAN, G. (1974). Characterization of the

3GY VOLUME 96, 1983

steady state population of messenger RNA and its poIy(adenylie acid) segment in mammalian cells. Biochemistry 13, 4633-4637.

JEFFERY, W. R., and CA~CO, D. G. (1978). Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the aseidian St&a. Ilev BioL 67,152-166.

JEFFERY, W. R. (1982). Calcium ionophore polarizes ooplasmic segregation in ascidian eggs. Science 216, 545-547.

LAZARIDES, E., and LINDBERG, U. (19’74). Actin is the naturally oe- curring inhibitor of deoxyribonuclease I. Proc. Nat. Acad Sci. USA X4742-4746.

LAUFER, J. S., and VON EHRENSTEIN, G. (1980). Nematode development after removal of egg cytoplasm: Absence of localized unbound determinants. Science 211,402-405.

LEHTO~EN, E., and BADLEY, R. A. (1980). Localization of eytosketetal proteins in preimplantation mouse embryos. J. Embryo1 Exp. Mar- phoL 55, 211-225.

LENK, R., RANSOM, L., KAIJFMANN, Y., and PENMAN, S. (1977). A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. C&l 10, 67-78.

LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. BioL Chem. 193, 265-275.

MESLAND, D. A. M., SPIEL&, H., and ROOS, E. (1981). Membrane-associated cytoskeleton and coated vesicles in cultured hepatocytes visualized by dry-cleaving. Eq. Cell Res. 132, 169-184.

MONROY, A., ORTOLANI, G., O’DELL, D. S., and MILLONIG, G. (1973). Binding of concanavalin A to the surface of unfertilized and fertilized ascidian eggs. Nature ~~~) 242,409-410.

MORGAN, T. H. (1935). Centrifuging the egg of Ilyanmsa in reverse. BioL Bull. 68, 268-279.

NERMUT, M. V. (1981). Visualization of the “membrane skeleton” in human erythroeytes by freeze etching. Eur. J. Cell BioL 25, 265- 271.

O’DELL, D. S., ORTOLANI, G., and MONROY, A. (1974). Increased binding of concanavalin A during maturation of ascidian eggs. Exp. Cell Res. 83, 408-411.

O’FARRELL, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J: BioZ. Chem. 250, 4007-4021.

ORTOLANI, G. (1955). I movementi corticali dell uovo di Ascidie alla fecondaziona. Riv. Bid 47, 169-181.

ORTOLANI, G., O’DELL, D. S., and MONROY, A. (1977). Localized binding of Dolichos lectin to the early Ascidia embryo. Exp. Cell. Res. 106,

402-404. &BORN, M., BORN, T., KOITZSCH, H., and WEBER, K. (1978). Stereo

immunofluoreseence microscopy: Three dimensional arrangement of microfilaments, microtubules, and tonofilaments. CeU 14, 4?7- 488.

RAJU, T. R., STEWART, M., and BUCKLEY, I. K. (1978). Selective extraction of cytoplasmic actin-containing filaments with DNA-ase I. cyt~o~gy 17,307-311.

REVERBERI, G. (1975). On some effects of cytochalasin B on the eggs and tadpoles of ascidians. Acta EndnyoL Ezp. 2, 137-158.

SAWADA, T., and OSANAI, K. (1981). The cortical contraction related to ooplasmic segregation in Ciona intesfinalti eggs. Wilhelm Roux’s

Arch. Dev. B&L 190,208-214. SHEETZ, M. P. (1979). Integral membrane protein interaction with

Triton cytoskeletons of erythrocytes. B&him. Biophys. Acta 557, 122-134.

SHEETZ, M. P., and SAWYER, D. (1978). Triton shells of intact erythrocytes. J. Supra. Mot Struck. 8, 399-412.

SCHLIWA, M., VAN BLERKOM, J., and PORTER, K. R. (1981). Stabilization of the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition. Proc. Nat. Acad Ski, USA 78, 4323-4333.

SMALL, J. V., and CELIS, J. E. (1978). Direct visualization of the lo-

JEFFERY AND MEIER Yellow Crescent Cytoskeleton 143

nm (lOOA)-filament network in whole and enucleated cultured cells. tunicate Styela plicata by variations in a natural light regime. J. J. Cell Sci. 31, 393-409. Exp. ZooL 195, 265-2’70.

SPUDICH, A., and SPUDICH, J. A. (1979). Actin in Triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs. J. Cell Biol. 82, 212-226.

TOH, B. H., and HARD, G. C. (1977). Actin co-caps with convanavalin A receptors. Nature (London) 269, 695-696.

TSUKITA, S., TSUKITA, S., and ISHIKAWA, H. (1980). Cytoskeletal network underlying the human erythrocyte membrane. Thin-section electron microscopy. J. Cell Biol. 85, 567-576.

VAN HANDEL, J. (1965). Microseparation of glycogen, sugars, and lipids. Anal Biochem. 11, 266-271.

WHITTAKER, J. R. (1980). Acetylcholinesterase development in extra cells caused by changing the distribution of myoplasm in ascidian embryos. J. EmbyoL Exp. McwphoL 55, 343-354.

WILSON, E. B. (1925). “The Cell in Development and Heredity,” 3rd ed. Macmillan, New York.

WOLOSEWICK, J. J., and PORTER, K. R. (1979). Stereo high-voltage electron microscopy of whole cells of human diploid line, WI-38. Amer. J. Anat. 147, 303-324.

VAN VENROOIJ, W. J., SILLEKENS, P. T. G., VAN EEKELEN, C. A. G., and REINDERS, R. J. (1981). On the association of mRNA with the cytoskeleton in uninfected and adenovirus-infected human KB cells. Exp. Cell Res. 135, 79-91.

WRAY, W., BOULIKAS, T., WRAY, V. P., and HANCOCK, R. (1981). Silver staining of proteins in polyacrylamide gels. Anal. B&hem. 118,197- 203.

ZALOKAR, M. (1974). Effect of colchicine and cytochalasin B on ooplasmic segregation of ascidian eggs. Wilhelm Roux’s Arch. Dev. BioL 175, 243-248.

VERDONK, N. H. (1968). The effect of removing the polar lobe in centrifuged eggs of Dent&urn. J. Embryo1 Exp. MorphoL 19, 33-42.

WANG, Y., and TAYLOR, D. L. (1979). Distribution of fluorescently labeled actin in living sea urchin eggs during early development. J Cell Biol 82, 672-679.

ZALOKAR, M. (1980). Activation of ascidian eggs with lectins. Dew. BioL 79, 232-237.

WEST, A. B., and LAMBERT, C. C. (1975). Control of spawning in the

ZBLLNER, N., and KIRSCH, K. (1962). Uber die quantitative Bestim- mung von Lipoiden (Mikromethode) mettels der vielen natrulichen Lipoiden (allen bekannten plasmalipoiden) gemeinsamen Sulpho- phosphovanillin-Reaktion. 2. Gesamte Exp. Med 135, 545-561.

A yellow crescent cytoskeletal domain in ascidian eggs and its role in early development

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