The apical lamina of the sea urchin embryo: Major glycoproteins associated with the hyaline layer

DEVELOPMENTAL

BIOLOGY

The Apical

168-178 (1982)

89,

Lamina of the Sea Urchin Embryo: Major Glycoproteins Associated with the Hyaline Layer H. GLENN HALL’

Marine Biology Research Division, A-002, Scripps Institution

of

AND VICTOR

D. VACQUIER

Oceanography, University of California-San

Diego, Lo Jolla, California 92098

Received April IS, 1981; accepted in revised form July 21, 1981 The hyaline layer (HL) surrounding the sea urchin blastula appears to dissolve in 1 M glycine. However, after this treatment, there persists over the surfaces of the blastomeres a layer of material, referred to here as the apical lamina (AL), that sloughs off as an adhesive convoluted bag upon gradual dissociation of the embryo. Isolated hyaline layers, referred to as HL-AL complexes, were analyzed by urea-SDS-polyacrylamide gel electrophoresis. A major protein of the HL-AL complex, hyalin, bands or precipitates in the stacking gel. Two other major proteins, both strongly PAS positive, migrate with apparent molecular weights of 175K and 145K daltons. As with intact embryos, the glycine wash removes the hyalin protein from the isolated HL-AL complex, leaving the undissolved AL which consists primarily of the 1’75K- and 145K-dalton proteins. The embryo’s own perivitelline-localized cortical granule peroxidase heavily radioiodinates the proteins of the HL-AL complex, further verifying their apical, extracellular location. Unlike hyalin, the AL proteins do not precipitate with calcium ions. Compared to the entire HL-AL complex, the AL contains a greater percentage of carbohydrate. No sialic acid is associated with the HL-AL complex, but the AL contains some sulfate. In contrast to a published report based on ultrastructural staining, no biochemical evidence was found in this study for the presence of collagen or significant glycosaminoglycan within the HL-AL complex. No developmental differences were observed in AL proteins from 1-hr-old embryos compared to those from blastulae. However, there is evidence suggesting heterogeneity and developmental differences in hyalin. The possible organization of hyalin and the AL proteins into separate layers surrounding the embryo is discussed. The influence of the AL proteins in morphogenesis and cell adhesion is considered, and hypothetical roles attributed to the HL and hyalin are critically questioned. INTRODUCTION

Stephens and Kane, 1970; Kane, 1970; Citkowitz, 1971, 1972). Ultrastructural studies, however, have revealed that the HL is not a single homogeneous layer, and that visibly distinct components are segregated as separate layers within the HL (Wolpert and Mercer, 1963; Lundgren, 1973; Spiegel and Spiegel, 1979). This paper presents biochemical evidence for the existence of major glycoproteins, distinct from hyalin, which persist as a fibrous layer surrounding the embryo after removal of hyalin. This remaining layer is designated the apical lamina (AL). Hyalin and the AL proteins may together form a single homogeneous layer in the intact embryo. However, it is equally plausible that a layer predominantly composed of the AL proteins is distinct from a layer predominantly composed of hyalin, and the two layers correspond to the different regions of the HL seen by electron microscopy. In this paper the term hyaline layer (HL) is used in its traditional sense, referring to the entire layer surrounding the embryo visible by microscopy. Hyaline layer is also used when referring to the structure isolated and described as such by other investigators. The hyalin-apical lamina complex (HL-AL complex) is the isolated structure characterized in this study, that ideally corresponds to the entire visible HL.

Upon fertilization of the sea urchin egg, lamellar material is released from the cortical granules and becomes incorporated into the fertilization envelope. Separate contents of the cortical granules, the extralamellar bodies, remain associated with the egg plasma membrane, spread and flow together to form the hyaline layer (HL) over the egg surface (Endo, 1961; Runnstrom, 1966). As the fertilized egg undergoes cleavage, the HL serves as a rigid spherical enclosure that is required to keep the dividing blastomeres together (Herbs& 1900). As the epithelium of the blastula wall is established, the cells remain tightly attached to the HL through microvillar processes extending from their apical surfaces (Dan, 1960). It has been proposed that this tight cellular adhesion to the HL plays a role in directing morphogenetic events of the embryo (Gustafson and Wolpert, 1967). From the HL a single major protein component, known as hyalin, has been biochemically characterized (Yazaki, 1968; Vacquier, 1969; Kane and Stephens, 1969; 1 Current address to which reprint requests should be sent: Laboratory of Cell Biology (83), Division of Biology and Medicine, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720.

0012-1606/82/010168-11$02.00/O Copyright All rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

168

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AND VACQUIER

Apical Laminu and Hyaline Layer Glycqwroteins

MATERIALS ANDMETHODS Sea urchin gametes, Strongylocentrotus pwpuratus, were collected, fertilized, and allowed to develop as previously described (Hall, 1978). Hyalin-apical lamina complexes (HL-AL) were isolated from 1-hr-old embryos and from swimming, hatched blastulae (16 hr). Isolations from hour-old embryos were preceded by treatment of the unfertilized egg with 10 mM dithiothreitol (DTT) in Millipore-filtered seawater (FSW), pH 9.2, for 10 min to prevent fertilization envelope formation (Epel et al., 1970). HL-AL complexes were isolated from a 10% or less suspension of embryos in icecold FSW containing 1% Triton X-100. The suspension was passed two times through a Yeda Press (Yeda Research and Development Co., Rehovot, Israel) under 1200 psi of nitrogen. HL-AL complexes were lightly pelleted using a clinical or preferably a hand-cranked centrifuge at about 180~ for 1 min. After removing clumps of HL-AL complexes floating due to trapped air bubbles, the supernatant was removed and discarded. The complexes from the top together with those comprising the pellet were resuspended by vigorous pipetting in a small volume of the FSW Triton X-100 solution and diluted further to about 50 pellet vol. This washing step was repeated three times. The preparation was examined by phase-contrast microscopy for visible contamination. To isolate the apical lamina (AL), HL-AL complexes were gently suspended in 20 to 30 vol of ice-cold 1.1 M glycine, 2 m&f EGTA, adjusted to pH 8.0 with NaOH, and allowed to stand for 30 min on ice, with periodic gentle mixing. The undissolved AL were pelleted at 3000g for 20 min (4°C). The glycine wash was dialyzed against distilled water, frozen, concentrated by lyophilization, and redissolved in cold electrophoresis sample solution: 8 M urea, 10 mlM triethanolamine-HCl, 0.1% SDS, 2 mMEDTA, 0.1% 2-mercaptoethanol, and 0.001% bromophenol blue, pH 7.2. Samples of AL and HL-AL complex were suspended and dialyzed directly against the sample solution. Hyalin protein was recovered as the calcium precipitate from a glycine wash of embryos using practically the same method described by Kane (1973). Hatched blastulae were gently suspended in the glycine solution described in the previous paragraph for 10 min without dissociation. The embryos were removed by hand centrifugation, and the wash recentrifuged at 25,000g for 30 min to remove possibly remaining single cells and cell fragments. The supernatant wash was dialyzed against several changes of distilled water, brought to 20 mM CaClz, 20 mM Tris-HCl, pH 8.0, and allowed to stand on ice for several hours. The calcium precipitate was collected by centrifugation at 1OOOgfor 5 min, dis-

169

solved by dialysis against glycine solution, and dialyzed against distilled water. The calcium precipitation and subsequent dialysis were repeated. As previously described (Hall, 1978), the perivitellinelocalized proteins were radioiodinated in situ by the embryos’ released cortical granule peroxidase with added radioactive iodide and a hydrogen peroxide-generating system. The proteins from embryos at two stages, 1 hr after fertilization and 1 hr prior to hatching, were labeled. Portions of the embryos labeled at each stage were allowed to hatch, collected by hand centrifugation, kept gently suspended in glycine solution for 30 min, and were either allowed to settle before dissociation or dissociated by gentle pipetting. Cells were pelleted from the dissociated sample at SOOgfor 4 min. The glycine wash was recentrifuged at 25,000~ for 20 min, brought to 10% TCA, dialyzed against water, concentrated, and dissolved in electrophoresis solution as described above. Samples of the labeled embryos and cells were homogenized (Teflon-glass) in 1 ml of 10 mM Tris-HCl, 2 mM EDTA, 2 mM EGTA, 1% 2-mercaptoethanol, pH 8.0, then brought to 10% TCA, and dissolved by dialysis against electrophoresis solution. Samples were analyzed by polyacrylamide slab gel electrophoresis in the presence of 8 M urea and 0.1% sodium dodecyl sulfate using the anionic glycine system described by Wu and Bruening (1971). The amounts of acrylamide:bis-acrylamide were 7%:0.19% in the resolving gel and 5%:0.29% in the stacking gel. Electrophoresis was run at 25 mA per gel until the tracking dye migrated 9 cm. Gels were fixed and stained in 0.08% Coomassie brilliant blue R-250, 25% methanol, 7.5% acetic acid, and destained in 25% methanol, 7.5% acetic acid, or the gels were stained for carbohydrate by the periodic acid-Schiff reaction according to Fairbanks et al. (1971). The destained gels were soaked overnight in 2% glycerol, 5% acetic acid, and while submersed, sandwiched between two soaked dialysis sheets (Hoefer, San Francisco, Calif.), clamped to a plate of glass with plastic strips around the periphery, and allowed to air-dry 2 to 3 days. For autoradiography, dried gels were clamped against sheets of Kodak X-Omat film between two plates of glass, kept in the dark for 2 to 4 weeks, and developed with Diafine (Acufine, Chicago, Ill.). Human erythrocyte ghosts were prepared according to Fairbanks et al. (1971) and used as a source of molecular weight standards. Blastulae washed in glycine solution (to remove the HL and to expose the underlying AL) were prepared for scanning electron microscopy. After suspension in 1.1 Mglycine, NaOH to pH 8.0, blastulae were allowed to settle onto a submersed piece of positively charged plastic membrane (derivatized with quarternary am-

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monium groups, Type AR 103, Ionics Inc., Watertown, Maine, as a gift from Mr. Bob Gentry, F & F Industries, La Jolla, Calif.). The solutions were gently removed and replaced in the following order keeping the membrane with attached embryos submersed: briefly in wash solution-0.55M NaCl, 50 mM boric acid with NaOH added to pH 8.5; overnight in wash solution with 2.5% glutaraldehyde; briefly again in wash solution; 1 hr in wash solution with 1% 0~0,; distilled water; a graded series of increasing ethanol; and Freon 113. The plastic membranes with attached embryos were critical-point dried in Freon 13, mounted on aluminum stubs with conductive glue, coated with about 200 A of a 6:4 goldpalladium mixture in a Technics Hummer II evaporator, and examined with a Cambridge S4 scanning electron microscope. Embryos would become detached from the plastic membrane if allowed to stay too long in glycine solution or wash solution before fixation. Samples of isolated HL-AL complexes were tested for susceptibility to digestion by purified bacterial collagenase (Type CLSPA, Worthington Diagnostics, Freehold, N. J.) HL-AL complexes were washed 5X with FSW to remove the Triton X-100 and suspended (about 0.25 mg protein) in 1 ml of 25 mM Tris-HCl, 10 mM calcium acetate, pH 7.4. About 50 units (125 pg) of dissolved collagenase (1 unit defined as the amount that releases 1 hmole of amino acids from undenatured tendon collagen within 5 hr at 37°C) was added, and the sample was incubated at 37°C for 24 hr. To 0.2 ml of the digestion mixture was added 25 ~1 of concentrated sulfuric acid diluted 1:20, and the precipitate removed by centrifugation. The supernatant was tested for reactivity with 0.1 ml ninhydrin reagent according to Schiffman et al. (1964). Controls included samples of HL-AL complexes incubated without enzyme, but with the enzyme added just prior to acid addition. To verify enzyme activity, 0.5 mg of tendon collagen, and collagen plus HL-AL complexes, were incubated with and without collagenase. The HL-AL collagenase reaction mixtures were concentrated by lyophilization, dissolved by dialysis against electrophoresis sample solution, and analyzed by electrophoresis. Analyses were performed on suspensions of samples after extensive dialysis against ice-cold distilled water. Protein determinations were according to Lowry et al. (1951) using bovine serum albumin as a standard. Nitrogen was assayed by the ninhydrin procedure according to Schiffman et al. (1964) using ammonium sulfate as a standard. Carbohydrate was determined, using galactose as a standard, by the phenol-sulfuric method of DuBois et al. as outlined by Ashwell (1966), scaled down to 0.2~ the volume and warming the samples to 90°C before adding the sulfuric acid. Uranic acid was

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tested for according to Blumenkrantz and Asboe-Hansen (1973) using galacturonic acid as a standard. Sialic acid was assayed by both the thiobarbituric acid method of Warren and the resorcinol procedure of Svennerholm as outlined by Spiro (1966) using N-acetylneuraminic acid as a standard. Sulfate was determined according to Terho and Hartiala (1971) using sodium sulfate as a standard. The precipitates of protein formed under the acidic conditions of hydrolysis preceding some of the assays were removed by centrifugation after hydrolysis before continuing with the reactions. Hydroxyproline was tested for by the method of Blumenkrantz and Asboe-Hansen (1975) after hydrolyzing samples in 6 N HCl, in the presence of 1 mg phenol/ml at 110°C under vacuum for 24 hr. RESULTS

Hatched sea urchin blastulae suspended in a solution of 1.1 M glycine and examined by phase-contrast microscopy appear to have lost the hyaline layer (HL) that surrounds the exterior of the embryo (Kane, 1973). As the cells round up, active ciliated movements assist in their dissociation. Occasionally, a thin web of persisting extracellular material can be seen stretching across the apical ends of the cells. Visualized more clearly by scanning electron microscopy, this material, referred to here as the apical lamina (AL), appears as a fibrous network (Fig. 1). If the embryos are kept for several hours in glycine solution without agitation and allowed to slowly dissociate by their own movements, the AL sloughs off as a very adhesive convoluted bag. The detached AL is visible under phase-contrast microscopy but is only slightly darker than the background. The AL stick together as aggregates which can be collected by slow centrifugation of “self-dissociated” embryos resuspended in glycine solution containing 1% Triton X-100. Dissociation of blastulae by mechanical assistance, for example, stirring, homogenizing, or pipetting, shreds the AL so that it cannot be recovered as aggregates. One-cell-stage embryos lyse within an hour in glycine solution, before which time no AL is seen to slough off, although there is biochemical evidence for the presence of an AL (described below). The AL is so adhesive that its isolation proved difficult. Within centrifuged AL aggregates were many trapped contaminants, especially nuclei, basal laminae, and cilia. Furthermore, the material was quickly lost by sticking to tube surfaces. Attempts to obtain cleaner AL preparations through sucrose density sedimentation were only partly successful, but the preparations were adequate to identify the most abundant components by electrophoresis. A more successful preparation of the AL was accomplished through the isolation of

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FIN3. 1. Scanning electron micrograph of a sea urchin blastula after being washed in glycine solution. The HL has been dissolved and the cells partly dissociated. The remaining AL is seen as a fibrous network covering the cells.

a HL-AL complex. For the isolation, a Yeda press was used to disrupt the embryos. Low pressures, less than 1000 psi, just sufficient to break apart the embryos, left HL-AL complexes as intact spheres, but which enclosed visible contaminating debris. High pressures ripped apart the layer complex, releasing the enclosed contaminants. However, the fragmented HL-AL complexes tended to be more sticky, perhaps as a result of exposing the internal adhesive AL, and would entrap contaminants upon aggregation. A pressure that compromised these effects, about 1200 psi, was selected for the isolation procedure. HL-AL complexes were suspended in a glycine wash, solubilizing much of the HL-AL complex material but leaving behind the delicate insoluble AL, recoverable as a low-speed pellet. The AL material remaining did not tend to aggregate as readily as did AL remaining after glycine dissociation of embryos. After a satisfactory isolation procedure was established, the protein components of HL-AL complexes and AL from hour-old embryos and from hatched blastulae were analyzed by SDS-urea-polyacrylamide gel electrophoresis (Fig. 2). The HL-AL complex from both developmental stages exhibits Coomassie blue-staining material that precipitates as a light diffuse band within

the stacking gel, extending as a smear to the top of and slightly penetrating the resolving gel. This material is probably the very acidic protein hyalin, a major component of the HL (Stephens and Kane, 1970). Hyalin isolated by established methods precipitates and smears on the gel tops in the same manner (Fig. 3), behavior characteristic of this protein in the presence of SDS (R. E. Stephens, personal communication). The HL-AL complex also contains two major diffuse protein bands with apparent molecular weights of 175K and 145K daltons and a minor band at 1lOK daltons (Fig. 2), all three of which stain intensely by the periodic acid-Schiff reaction. In some preparations, there is an additional band between the two larger proteins. Inclusion of urea in the samples and gels enhances solubilization of the complexes and sharpening of the bands. In comparison to the entire HL-AL complex, AL samples contain primarily the 175K-, 145K-, and llOK-dalton proteins and very little hyalin. No differences are observed in the AL proteins from the two developmental stages, except that the llOK-dalton band is more diffuse from the later stage. Upon washing the HL-AL complexes with glycine solution, some fragmentation of the AL probably occurs because all of the AL protein is not re-

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I I 12 I3 I4 I5

16 17

-d (175K) (145K) (I IOK)

FIG. 2. Photograph of an SDS-urea-polyacrylamide gel. Left half of gel, Lanes 1-9, Coomassie blue-stained proteins; right half of gel, Lanes 10-1’7, autoradiograph of proteins radioiodinated by the cortical granule peroxidase. (1) Human erythrocyte ghosts as molecular weight standards. (2) and (3) HL-AL complexes from I-hr-old embryos. (4) and (5) AL from I-hr-old embryos. (6) and (7) HL-AL complexes from hatched blastulae. (8) and (9) AL from hatched blastulae. (10) Proteins radioiodinated in embryos 1 hr after fertilization and prepared for electrophoresis immediately afterwards. (11) Embryos labeled as sample in Lane 10, except allowed to develop and hatch before preparing for electrophoresis. (12) Cellular pellet after glycine solution dissociation of hatched blastulae from sample in Lane 11. (13) Glycine wash in which the cells in Lane 12 were dissociated and removed. (14) Proteins radioiodinated in embryos 1 hr prior to hatching and prepared for electrophoresis immediately afterwards. (15) Embryos labeled as sample in Lane 14, except allowed to develop and hatch before preparing for electrophoresis. (16) Embryo pellet after gentle glycine wash of hatched blastulae from sample in Lane 15. (17) Glycine wash in which the embryos in Lane 16 were suspended and removed.

covered in the low-speed pellet. Samples of the glycine wash analyzed by electrophoresis contain, along with the dissolved hyalin, some AL protein. The existence and location of the AL proteins is also revealed through their radioiodination by the sea urchin embryo’s endogenous cortical granule peroxidase in the presence of radioactive iodide. Upon fertilization, a peroxidase released from the egg’s cortical granules is responsible for hardening the fertilization envelope by crosslinking tyrosyl residues (Foerder and Shapiro, 19’79; Hall, 1978). In the presence of radioactive iodide and an HzOz-generating system, the peroxidase heavily labels the fertilization envelope and components within

the perivitelline space (Hall, 1978). To make a direct comparison, the labeled proteins were electrophoretitally separated on the same gel alongside samples of the isolated HL-AL complexes, the right half of the gel shown as an autoradiograph (Fig. 2). Embryos labeled 1 hr after fertilization and 1 hr before hatching show almost identical patterns of labeled proteins (Lanes 10 and 14). There is a large amount of label within the stacking gel and on top of the resolving gel, and there are two heavily labeled bands that coincide with the 175K- and 145K-dalton AL proteins e and f. These iodinated proteins remain with the embryo after hatching, whereas, a labeled band corresponding to the llOK-

HALLAND~ACQUIER

Apical

Lamina and Hyaline

dalton protein g as well as several lower-molecularweight labeled proteins are lost upon hatching (Lanes 11 and 15). The lost proteins have been shown to coincide with fertilization envelope precursor material that probably fails to incorporate with the envelope and remains within the perivitelline space until after hatching (Hall, 1978). The variability in the pattern of the lower-molecular-weight bands seen between Lanes 10 and 14 is due to different batches of embryos and not to differences in developmental stages. After a gentle glycine wash without embryo dissociation, the 175K- and 145K-dalton labeled bands remain with the embryo (Fig. 2, Lane 16), but upon mechanical dissociation, these bands are recovered from the glycine wash (Lane 13). This distribution after the different treatments is consistent with the behavior of the visible AL from both isolated HL-AL complexes and embryos treated with glycine solution. No differences are observed between the AL proteins labeled at the two developmental stages, except that the earlier labeled AL proteins tend to partition more completely into the glycine wash upon embryo dissociation. AL proteins labeled at the older stage are often found associated with both the wash and with the pellet of dissociated cells. The llOK-dalton band is intensely labeled by the peroxidase but is released from the embryo after hatching. However, this protein is a minor component of HL-AL complexes isolated from both 1-hr-old embryos and hatched blastulae. Perhaps, this represents different distributions of the protein within the HLAL complex. A fraction of the llOK-dalton protein most accessible to the peroxidase and therefore radioiodinated may be that which was lost at hatching, whereas the fraction that remains with the isolated HL-AL complex may not be accessible to in viva labeling by the peroxidase. The diffuse label at the top of the gels corresponds to the distribution and poor resolution of isolated hyalin seen in the Coomassie blue-stained gels. This labeled material is removed from the embryos by a glycine wash, again consistent with the behavior of hyalin. However, the labeled material removed by the wash and electrophoresed separately exhibits better resolution and heterogeneity. The improved resolution may be due to the lower amount of protein loaded onto the gels compared to samples of entire labeled embryos and to samples of Coomassie blue-stained hyalin. The glytine wash in which early labeled embryos are dissociated (Fig. 2, Lane 13) shows one major band, a, and two minor bands, b and c, in the stacking gel, label on top of the resolving gel, a band close to the top of the resolving gel, d, as well as the AL corresponding bands e and f. Several of the labeled proteins within this glytine wash are distinguished on the basis of their pre-

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cipitability in the presence of calcium ions, a known characteristic of hyalin (Stephens and Kane, 1970). Aliquots (100 ~1) of the wash were dialyzed against 10 m&f Tris-HCl buffer, pH 8.0, and brought to either 20 mM CaC12, 20 mM CaCIZ, and 50 mM MgClz, or 20 mM CaCl, with 0.05 mg added unlabeled hyalin, and the precipitates removed by centrifugation. Only the labeled proteins that band within the stacking gel (Fig. 3, Bands a, b, and c) are found in the precipitates, as would be expected of the protein hyalin. Magnesium ions or cold carrier hyalin included in the samples reduce the amount of stacking gel bands remaining in the supernatant and correspondingly increase the amount recovered in the pellet. The AL proteins as well as the band just within the resolving gel d remain in the supernatant, distinguishing these proteins from hyalin. Protein d shows behavior distinctive from the AL proteins; it is removed along with hyalin by gentle a glycine wash that does not dissociate blastulae (Fig. 2, Lane 17). The apparent heterogeneity of the calcium-precipitable material may be an artifact due to the precipitation of hyalin upon stacking in the presence of SDS. Since the precipitability of hyalin makes it difficult to resolve by SDS-polyacrylamide gel electrophoresis, other methods of separation and identification will be necessary to determine the relation of these separate bands to hyalin. Whatever the cause of the heterogeneity, however, it is found consistently and there does appear to be a developmental difference. In the glycine wash from embryos labeled as blastulae, there is a loss of the major band a in the stacking gel and a greater accumulation of label at the top of the resolving gel (Fig. 2, Lane 17). This difference is observed from several batches of embryos. It must be emphasized that glycine washes from identical stages, hatched blastulae, are compared in the gel Lanes 13 and 17, but it is the labeling period that is different. The change would not, therefore, reflect a modification of the earlier labeled material but may reflect different proteins with changing accessibility to the peroxidase. Other cellular components may interact with hyalin and cause it to precipitate higher up in the stacking gel. Since identical stages are compared, the developmental stage would not be responsible for the change in the presence of such a component. However, the extent of embryo dissociation, complete in the glycine wash in Lane 13, incomplete in Lane 17, could affect the amount of interfering material released into the wash. However, the change does not appear to be due to the absence of the AL proteins in Lane 17. Glycine washes in which later labeled embryos are dissociated contain more AL protein but do not have the heavy band a in the stacking gel. Furthermore, early labeled material separated by

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calcium ion precipitation from the AL proteins present in the glycine wash and from other possible non-calcium-precipitable compounds continue to have band a in the stacking gel (Fig. 3). In comparison to the HL-AL complex, the AL alone contained a greater percentage of carbohydrate (Table 1); a finding compatible with the low carbohydrate content of hyalin (Stephens and Kane, 1970) and the heavy PAS staining of the AL proteins. Standardized to total I

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TABLE COMPARISON

OF HL-AL

1

COMPLEX

HL-AL Carbohydrate Uranic acid Hydroxyproline Sialic acid (TBA assay) Sialic acid (resorcinol assay) Sulfate

AND AL ALONE

complexes

3.74 f 0.44 0.08 + 0.02 <0.005
AL 11.56 + 1.74 0.55 * 0.03 10.0038
a Figures are weight percentage of total protein. The estimate for total protein is derived by dividing the value from the ninhydrin reaction by a factor of 0.152 (fraction of nitrogen in bovine serum albumin).

nitrogen, determined by the ninhydrin reaction (Schiffman et al., 1964), the HL-AL complex and AL showed a stronger reaction than did bovine serum albumin in the Folin assay for protein (Lowry et al., 1951). The figures in Table 1 were standardized to total nitrogen. Less than 0.1% sialic acid, by weight, was found associated with either the HL-AL complex or AL. Less than 1% uranic acid was detected. Some sulfate was found in the HL-AL complex and in greater proportion in the AL fraction. The quantification of sulfate was not accurate due to interfering yellow color produced in the assay. In contrast to a report that the hyaline layer contains collagen (Spiegel and Spiegel, 1979), no biochemical evidence was found for the existence of collagen in the AL. To a detection accuracy of less than O.Ol%, there was no indication of the presence of hydroxyproline. The proteins of the HL-AL complex were insensitive to digestion by purified bacterial collagenase as determined by the inability of the enzyme to increase the amount of ninhydrin-positive material in the incubation mixture and the failure of the enzyme to degrade the AL proteins characterized by electrophoresis. Collagenase activity was verified by the release of ninhydrin-positive material when bovine tendon collagen is included in the reaction mixture. DISCUSSION

FIG. 3. Autoradiograph of fractions of a glycine wash in which hatched blastulae, labeled 1 hr after fertilization, were dissociated. The entire glycine wash is shown in Fig. 2, Lane 13. (1) and (2) Coomassie blue-stained hyalin. (3) Precipitate formed upon bringing the labeled glycine wash to 20 mMCaC1z. (4) Supernatant remaining after removal of precipitate in Lane 3. (5) Precipitate formed upon bringing the labeled glycine wash to 20 mM CaClz and 50 mM MgClz. (6) Supernatant remaining after removal of precipitate in Lane 5. (7) Precipitate formed upon bringing the labeled glycine wash to 20 mkf CaCh after addition of unlabeled hyalin. (8) Supernatant remaining after removal of precipitate in Lane 7.

Several investigators have identified a single protein, referred to as hyalin in the later studies, as the principle component of the sea urchin embryonic hyaline layer (HL) (Yazaki, 1968; Vacquier, 1969; Kane and Stephens, 1969; Stephens and Kane, 1970; Kane, 1970; Citkowitz, 1971, 1972). This study provides biochemical evidence for the existence of other major glycoproteins associated with the HL. These glycoproteins are found in isolated hyaline layer complexes from 1-hr-old embryos and from hatched blastulae. As a separate line of evidence, the existence of the glycoproteins is revealed through their radioiodination by the perivitelline-lo-

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Apical Luminu and Hyaline Layer Glycuproteins

calized cortical granule peroxidase in the presence of radioactive iodide and hydrogen peroxide (Hall, 1978). That these glycoproteins are obtained from complexes isolated from 1-hr-old embryos and are labeled by the peroxidase at this early stage demonstrates they are located apically and do not originate from intercellular spaces or from the blastocoel. The glycoproteins described here are distinguished from hyalin on the basis of electrophoretic migration (migrating with apparent molecular weights of 1’75K and 145K daltons), precipitability with calcium ions, and solubility in glycine solution. Glycine solution removes hyalin and other proteins from the surface of embryos. The l%K- and 145K-dalton glycoproteins are insoluble and are left behind as an apparent separate layer, the apical lamina (AL). Hyalin and the AL glycoproteins may appear to exist as separate layers only as a result of their differential solubility in glycine solution, whereas in the intact embryo, these components may be enmeshed as a single layer. By light microscopy, however, the sea urchin embryo does appear to be surrounded by two layers. The appearance of an inner layer has been attributed to the numerous cell processes that traverse a fluid-filled region of varying thickness and which attach to the inside of an outer gelled layer of constant thickness, the HL (Dan, 1960). By electron microscopy, Wolpert and Mercer (1963) observed a similar organization of two regions around the exterior of hatched blastulae. The cell processes appeared to cross an inner region consisting of electron-dense material rather than simply seawater or other fluid. The outer region was distinguished as an area of contrast separated slightly from the inner region and associated with the ends of the cell processes. Wolpert and Mercer (1963) referred to the regions as HLs one and two. In unhatched embryos there was less distinction between the two regions, so together they appeared as one. Lundgren (1973) provided further evidence for the existence of separate regions. Using ruthenium red staining, he identified an outer zone, an inner zone, and a “support layer” junction between the two. This organization was observed in both unhatched and hatched embryos but with slightly fainter staining in unhatched embryos, which Lundgren attributed to interference by the surrounding fertilization envelope. Improved fixation and staining upon loss of the fertilization envelope may have been the reason it appeared to Wolpert and Mercer (1963) that the HL differentiates into two layers at hatching rather than the two layers existing well before that time. Expanding upon Lundgren’s observations, using a modified procedure for fixation and ruthenium red staining, Spiegel and Spiegel (1979) were able to recognize with greater clarity spe-

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cific structures associated with the different regions, most notably striated fibers within the inner region. The material removed by glycine, largely hyalin, and the AL material remaining, may, in the intact embryo, predominantly exist within separate layers corresponding to the different regions seen by electron microscopy. Recent evidence has shown that immunofluorescent localization of hyalin is confined to the outer region of the HL. Between this outer region and the apical surface is a space without detectable hyalin (D. R. McClay, personal communication). This space may correspond to a region comprising the AL proteins reported in this paper. The persisting AL exposed after hyalin removal appears as a fibrous network which may be the fibers of the inner region seen by Spiegel and Spiegel (1979). To obtain the AL as an intact layer from embryos treated with glycine solution, it is necessary to allow the AL to gradually slough off. Since an AL does not slough off one-cell-stage embryos, separation of the layer from the large single cell may occur by a different mechanism than from the many smaller cells of the blastula, or the lysing fertilized egg may shred the AL before it becomes completely detached from the surface. After separating from the cells, the highly convoluted AL is of a much smaller diameter than the intact blastula. The AL behaves as if it contracts and condenses into a more rigid or stable structure after being detached from the cell surface. It is possible that the observed fibers are the result of condensation of less dense and less structured material. Indeed this is also suggested by an observation of Spiegel and Spiegel (1979) that an amorphous crystalline lattice, described as another component of the inner layer, has the same crystalline periodicity as do the fibers. While Spiegel and Spiegel report that the fibers are collagen because their morphology is altered by collagenase, the present study fails to find any biochemical evidence that collagen is associated with the HL-AL complexes. The layer complexes contain no hydroxyproline (Table l), have an amino acid composition unlike collagen (H. G. Hall, preliminary unpublished results), and are insensitive to digestion by bacterial collagenase. The high concentration of collagenase used by Spiegel and Spiegel (1 mg/ml) may have contained sufficient nonspecific contaminating proteolytic activity to degrade the fibers, or, conceivably, may have altered the fiber morphology by a nonenzymatic mechanism. Alternately, the isolated HL-AL complexes may not contain the fibers seen by Spiegel and Spiegel in intact embryos. In such a case, unless the postulated collagen fibers are readily soluble in glycine solution, the fibrous net of material seen in Fig. 1 would have to consist of two populations of fibers, one that separates with the

176

DEVELOPMENTALBIOLOGY VOLUME89,1982

isolated complex, the AL fibers, and one that would not. This does not seem likely. Furthermore, if components other than those in the isolated HL-AL complex exist in significant amounts on the surface, they would have escaped radioiodination by the peroxidase. Due to the lack of tyrosines, collagen is a molecule that could escape iodination. The intense labeling of the AL proteins, indicating significant tyrosine content, further distinguishes these proteins from collagen. On the basis of ruthenium red staining, Spiegel and Spiegel (1979) suggest that the HL contains glycosaminoglycans. This stain detects the presence of polyanions (Luft, 1971). The major component of the HL, hyalin, is notable for its polyanionic composition (Stephens and Kane, 1970), and, therefore could be stained by ruthenium red. In the present study, very little uranic acid was found in HL-AL complexes (Table 1). However, some sulfate was found in the AL. Therefore, if glycosaminoglycans are present in the HL, they may be of the keratan sulfate type lacking uranic acid. Sulfated glycoproteins have been found to be synthesized by sea urchin embryos during gastrulation (Heifetz and Lennarz, 1979). So, it is possible that the sulfate found here may be covalently associated with the AL glycoproteins rather than with a separate polysaccharide component. Citkowitz (1972) analyzed the components of the isolated HL by electrophoresis. Depending upon sample treatment and the type of gel, variation was observed in the pattern of a major band, presumably hyalin, and two closely migrating minor bands. The AL proteins were not detected. The HL isolation procedure, employing concentrated salt solutions, differed from the HL-AL complex isolation procedure described here. During the initial stages of this study, HLs were isolated by the Citkowitz method (1971) and were found to contain variable amounts and considerably less detectable AL proteins. However, small amounts, even if minor, were usually seen, so it is perplexing why the AL proteins were not found in the earlier study. The amino acid composition of isolated HL-AL complexes (H. G. Hall, unpublished preliminary results) is similar to that reported by Citkowitz for isolated HLs. In contrast to Citkowitz’s results, no sialic acid was found associated with HL-AL complexes, although similar amounts of carbohydrate were found. Citkowitz’s studies suggest heterogeneity in hyalin protein. Hyalin, purified by repeated cycles of calcium precipitation, has been shown to sediment as a hypersharp peak, suggesting a single component, and it tends to self-aggregate (Vacquier, 1969; Stephens and Kane, 1970; Kane, 1970; Citkowitz, 1972). Citkowitz (1972) points out that several components which tend to strongly self-aggre-

gate could also sediment as a single peak. Results from the present study also suggest heterogeneity and possible developmental differences in hyalin. However, because of the difficulty in analyzing hyalin by SDSgel electrophoresis, further study employing other methods of separation and identification will be necessary before heterogeneity can be firmly established. By removing the HL in calcium-free seawater, Herbst (1900) first demonstrated that this layer is necessary to hold together cleaving blastomeres. Dan (1960) showed that processes extending from the apical cell surfaces form a tight mechanical attachment to the HL. From these observations, Gustafson and Wolpert (1967) proposed that morphogenetic events of the sea urchin embryo may be generated by modifications in cell to cell adhesions in conjunction with changing cellular affinities for the HL. While the proposal by Gustafson and Wolpert is valuable in presenting conceivable and, perhaps, universal mechanisms by which an extracellular layer may direct morphogenesis, such a role suggested for the HL is still hypothetical. Certainly, natural and artificial substrata have been shown to affect cellular activities such as growth, attachment, movement, and differentiation (reviewed by Grinnel, 1978; and Gospodarowicz et al., 1978). Although, in the sea urchin, the HL seems a likely candidate as an extracellular layer that could direct morphogenesis, it has not yet been shown to have a function other than to encircle the blastomeres. Participation by the HL cannot be generally relevant to animal morphogenesis simply because an HL is found in only two echinoderm classes (Holland, 1981). In the starfish, an echinoderm lacking an HL and closely related to the sea urchin, the interaction of the cells alone without the aid of an extracellular layer seems to be sufficient in organizing the cells into a blastula form (Dan-Sohkawa and Fujisawa, 1980). If the extracellular layer surrounding the embryo does play a significant role in morphogenesis, then hyalin and the AL glycoproteins either as a single layer or as separate layers would probably serve this function in concert. If the AL material does form a layer beneath a separate hyalin-containing layer, then it may mediate cellular attachment to the outside layer. Such a function is not obligatory, however, since cell processes pass through the inner layer to make direct contact with the outer. Some investigators have suggested that hyalin is a cell adhesion factor. Ultrastructural studies (Spiegel and Spiegel, 1978) describe the presence of “wispy granular material” between cells that looks like hyalin, but it was not identified biochemically. Other studies (Timourian and Watchmaker, 1975, Kondo and Sakai, 1971), showing aggregation of cells with hyalin in so-

HALL AND VACQUIER

Apical

Lamina

lution, did not eliminate nonspecific binding due to the polyanionic nature of hyalin or to simple entrapment of cells within a hyalin precipitate. Although hyalin may influence the aggregation of cells, there is yet no evidence that it promotes specific recognition. Tonegawa (1973) identified a cell aggregating substance from sea urchin embryos distinct from hyalin. This substance was recovered as a particulate from mechanically dissociated blastulae, after removal of the HL. Judging from the method of preparation, this particulate material most likely contained AL fragments which may have had the aggregating activity. Because of its very adhesive nature, the presence of AL fragments in dissociated cell suspensions may influence the reaggregation patterns of blastomeres, and, therefore, must be considered in studies attempting to evaluate this phenomenon. The HL appears to originate from the cortical granules (Hylander and Summers, 1980; and see the introduction), a likely source also for the AL proteins. Hyalin has been shown to be regenerated after its removal from blastulae by a treatment that does not remove the AL (Kane, 1973). Although the AL is not removed, a common vesicle source of hyalin and AL proteins may result in their concomitant and proportional replacement. It would be of interest to analyze the mutual associations and distributions of these proteins over the cell surfaces to see if they become separated into distinct layers after fertilization and upon regeneration. The authors would like to thank Drs. Mina J. Bissell, Raymond E. Stephens, Stephen C. Benson, and David R. McClay for their helpful advice. This work was supported by an NIH Postdoctoral Fellowship to H.G.H. and NIH Grant HD 12986 to V.D.V. REFERENCES G. (1966). New calorimetric methods of sugar analysis. In “Methods in Enzymology” (Neufeld, E. F., and Ginsburg, V., eds.), Vol. 8, pp. 85-95. Academic Press, New York. BLUMENKRANTZ, N., and ASBOE-HANSEN, G. (1973). New method for quantitative determination of uranic acids. Anal. Biochem 54,48& ASHWELL,

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TONEGAWA, Y. (1973). Isolation and characterization of a particulate cell-aggregation factor from sea urchin embryos. Deuelop. Orowth Differ. 14, 337-352. VACQUIER, V. D. (1969). The isolation and preliminary analysis of the hyaline layer of sea urchin eggs. Exp. Cell Res. 54,140-142. WOLPERT, L., and MERCER, E. H. (1963). An electron microscope study of the development of the blastula of the sea urchin embryo and its radial polarity. Exp. Cell Res. 30, 280-300. WV, G. -J., and BRUENING, G. (1971). Two proteins from cowpea mosaic virus. VGrology 46, 596-612. YAZAKI, I. (1968). Immunological analysis of the calcium precipitable protein of sea urchin eggs. 1. Hyaline layer substance. Emt~ologia 10,131-141.