Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.     

Exogenous hyalin and sea urchin gastrulation, Part II: hyalin, an interspecies cell adhesion molecule.

The 330 kDa fibrillar glycoprotein hyalin is a well known component of the sea urchin embryo extracellular hyaline layer. Only recently, the main component of hyalin, the hyalin repeat domain, has been identified in organisms as widely divergent as bacteria and humans using the GenBank database and therefore its possible function has garnered a great deal of interest. In the sea urchin, hyalin serves as an adhesive substrate in the developing embryo and we have recently shown that exogenously added purified hyalin from Strongylocentrotus purpuratus embryos blocks a model cellular interaction in these embryos, archenteron elongation/attachment to the blastocoel roof. It is important to demonstrate the generality of this result by observing if hyalin from one species of sea urchin blocks archenteron elongation/attachment in another species. Here we show in three repeated experiments, with 30 replicate samples for each condition, that the same concentration of S. purpuratus hyalin (57 microg/ml) that blocked the interaction in living S. purpuratus embryos blocked the same interaction in living Lytechinus pictus embryos. These results correspond with the known crossreactivity of antibody against S. purpuratus hyalin with L. pictus hyalin. We propose that hyalin-hyalin receptor binding may mediate this adhesive interaction. The use of a microplate assay that allows precise quantification of developmental effects should help facilitate identification of the function of hyalin in organisms as divergent as bacteria and humans.     

CELL-BINDING SUBSTANCES IN SEA URCHIN EMBRYOS*

Properties of ovacquenin, a reaggregation-promoting substance from sea urchin embryos, were further studied and compared with those of hyalin, a calcium-insoluble protein of the hyaline layer surrounding the sea urchin embryo. Properties of hyalin were basically in agreement with previous reports, but differed in some aspects. Hyalin and ovacquenin were very similar in various aspects and were hard to distiguish generally, but they were separated by ammonium sulfate fractionation. Hyalin was precipitated at a low salt concentration, while ovacquenin remained soluble until the salt concentration exceeded half saturation. Therefore it was concluded that hyalin and ovacquenin are very much alike but distinct from each other. Probable relation of ovacquenin to hyalin was discussed.     

Polypeptide composition and organization of the sea urchin extraembryonic hyaline layer.

The protein composition and organization of the sea urchin extraembryonic hyaline layer was examined. Hyalin and a polypeptide of 45 kilodaltons (kDa) were present in hyaline layers isolated from 1-h-old embryos through to the pluteus larva stage. In contrast, several polypeptide species ranging in size from 175 to 32 kDa either decreased in amount or disappeared from the layer as embryonic development proceeded. Concomitant with the changes in composition, hyaline layers became progressively more refractory to dissolution by washing in Ca2+, Mg2(+)-free seawater. Incubation of intact layers, isolated from 1-h-old embryos, with proteinase K resulted in the selective digestion of hyalin and was accompanied by release of the 45-kDa polypeptide from the layers. Washing intact layers in 20 mM Tris (pH 8.0) also resulted in the selective removal of hyalin and the 45-kDa polypeptide. The Ca2(+)-precipitable protein hyalin, alone among the hyaline layer polypeptides, bound the Ca2(+)-antagonist ruthenium red. These results suggest a structural organization within the hyaline layer that is both heterogenous and dynamic throughout embryonic development.     

Jun N‐terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N‐terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125‐treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation. genesis 53:762–769, 2015. © 2015 Wiley Periodicals, Inc.     

An ultrastructural immunocytochemical localization of hyalin in the sea urchin egg

The fertilized sea urchin egg is invested by the hyaline layer, a thick extracellular coat which is necessary for normal development. On the basis of ultrastructural studies and the fact that hyalin is released during the time of the cortical reaction, it has been generally accepted that hyalin is derived from the cortical granules. However, this has never been proven definitely, and recently, it has been reported that hyalin is a membrane and/or cell surface protein. To determine where hyalin is stored, we carried out an ultrastructural immunocytochemical localization of hyalin in the unfertilized egg. Hyalin purified from isolated hyaline layers was used to immunize rabbits. Antisera so obtained were shown to be hyalin specific following absorption with a combination of sea urchin proteins. Immunocytochemical localizations were carried out on sections of Epon-embedded material using protein A-coated gold particles as an antibody marker. Our results demonstrate that, prior to fertilization, hyalin is stored in the homogeneous component of the cortical granule in Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus. Labeling of small cortical vesicles in both unfertilized and fertilized eggs, suggests that these vesicles may contain a secondary reservoir of hyalin.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

On the ultrastructure of hyalin, a cell adhesion protein of the sea urchin embryo extracellular matrix

Hyalin is a large (ca. 350 x 10(3) kD by gel electrophoresis) molecule that contributes to the hyalin layer surrounding the sea urchin embryo. In previous work a mAb (McA Tg-HYL), specific for hyalin, was found to inhibit cell-hyalin adhesion and block morphogenesis of whole embryos (Adelson, D. L., and T. D. Humphreys. 1988. Development. 104:391-402). In this report, hyalin ultrastructure was examined via rotary shadowing. Hyalin appeared to be a filamentous molecule approximately 75-nm long with a globular "head" about 12 nm in diameter that tended to form aggregates by associating head to head. Hyalin molecules tended to associate with a distinct high molecular weight globular particle ("core"). In fractions containing the core particle often more than one hyalin molecule were seen to be associated with the core. The core particle maintained a tenacious association with hyalin throughout purification procedures. The site(s) of McA Tg-HYL binding to the hyalin molecule were visualized by decorating purified hyalin with the antibody and then rotary shadowing the complex. In these experiments, McA Tg-HYL attached to the hyalin filament near the head region in a pattern suggesting that more than one antibody binding site exists on the hyalin filament. From the ultrastructural data and from the cell adhesion data presented earlier we conclude that hyalin is a filamentous molecule that binds to other hyalin molecules and contains multiple cell binding sites. Attempts were made to demonstrate the existence of lower molecular weight hyalin precursors. Whilst no such precursors could be identified by immunoprecipitation of in vivo labeled embryo lysates, immunoprecipitation of in vitro translation products suggested such precursors (ca 40 x 10(3) kD) might exist.     

Sea urchin morphogenesis and cell-hyalin adhesion are perturbed by a monoclonal antibody specific for hyalin

We have generated and characterized a monoclonal antibody (McA Tg-HYL) that recognizes sea urchin hyalin as evidenced by immunofluorescence staining of the hyaline layer (HL) and immunoblot staining of the hyalin protein band. On immunoblots of HL extracts only the hyalin protein reacted with McA Tg-HYL. Immunoprecipitates of radioactive proteins from embryos incubated with [35S]methionine yielded radioactive hyalin and 190, 140 and 105 x 10(3) Mr proteins associated with hyalin. McA Tg-HYL was generated against Tripneustes gratilla embryos but reacts with hyalin from the distantly related sea urchin species, Colobocentrotus atratus, Strongylocentrotus purpuratus, Arbacia punctulata, Lytechinus variegatus and Lytechinus pictus. Developing embryos of the above-mentioned six species were treated with McA Tg-HYL and did not gastrulate or form arms. Observations of treated embryos revealed areas of separation of the hyaline layer from the underlying embryonic cells, suggesting that McA Tg-HYL was interfering with binding of the cells to the HL. Using the centrifugation-based adhesion assay of McClay et al. (Proc. natn. Acad. Sci. U.S.A. 78, 4975-4979, 1981), Fab' fragments of McA Tg-HYL were found to inhibit cell-hyalin binding. McA Tg-HYL did not inhibit hyalin gelation in vitro or the reaggregation of dissociated blastula cells. We postulate that McA Tg-HYL recognizes an evolutionarily conserved hyalin domain involved in cell-hyalin binding and required for normal epithelial folding.     

Hyalin release during normal sea urchin development and its replacement after removal at fertilization

All stages of sea urchin embryos through pluteus can be dissociated to their component cells through the use of a 1 M solution of glycine containing EDTA, and a similar glycine solution can be used to prevent the formation of the fertilization membrane and remove the hyaline layer material released at fertilization. The protein hyalin, which makes up the bulk of the hyaline layer, can be recovered from these glycine solutions by calcium addition and quantitative agreement was found between the hyalin released at fertilization and the hyalin present at all later developmental stages. However, embryos stripped of their hyalin at fertilization often develop normally, which is unexpected in view of the apparent involvement of the hyaline layer in developmental mechanics. Such embryos are found to have regenerated an appreciable fraction of the hyalin removed at fertilization and this regeneration occurs at the time of blastulation. Thus the regeneration appears to be stimulated by hyaline layer removal at fertilization, but it does not take place until several hours later, at the time this layer has been postulated to play a role in development.     

A cyto-embryological study of gastrulation in the sand dollar, Scaphechinus mirabilis

Processes of gastrulation in the sand dollar Scaphechinus mirabilis were compared with those in the sea urchin Hemicentrotus pulcherrimus , which seemed to show a typical pattern of gastrulation. Measurement of the archenteron length clearly demonstrated that invagination processes in H. pulcherrimus are divided into two phases, the primary and secondary invagination. On the other hand, invagination in S. mirabilis was revealed to continue at a constant rate. To see the movement of cells during gastrulation, embryos were labeled with Nile blue. In H. pulcherrimus embryos, labeled cells were observed along the full length of the archenteron, if the embryos had been labeled before and during the primary invagination. Labeled cells were never observed in the embryos stained after the primary invagination. In contrast, labeled cells were always discerned at the basal part of the archenteron in S. mirabilis , even if the embryos were stained after invagination had undergone considerable progress. The number of cells in the archenteron of S. mirabilis embryos increased with the advancement of gastrulation, while the numbers were almost constant in H. pulcherrimus . These results suggest that the cellular basis of gastrulation in S. mirabilis is quite different from that in well-known species of sea urchins.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

Archenteron elongation in the sea urchin embryo is a microtubule-independent process

Earlier studies using colchicine (L. G. Tilney and J. R. Gibbins, 1969, J. Cell Sci. 5, 195-210) had suggested that intact microtubules (MTs) are necessary for archenteron elongation during the second phase of sea urchin gastrulation (secondary invagination), presumably by allowing secondary mesenchyme cells (SMCs) to extend their long filopodial processes. In light of subsequently discovered effects of colchicine on other cellular processes, the role of MTs in archenteron elongation in the sea urchin, Lytechinus pictus, has been reexamined. Immunofluorescent staining of ectodermal fragments and isolated archenterons reveals a characteristic pattern of MTs in the ectoderm and endoderm during gastrulation. Ectodermal cells exhibit arrays of MTs radiating away from the region of the basal body/ciliary rootlet and extending along the periphery of the cell, whereas endodermal cells exhibit a similar array of peripheral MTs emanating from the region of the apical ciliary rootlet facing the lumen of the archenteron. MTs are found primarily at the bases of the filopodia of normal SMCs. beta-Lumicolchicine (0.1 mM), an analog of colchicine which does not bind tubulin, inhibits secondary invagination, indicating that the effects previously ascribed to the disruption of MTs are probably due to the effects of colchicine on other cellular processes. The MT inhibitor nocodazole (5-10 micrograms/ml) added prior to secondary invagination does not prevent gastrulation or spontaneous exogastrulation, even though indirect immunofluorescence indicates that cytoplasmic MTs are completely disrupted in drug-treated embryos. Transverse tissue sections indicate that a comparable amount of cell rearrangement occurs in nocodazole-treated and control embryos. Significantly, SMCs in nocodazole-treated embryos often detach prematurely from the tip of the gut rudiment and extend abnormally large broad lamellipodial protrusions but are also capable of extending long slender filopodia comparable in length to those of control embryos. These results indicate that cytoplasmic MTs are not essential for either filopodial extension by SMCs or for the active epithelial cell rearrangement which accompanies elongation during sea urchin gastrulation.     

Pattern of Brachyury gene expression in starfish embryos resembles that of hemichordate embryos but not of sea urchin embryos.

Echinoderms, hemichordates and chordates are deuterostomes and share a number of developmental features. The Brachyury gene is responsible for formation of the notochord, the most defining feature of chordates, and thus may be a key to understanding the origin and evolution of the chordates. Previous studies have shown that the ascidian Brachyury (As-T and Ci-Bra) is expressed in the notochord and that a sea urchin Brachyury (HpTa) is expressed in the secondary mesenchyme founder cells. A recent study by [Tagawa et al. (1998)], however, revealed that a hemichordate Brachyury (PfBra) is expressed in a novel pattern in an archenteron invagination region and a stomodaeum invagination region in the gastrula. The present study demonstrated that the expression pattern of Brachyury (ApBra) of starfish embryos resembles that of PfBra in hemichordate embryos but not of HpTa in sea urchin embryos. Namely, ApBra is expressed in an archenteron invagination region and a stomodaeum invagination region.     

SOME PROPERTIES OF HYALIN : The Calcium-Insoluble Protein of the Hyaline Layer of the Sea Urchin Egg

The principal protein component of the hyaline layer of sea urchin eggs is the calcium-insoluble protein first described by Kane and Hersh. The protein hyalin is abnormally high in acidic amino acids, almost devoid of basic amino acids, and characteristically rich in valine and proline. Essentially all of the cysteine present is found in the disulfide form; no evidence points to intermolecular disulfide linkages. Hyalin from several species has a minimal subunit weight of about 100,000, though evidence exists for a particle three times this weight in urea or guanidine hydrochloride from one species. Optical rotatory dispersion measurements indicate no α-helix content, though the dispersion has unique characteristic features. Addition of small quantities of calcium causes hyalin to gel to a birefringent fibrous form. The fibrous, birefringent form of hyalin is rendered isotropic upon addition of EDTA, but the birefringence is restored with re-addition of divalent cation.     

RhoA regulates initiation of invagination, but not convergent extension, during sea urchin gastrulation

During gastrulation, the archenteron is formed using cell shape changes, cell rearrangements, filopodial extensions, and convergent extension movements to elongate and shape the nascent gut tube. How these events are coordinated remains unknown, although much has been learned from careful morphological examinations and molecular perturbations. This study reports that RhoA is necessary to trigger archenteron invagination in the sea urchin embryo. Inhibition of RhoA results in a failure to initiate invagination movements, while constitutively active RhoA induces precocious invagination of the archenteron, complete with the actin rearrangements and extracellular matrix secretions that normally accompany the onset of invagination. Although RhoA activity has been reported to control convergent extension movements in vertebrate embryos, experiments herein show that RhoA activity does not regulate convergent extension movements during sea urchin gastrulation. Instead, the results support the hypothesis that RhoA serves as a trigger to initiate invagination, and once initiation occurs, RhoA activity is no longer involved in subsequent gastrulation movements.     

Sea urchin egg hyaline layer: evidence for the localization of hyalin on the unfertilized egg surface

The sea urchin embryo hyaline layer is an extracellular investment which develops within 20 min postinsemination of Strongylocentrotus purpuratus eggs and contains a single calcium-precipitable subunit termed hyalin. Other ultrastructural and biochemical studies have suggested that hyalin is localized in the cortical granules. We have examined the hypothesis that hyalin is a cell surface protein of the unfertilized egg using vectorial lactoperoxidase-catalyzed radioiodination. Extracts of labeled unfertilized eggs contained several labeled proteins, one of which was electrophoretically indistinguishable from authentic hyalin isolated by each of three different procedures. Pronase digestion of labeled unfertilized eggs removed 75% of the label, but the labeled hyalin-like molecule was still present in whole cell extracts. Upon insemination, pronase-digested, labeled eggs formed an apparently normal hyaline layer and whole cell extracts contained the labeled hyalin-like molecule. Denuded, labeled eggs were inseminated and the hyaline layer was selectively solubilized in calcium- and magnesium-free artificial seawater. Labeled hyalin was purified from this crude hyalin preparation to constant specific radioactivity and apparent homogeneity as shown by gel electrophoresis. These data strongly suggest that hyalin or a precursor is a cell surface protein of the unfertilized sea urchin egg.     

Role of calcium in stabilizing the structure of hyalin, a major protein component of the sea urchin extraembryonic hyaline layer

The interactions of NaCl and CaCl2 with the sea urchin embryo coat protein hyalin were investigated. Endogenous protein tryptophan fluorescence was enhanced by almost 45% in the presence of 200mM NaCl while 1mM CaCl2 reversed this effect and brought the intensity of fluorescence back close to that of the native protein. Half-maximal concentrations of 53 and 0.32mM were determined for NaCl and Ca+2, respectively. Hyalin conformation, as measured by circular dichroic spectroscopy, was altered by NaCl and CaCl2 in a fashion parallel to the effects of these salts on tryptophan fluorescence. Sodium chloride disrupted hyalin secondary structure while CaCl2 affected the return of hyalin to its native conformation. The interactions of NaCl and CaCl2 with hyalin were not modulated by MgCl2. These results suggest a role for CaCl2 in stabilizing hyalin against the disruptive effects of the high concentration of NaCl present in sea water.     

Tissue-specific, temporal changes in cell adhesion to echinonectin in the sea urchin embryo.

Echinonectin is a dimeric, glycoprotein found in the hyaline layer of the developing sea urchin embryo. It was found that echinonectin supports adhesion of embryonic cells in vitro. Previous studies have shown that the protein hyalin also supports adhesion. The purpose of this study was to examine the specificity of cell-echinonectin interactions during sea urchin development. Primary mesenchyme cells (PMCs) ingress into the blastocoel during gastrulation. In the process the PMCs lose contact with the hyaline layer. It was found experimentally that differentiating PMCs decreased their adhesion to hyalin at the time of ingression. It was of interest, therefore, to determine whether there was a coordinate loss of adhesion to echinonectin at ingression as well. When cell-echinonectin interactions were quantified using a centrifugal force-based adhesion assay, it was shown that micromeres adhered well to echinonectin. At the time of ingression, PMCs displayed reduced adhesion to echinonectin just as had been found when hyalin was tested as a substrate. There was no change in adhesion of presumptive ectoderm or endoderm to echinonectin over the same time period. Early in gastrulation presumptive ectoderm and endoderm adhered to echinonectin only half as strongly as to equimolar concentrations of hyalin. After gastrulation endoderm cells were observed to retain the same relative affinity to hyalin and echinonectin, while ectoderm cells became equally adhesive for both hyalin and echinonectin. Quantitatively, this represents an overall increase in the affinity of ectodermal cells for echinonectin. Adhesion to combined substrata of echinonectin and hyalin was reduced but not abolished by monoclonal antibodies specific for echinonectin. The antibodies did not cross-react with hyalin. We conclude that both echinonectin and hyalin independently act as adhesive substrata for the developing sea urchin embryo. PMCs lose an affinity for echinonectin and ectodermal cells later increase their affinity for this substrate.     

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

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 175K- 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.