A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.     

Spiculogenesis in the sea urchin embryo: Studies on the SM30 spicule matrix protein

When proteins isolated from spicules of Strongylocentrotus purpuratus embryos were examined by western blot analysis, a major protein of approximately 43 kDa was observed to react with the monoclonal antibody, mAb 1223. Previous studies have established that this antibody recognizes an asparagine-linked, anionic carbohydrate epitope on the cell surface glycoprotein, msp130. This protein has been shown to be specifically associated with the primary mesenchyme cells involved in assembly of the spicule. Moreover, several lines of evidence have implicated the carbohydrate epitope in Ca²⁺ deposition into the growing spicule. The 43 kDa, spicule matrix protein detected with mAb 1223 also reacted with a polyclonal antibody to a known spicule matrix protein, SM30. Further characterization experiments, including deglycosylation using PNGaseF, two-dimensional electrophoresis, and immunoprecipitation, verified that the 43 kDa spicule matrix protein had a pl of approximately 4.0, contained the carbohydrate epitope recognized by monoclonal antibody mAb 1223 and reacted with anti-SM30. Electron microscopy confirmed the presence of proteins within the demineralized spicule that reacted with mAb 1223 and anti-SM30. We conclude that the spicule matrix protein, SM30, is a glycoprotein containing carbohydrate chains similar or identical to those on the primary mesenchyme cell membrane glycoprotein, msp130.     

Immunocytochemical evidence suggesting heterogeneity in the population of sea urchin egg cortical granules

Unfertilized eggs of many species of animals contain cortical granules, which are specialized secretory granules that upon fertilization release their contents from the egg. The unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, contain cortical granules that all display an identical and elaborate internal morphology. It has been assumed that they all contain identical components. In this report we present immunocytochemical data which indicate that the cortical granule population of S. purpuratus eggs is heterogeneous. Two monoclonal antibodies are shown to react to the spiral lamellae region of approximately 20% of the cortical granules, implying that the contents of the reactive granules differ from the contents of the majority of the population. An egg protein of greater than 320 kDa is recognized by the antibody. These antibodies also stain a 130-kDa protein expressed on the surface of primary mesenchyme cells in later development. Both antibodies recognize a post-translational modification of this protein. This suggests that an antigenically similar epitope is present both on the 130-kDa primary mesenchyme cell-specific protein and in the cortical granules. To determine if the primary mesenchyme and cortical granule proteins are related, a fusion protein antibody specific for a region of the 130-kDa protein was used to stain unfertilized eggs. This antibody did not stain cortical granules. Thus, 20% of the cortical granules contain a molecule that has an epitope antigenically similar to the post-translational modification recognized in primary mesenchyme cells by the monoclonal antibodies.     

Characterization of post-translational modifications common to three primary mesenchyme cell-specific glycoproteins involved in sea urchin embryonic skeleton formation

Previous studies have established the importance of a complex, N-linked oligosaccharide chain, recognized by a monoclonal antibody (mAb 1223), in the formation of sea urchin embryonic skeletal components known as spicules. To further investigate the function of this epitope, mAb 1223 was added to primary mesenchyme (PM) cell cultures prior to spiculogenesis. The antibody did not inhibit cell migration, cell attachment, or synthesis of the filapodial networks upon which the spicules are deposited. However, it did block deposition of mineralized CaCO3 along these filapodia, strongly supporting the previously proposed role for the 1223 epitope in calcium accumulation and/or deposition. Previously the 1223 epitope has been most extensively studied in association with a mesenchyme-specific protein of 130 kDa (msp 130). It has now been established, by Western blot analysis of whole embryo and PM cell extracts using mAb 1223, that two other proteins of 205 and 250 kDa contain the 1223 epitope. A study of the developmental profiles of expression of these glycoproteins revealed that all three were first expressed just prior to spiculogenesis, consistent with a role for any or all of these proteins in this process. Additionally all three proteins incorporated ethanolamine, myristate, and palmitate, the precursors of the glycosylphosphatidylinositol (GPI) anchor. Further labeling studies revealed differences in the metabolic lability of the GPI anchor in the three proteins; pulse-chase studies demonstrated that the ethanolamine moiety was stable in msp 130, but was rapidly chased from the 205-kDa protein (T1/2 = 14 hr). Phosphatidylinositol-specific phospholipase C partially released (50%) msp 130 from the PM cell surface, whereas it had no effect on release of the 205- and 250-kDa proteins. Studies with 35SO4 labeling and PNGase F treatment directly established that all three proteins are sulfated, and that most of the sulfate is attached to the N-linked oligosaccharide chains. Thus, the three major mAb 1223-reactive glycoproteins in PM cells are also the three major proteins containing both sulfated N-linked oligosaccharide chains and GPI anchors. Further investigation of this intriguing correlation may help to define the precise function of the 1223 epitope in the process of spicule formation.     

Sea urchin primary mesenchyme cells: relation of cell polarity to the epithelial-mesenchymal transformation

In euechinoid sea urchin embryos, a subset of epithelial cells in the wall of the blastula become pulsatile, elongate, lose connections with their neighboring cells, and move into the blastocoel to form the primary mesenchyme cells. The Golgi apparatus and microtubule organizing center (MTOC) are located at the apical end of these epithelial cells. We show that as primary mesenchyme cells begin to move into the blastocoel, the Golgi apparatus and MTOC move to a new position adjacent to the apical side of the nucleus. They do not move to a position between the nucleus and the leading (i.e., basal) end of the cell as they do in cultured fibroblasts undergoing directed migration. In addition, we have inhibited the movement of membranous vesicles to the cell surface by incubating embryos in the ionophore monensin. We have used antibodies to msp130, a primary mesenchyme cell surface-specific glycoprotein, to demonstrate that monensin inhibits the movement of msp130-containing vesicles to the cell surface. Despite the inhibition of membrane shuttling by monensin, primary mesenchyme cells ingress on schedule and display normal cell-shape changes. We draw two conclusions from our data. First, the cellular elongation that characterizes ingression is not due to the local insertion of membrane at the leading (basal) end of the cell. Second, ingression does not depend upon establishment of the same cell polarity required for fibroblasts to carry out directed cell migration.     

Molecular analysis of heterochronic changes in the evolution of direct developing sea urchins

The sea urchin Heliocidaris erythrogramma is a direct developer; it progresses directly from the gastrula to the juvenile adult without forming a pluteus larva. No larval skeleton is formed by mesenchyme cells, but formation of the juvenile skeleton is accelerated. We have examined two alterations in mesenchyme cell behavior that accompany this striking change in developmental pattern. 1) Rapid cell proliferation produces 1700–2200 mesenchyme cells by mid-gastrula, compared to 30–60 primary mesenchyme cells in species with typical larval development. This change may reflect the accelerated production of adult structures in H. erythrogramma. 2) B2C2 is a monoclonal antibody that recognizes primary (Anstrom et al., 1987) and adult mesenchyme cells associated with skeleton formation in typical developers. The altered pattern of B2C2 staining in H. erythrogramma (e.g., a later initial appearance of the B2C2 antigen) suggests that H. erythrogramma has deleted part of a larval program of development and accelerated its adult program of development. These results indicate that cellular and molecular heterochronies accompany the morphological changes in H. erythrogramma development.     

Micromere Differentiation in the Sea Urchin Embryo: Immunochemical Characterization of Primary Mesenchyme Cell-Specific Antigen and Its Biological Roles

Primary mesenchyme cell (PMC)-specific antigens in developing sea urchin embryos of five different species have been studied by using two different monoclonal antibodies, P4 and B2C2. Like B2C2 in Strongylocentrotus purpuratus (Anstrom et al. , 1987) P4 reacted with the N-linked carbohydrate in Strongylocentrotus intermedius embryo. Although both antibodies recognize the same group of glycoproteins in S. intermedius , P4 epitopes appeared earlier than B2C2 epitopes in Clypeaster japonicus embryo. PMCs of Anthocidaris crassispina blastulae raised in sulfate-deficient sea water were immuno-reactive with P4 but not with B2C2, although the embryos raised in normal sea water reacted with both antibodies at similar intensity. These results suggest that the epitopes of P4 and B2C2 are formed by glycosylation and sulfation, respectively. PMCs may display differential modification in their surface glycoprotein synthesis during differentiation. Furthermore, P4 inhibited cultured micromere descendant cells of Hemicentrotus pulcherrimus from attaching to the plastic dishes and forming spicules in vitro without detectable cytotoxic effect. P4-reactive glycoproteins may play important roles in cell-substrate interaction and spicule formation.     

Skeletogenesis by transfated secondary mesenchyme cells is dependent on extracellular matrix-ectoderm interactions in Paracentrotus lividus sea urchin embryos

In the sea urchin embryo, primary mesenchyme cells (PMCs) are committed early in development to direct skeletogenesis, provided that a permissive signal is conveyed from adjacent ectoderm cells. We showed that inhibition of extracellular matrix (ECM)-ectoderm cells interaction, by monoclonal antibodies (mAb) to Pl-nectin, causes an impairment of skeletogenesis and reduced expression of Pl-SM30, a spicule-specific matrix protein. When PMCs are experimentally removed, some secondary mesenchyme cells (SMCs) switch to skeletogenic fate. Here, for the first time we studied SMC transfating in PMC-less embryos of Paracentrotus lividus. We observed the appearance of skeletogenic cells within 10 h of PMCs removal, as shown by binding of wheat germ agglutinin (WGA) to cell surface molecules unique to PMCs. Interestingly, the number of WGA-positive cells, expressing also msp130, another PMC-specific marker, doubled with respect to that of PMCs present in normal embryos, though the number of SM30-expressing cells remained constant. In addition, we investigated the ability of SMCs to direct skeletogenesis in embryos exposed to mAbs to Pl-nectin after removal of PMCs. We found that, although phenotypic SMC transfating occurred, spicule development, as well as Pl-SM30-expression was strongly inhibited. These results demonstrate that ectoderm inductive signals are necessary for transfated SMCs to express genes needed for skeletogenesis.     

Cytoplasm calcium-binding proteins of germ cells and embryos of the sea urchin

Synchronous, demonstrative, easily reproducible fertilization with the following embryonic development makes the process in the sea urchin extremely attractive for studying many biological enigmas. In particular, germ and embryonic cells of the sea urchin present a wide opportunity for investigating different associated phenomena launched by an increase in concentration of Ca²⁺ in cells ([Ca²⁺]_i).Ca²⁺ ions participate in the activation of diverse processes of respiration and sperm motility (Shapiro et al., 1990; Brokaw, 1991), chemotaxis of spermatozoa to components of the egg jelly (Ward et al., 1985), acrosomal reaction (Trimmer et al., 1986; Shapiro et al., 1990), cortical reaction, formation of the fertilization membrane (Sasaki, 1984; Sardet and Chang, 1987), cellular division in the embryo (Poenie et al., 1985; Silver, 1986; Whitaker and Patel, 1990), their adhesion (McClay and Matranga, 1986), differentiation and formation of spicules (Mitsunaga et al., 1988) and metamorphosis (Carpenter et al., 1984).The present review combines information on the function of calcium-binding proteins and their targets, calmodulin regulation of NAD-kinase, exocytosis of cortical granules, Ca²⁺- and calmodulin-dependent protein phosphatase, Ca²⁺-dependent protein phosphorylation, regulation of ion-exchanger in the germ and embryonic cells as well as Ca²⁺- and calmodulin control of sperm motility in sea urchins.     

Disappearance of an epithelial cell surface-specific glycoprotein (Epith-1) associated with epithelial-mesenchymal conversion in sea urchin embryogenesis

Cell surface modification during mesenchyme ingression was examined using a monoclonal antibody (mAb), anti-Epith-1 mAb, raised against a protein (Epith-1) that was confined to the lateral surface of the epithelial cells in embryo of the sea urchin, Temnopleurus hardwicki. The mAb epitope was N-glycosylated oligosaccharides of 160 kDa monomeric Epith-1 protein. The glycoprotein was negatively charged, and its isoelectric point (IP) was 4.98. The mAb, however, is not immunologically cross-reactive with other sea urchin embryos including Hemicentrotus pulcherrimus, Strongylocentrotus nudus, and Scaphechinus mirabilis. Epith-1 is present initially in the cytoplasm of unfertilized eggs. Cytoplasmic Epith-1 shifted to the cell surface to be integrated in plasma membrane during the first cleavage, and remained there during early embryogenesis by retaining the same relative molecular mass (Mr). During primary and secondary mesenchyme ingression periods, however, Epith-1 disappears from the presumptive mesenchyme cell surface that was associated with internalization of the protein. In plutei, an additional anti-Epith-1 mAb-positive protein appears at the 142 kDa region, which was not associated with any visible alteration of the histologic localization of the protein in larvae. Anti-Epith-1 mAb IgG did not inhibit the reaggregation of epithelial cells in vitro, which suggests that either the protein is not involved in cell-cell adhesion or that the mAb is not recognizing the active site of the protein.     

Analysis of competence in cultured sea urchin micromeres.

Sea urchin embryo micromeres form the primary mesenchyme, the skeleton-producing cells of the embryo. Almost nothing is known about nature and timing of the embryonic cues which induce or initiate spicule formation by these cells. A related question concerns the competence of the micromeres to respond to the cues. To examine competence in this system we have exposed cultured sea urchin micromeres to an inducing medium containing horse serum for various periods of time and have identified a period when micromeres are competent to respond to serum and form spicules. This window, between 30 and 50 h after fertilization, corresponds to the time when mesenchyme cells in vivo are aggregating and beginning to form the syncytium in which the spicule will be deposited. The loss of competence after 50 h is not due to impaired cell health since protein synthesis at this time is not significantly different from controls. Likewise the accumulation of a spicule matrix mRNA (SM 50) and a cell surface glycoprotein (msp 130), both indices of micromere/mesenchyme differentiation, still occurs in cells that have lost competence to respond to serum by forming spicules. These experiments demonstrate that the acquisition and loss of competence in these cells are regulated developmental events and establish an in vitro system for the identification of the molecular basis for inductive signal recognition and signal transduction.     

Developmental distribution of a cell surface glycoprotein in the sea urchin Strongylocentrotus purpuratus

Recent studies from this laboratory have shown that an antigen recognized by a monoclonal antibody (MAb 1223) displays a bimodal distribution of expression in development of the embryo of Strongylocentrotus purpuratus. This molecule is specifically localized to the primary mesenchyme cells of the embryo, but is also found within the egg. In the current study, immunoelectron microscopy was used to determine the subcellular distribution of the antigen and to determine its fate during early stages of development of the embryo. In eggs, the epitope recognized by MAb 1223 was localized to the cortical vesicles. Immunoblot analysis of an isolated cell surface complex (CSC) that contained the cortical vesicles revealed the presence of a 130-kDa protein, as well as immunoreactive components of higher molecular weight. Upon fertilization, the antigen was exocytosed from the cortical vesicles and became associated with the hyaline layer, the fertilization envelope, and the plasma membrane. Subsequently, the epitope could be detected within small vesicles and yolk platelets. By 60 min postfertilization, the amount of epitope detected intracellularly or in the perivitelline compartment was greatly reduced. At later stages of development, when formation of the embryonic skeleton occurred, the 1223 antigen was principally localized to the Golgi complex and to the syncytial cell surface of the primary mesenchyme cells. Thus, the results of this study suggest that in S. purpuratus the 1223 antigen is stored and secreted from the cortical vesicles of the egg, degraded after fertilization, and then later expressed on the surface of the primary mesenchyme cells.     

Ontogeny and characterization of mesenchyme antigens of the sea urchin embryo

A monoclonal antibody, Sp12, binds to cortical granules, the hyaline layer, and skeletogenic, chromogenic, and blastocoelar mesenchyme of sea urchin eggs and embryos. Adult urchins also express Sp12 antigens in the dermal layer of the test and spines. Antigen is expressed on the surface of primary mesenchyme cells after they have entered the blastocoel, and by two secondary mesenchyme derivatives--the blastocoelar cells after they have been released from the tip of the archenteron, and the pigment cells in prism stage embryos. Immunogold localizations show antigen on the surfaces of mesenchyme, within membrane bounded vesicles, and associated with the Golgi apparatus. Western blots of antigens immunoprecipitated from seven developmental stages reveal twelve antigens ranging in Mr from 35 k to 240 k. Most of these antigens appear, disappear or change Mr over the first five days of development. Characterizations of this complex array of antigens show that the epitope recognized by Sp12 is eliminated by proteolytic enzymes and endoglycosidase F, while immunoreactivity is only reduced by periodate oxidation. As well, calcium magnesium free seawater extracts a subset of antigens different from that retained by crude membrane preparations. It is proposed that the mesenchyme of sea urchin embryos produces a family of developmentally regulated cell surface and extracellular matrix glycoproteins which all exhibit a carbohydrate epitope recognized by Sp12.     

Production of monoclonal antibodies against gliofibrillary acid protein (GFAP) and their specificity

Splenic Mice cells immunized with glial fibrillary acidic protein (GFAP) were fused with SP 2/0 myeloma cells. After screening and cloning we obtained two types of hybridomas. Some of them secrete IgG class antibodies, the others IgM class antibodies. The specificity of these antibodies has been tested by three immunoenzymatic methods. The results are that IgG monoclonal antibodies identify an astrocyte-GFAP specific epitope and IgM monoclonal antibodies cross-react with a common epitope to GFAP and vimentin.     

Primary mesenchyme cells of the sea urchin embryo require an autonomously produced, nonfibrillar collagen for spiculogenesis

A collagen molecule in the sea urchin embryo was characterized by analysis of a 2.7-kb cDNA clone. This clone, Spcoll, was obtained by screening a gastrula stage Strongylocentrotus purpuratus cDNA library with a 237-bp genomic clone encoding a collagen-like sequence previously isolated by Venkatesan et al. (1986). DNA sequence analysis of the cDNA clone demonstrated the nonfibrillar nature of the encoded molecule--13 interruptions of the Gly-X-Y repeat motif were found in the 85-kDa open reading frame. The mRNA of approximately 9 kb accumulated specifically in mesenchyme cells of the embryo through development to the pluteus larva. Polyclonal antibodies generated against a Spcoll-beta-galactosidase fusion protein were utilized to identify and localize the native Spcoll. This collagen molecule of approximately 210 kDa was deposited into the blastocoel by the primary mesenchyme cells. When primary mesenchyme cells were cultured in vitro, Spcoll was secreted into the media and accumulated at sites of cell-substrate interaction. Addition of anti-Spcoll antibodies to primary mesenchyme cell cultures selectively inhibited spiculogenesis, whereas other antibodies had no inhibitory effect. Since collagen is not a component of the organic matrix of spicules (Benson et al., 1986), these results suggest that the autonomous production of Spcoll by differentiating mesenchyme cells in turn influences the point in differentiation at which these cell initiate biomineralization.     

Migratory and invasive behavior of pigment cells in normal and animalized sea urchin embryos

Pigment cell precursors in the vegetal plate of late mesenchyme blastulae of the sea urchin Strongylocentrotus purpuratus begin to express a cell surface epitope recognized by the monoclonal antibody SP-1/20.3.1. When one-quarter gastrulae are dissociated into ectodermal and mesenchymal fractions, most SP-1/20.3.1 immunoreactive cells separate into the mesenchymal fraction, whereas at the full gastrula and all later stages almost all epitope-bearing cells are in the ectodermal fraction. Exposure of embryos to sulfate-free seawater p-nitrophenyl beta-D-xyloside, and tunicamycin, all of which prevent primary mesenchyme migration, does not inhibit SP-1/20.3.1 immunoreactive cells from distributing similarly to those in controls, although pigment synthesis is completely inhibited in sulfate-free conditions. Time-lapse video sequences reveal that pigment cells, and a small set of rapidly migrating, SP-1/20.3.1 immunoreactive amoeboid cells that appear in the pluteus, remain closely associated with the ectodermal epithelium during most of larval development. Transmission electron microscopy observations of plutei show pigment cells tightly apposed to the ectodermal epithelium at discontinuities in the basal lamina and sandwiched between the basal lamina and the epithelial cells. It is concluded that SP-1/20.3.1 immunoreactive mesenchymal cells invade the ectodermal epithelium and may use migratory substrates other than those used by primary mesenchymal cells.