Biomedical subjects

G M Wessel

Publications and source records attributed to G M Wessel.

At least 37 records · Page 2Linked to original sources

PubMed1997

The exocytosis regulatory proteins syntaxin and VAMP are shed from sea urchin sperm during the acrosome reaction.

Syntaxin is a cytoplasmically oriented plasma membrane protein and VAMP (vesicle-associated membrane protein; synaptobrevin) is a protein associated with the secretory vesicle membrane. These two proteins form part of a complex which is thought to mediate the fusion of plasma and vesicle membranes during exocytosis. This paper reports the identification of syntaxin and VAMP homologues in sea urchin sperm. During fertilization, sea urchin sperm release the contents of a single vesicle, the acrosomal vesicle, exposing the membrane destined to fuse with the egg. During acrosomal exocytosis, the plasma membrane over the acrosomal vesicle fuses at multiple points with the acrosomal membrane (vesiculation) and syntaxin and VAMP are shed with the resulting membrane vesicles. Sea urchin sperm syntaxin and VAMP are associated in a complex as detected by immunoprecipitation. Following acrosomal exocytosis, syntaxin and VAMP cosediment to denser fractions on sucrose gradients showing that they have undergone associative changes during or after the acrosome reaction. Syntaxin and VAMP localization and loss during acrosomal exocytosis support a role for these proteins in regulating the acrosome reaction.

Acrosome↗

PubMed1997

Cortical granules of the sea urchin translocate early in oocyte maturation.

Cortical granules are secretory vesicles poised at the cortex of an egg that, upon stimulation by sperm contact at fertilization, secrete their contents. These contents modify the extracellular environment and block additional sperm from reaching the egg. The role of cortical granules in blocking polyspermy is conserved throughout much of phylogeny. In the sea urchin, cortical granules accumulate throughout the cytoplasm during oogenesis, but in mature eggs the cortical granules are attached to the plasma membrane, having translocated to the cortex at some earlier time. To study the process of cortical granule translocation to the cell surface we have devised a procedure for maturation of sea urchin oocytes in vitro. Using this procedure, we examined the rate of oocyte maturation by observing the movement and breakdown of the germinal vesicle, the formation of polar bodies and the formation of the egg pronucleus. We find that oocyte maturation takes approximately 9 hours in the species used here (Lytechinus variegatus), from the earliest indication of maturation (germinal vesicle movement) to formation of a distinct pronucleus. We then observed the translocation of cortical granules in these cells by immunolocalization using a monoclonal antibody to hyalin, a protein packaged specifically in cortical granules. We found that the translocation of cortical granules in in vitro-matured oocytes begins with the movement of the germinal vesicle to the oocyte cell surface, and is 50% complete 1 hour after germinal vesicle breakdown. In the in vitro-matured egg, 99% of the cortical granules are at the cortex, indistinguishable from translocation in oocytes that mature in vivo. We have also found that eggs that mature in vitro are functionally identical to eggs that mature in vivo by four criteria. (1) The matured cells undergo a selective turnover of mRNA encoding cortical granule contents. (2) The newly formed pronucleus begins transcription of histone messages. (3) Cortical granules that translocate in vitro are capable of exocytosis upon activation by the calcium ionophore, A23187. (4) The mature egg is fertilizable and undergoes normal cleavage and development. In vitro oocyte maturation enables us to examine the mechanism of cortical granule translocation and other processes that had previously only been observed in static sections of fixed ovaries.

Animals↗

PubMed1996

Endoderm differentiation in vitro identifies a transitional period for endoderm ontogeny in the sea urchin embryo.

The vegetal plate of the sea urchin embryo is specified during early cleavage divisions of the embryo as shown by the classical experiments of Horstadius (reviewed in "Experimental Embryology of Echinoderms," 1973, Clarendon, Oxford). Not until gastrulation, though, do the cells within this territory differentiate into their characteristic cell types. Vegetal plate descendents comprise the coelomic epithelium, circumesophageal muscle, basal cells, pigment cells, and endodermal epithelium. We report here that cells of the endodermal lineage acquire the ability to differentiate autonomously several hours prior to gastrulation, between the late blastula and early mesenchyme blastula stages. Cells dissociated from whole embryos after the late blastula stage have the ability to differentiate in vitro, independent of cell contacts and of the embryonic environment. In contrast, preendoderm cells removed from the embryo prior to the late blastula stage show no ability to differentiate when cultured in vitro even though cells of other lineages, e.g., ectoderm and skeletogenic mesenchyme, show morphological and molecular differentiation in these same cultures. We have used the expression of the endoderm-specific gene products Endo 1 and LvN1.2, detected by RNase protection assays and by in situ immunolabeling, to quantify endoderm differentiation independent of embryonic or cellular morphology. These studies define a transitional period in the ontogeny of the endoderm, from cells reliant on interactions to promote fate specification and organization of territories to later events involved in morphogenesis that result from cell-type-specific gene expression.

Animals↗

PubMed1996

An extracellular matrix molecule that is selectively expressed during development is important for gastrulation in the sea urchin embryo.

The extracellular matrix is important in the regulation of many cellular events of early development including migration, shape change, proliferation and gene expression. In the sea urchin embryo, disruption of the extracellular matrix results in selective defects in each of these events during gastrulation. Here we describe a new molecule of the extracellular matrix in Lytechinus variegatus, referred to as ECM 18, that has several important features. First, antibody interference of ECM 18 results in a profound but reversible inhibition of primary mesenchyme cell organization and endoderm morphogenesis during gastrulation. Second, during gastrulation, ECM 18 mRNA accumulates to highest levels in the invaginating endoderm and the ECM 18 protein deposited in the basal lamina surrounding the archenteron as well as in other areas of the blastocoel wall. Immunolocalization by fluorescence and electron microscopy demonstrates the selective accumulation of ECM 18 in the extracellular matrix. Third, although the mRNA encoding ECM 18 is present throughout development, the protein accumulates only during gastrulation. ECM 18 protein is not detected in eggs or early embryos and analysis of polysome-associated mRNA suggests that at least part of the translational regulation of ECM 18 is at the level of ECM 18 mRNA-polysome formation. Finally, sequence analysis of ECM 18 shows that the protein contains a repeat sequence with a conserved cysteine motif, suggestive of involvement in protein-protein interactions. Thus, ECM 18 appears to be important in mediating select morphogenetic changes during gastrulation and the pattern of its expression in the embryo is unique among the extracellular matrix molecules known in this embryo.

Amino Acid Sequence↗

PubMed1995

A protein of the sea urchin cortical granules is targeted to the fertilization envelope and contains an LDL-receptor-like motif.

Cortical granules of eggs contain a population of heterogeneous proteins that are sequestered selectively within the cortical granule during oogenesis and that participate in the block to polyspermy during the fertilization reaction. To begin to understand the targeting mechanism of proteins to this organelle, we have identified cDNA clones that encode proteins of the cortical granule. Here we characterize the protein encoded by the cDNA, SFE 9, which is packaged specifically into the spiral lamellae of the cortical granules in the sea urchin Strongylocentrotus purpuratus. The SFE 9 protein showed a molecular mobility in PAGE of 150 kDa, close to the estimated 128 kDa predicted by cDNA sequence, and contained three regions of distinct repeating sequence. Each type of repeat sequence was of different length, frequency, and amino acid composition, but each type showed a high degree of conservation within a repeat type. A fourth region of sequence showed similarity to the low-density lipoprotein (LDL)-receptor motif. The N-terminal 2/3 of the protein contained an abundance of cysteine residues with regular and conserved spacings. Upon fertilization, SFE 9 was secreted and was rapidly incorporated into the fertilization envelope. The integration of SFE 9 into the fertilization envelope was complete within 5 min post-fertilization and was mediated by ovoperoxidase-catalyzed cross-linking. Incorporation of SFE 9 into the fertilization envelope was not uniform. Instead, it concentrated to the microvillar casts both in normal and in ovoperoxidase-inhibited fertilization envelopes. Thus, SFE 9 shows molecular heterogeneity within the fertilization envelope and the targeted integration is independent of ovoperoxidase activity. The strong repeat regions and the LDL-receptor motif of SFE 9 are postulated to be used in specific protein-protein interactions that occur either during traflicking to the cortical granules during biogenesis or to the fertilization envelope during the fertilization reaction.

Amino Acid Sequence↗

PubMed1995

A diversity of enzymes involved in the regulation of reversible tyrosine phosphorylation in sea urchin eggs and embryos.

Reversible tyrosine phosphorylation is involved in the fertilization reaction and early embryogenesis of the sea urchin (Foltz and Shilling, 1993; Ramachandran et al., 1993). To determine the enzymes present that may be involved in this regulation, we used a PCR screen to identify sequences that encode protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP). We identified five PTKs and eight PTPs using cDNA libraries from two sea urchin species at two different stages of development, and the similarities to known PTK and PTP amino acid sequences ranged from 70 to 95%. The cognate proteins represented both "receptor"-class and cytoplasmic enzymes. Using RNAse protection assays we found that the respective mRNAs showed many accumulation profiles that we have grouped into three basic patterns: (1) mRNA levels that do not vary by more than two to three times throughout development; (2) mRNA levels highest in eggs or ovaries; and (3) mRNA levels highest in gastrula or pluteus stages. mRNAs specific to adult somatic cells of the ovary were not found, nor were mRNAs that accumulated selectively at the blastula stage. The results show that a diversity of enzymes involved in the regulation of reversible tyrosine phosphorylation is present in eggs and embryos of the sea urchin and that the differential accumulation in development of each mRNA suggests specific functional responsibilities by members of these enzyme families.

Amino Acid Sequence↗

PubMed1994

Cortical granule biogenesis is active throughout oogenesis in sea urchins.

Cortical granules are secretory vesicles formed in the eggs of most animals and are essential for the prevention of polyspermy in these organisms. We have studied the biogenesis of cortical granules in sea urchin oocytes by identifying cDNA clones that encode proteins targeted selectively to the cortical granules. These cDNA clones were identified by an immunoscreen of a cDNA library using antibodies to proteins of the fertilization envelope. Four different mRNAs were identified, ranging from 4 kb to 13 kb in length, that encoded proteins targeted specifically to cortical granules. Accumulation of these mRNAs began very early in oogenesis, in oocytes approximately 10-15 microns in diameter, and continued throughout oogenesis. The mRNAs reached peak abundance (on a per cell basis) in germinal vesicle stage oocytes, and the accumulation of each mRNA was linear with respect to oocyte growth. During breakdown of the germinal vesicle these mRNAs were degraded so that in eggs the mRNA signals were at background levels. Antibodies generated to recombinant proteins made from each of these cDNA clones showed that in the oocyte each cognate protein appeared early in oogenesis. These proteins accumulated only in cortical granules: no accumulation was seen in the cytoplasm, in Golgi, or in other vesicles, and no heterogeneity of the contents was seen within the population of cortical granules. Using these antibodies we show that cortical granules accumulated linearly throughout oogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Transient, localized accumulation of alpha-spectrin during sea urchin morphogenesis.

The mRNA and protein of alpha-spectrin in the sea urchin embryo is shown here to be transiently overexpressed in cells initiating certain morphogenetic changes. This expression was detected by use of a 4.5-kb cDNA clone that encodes alpha-spectrin isolated from a Lytechinus variegatus cDNA library. DNA sequence analysis demonstrated 80% similarity of this cDNA to human nonerythroid alpha-spectrin. Sea urchin alpha-spectrin RNA accumulated to a uniform basal level in all cells of the embryo with a marked, transient increased accumulation in the primary mesenchyme cells of the vegetal plate just prior to gastrulation. Cells of the endoderm also express increased levels of spectrin during gastrulation and these levels remained high in the myoepithelial cells of the sphincter constrictions that separates the gut into foregut, midgut, and hindgut domains. Antibodies made to recombinant sea urchin spectrin synthesized in Escherichia coli were used to localize the protein in situ. These experiments showed that all cells of the embryo accumulated alpha-spectrin protein confined to the cortical regions of cell-cell contact. In epithelial cells, this localization showed a distinct honeycomb pattern. Primary mesenchyme cells and the myoepithelial cells of the gut sphincters showed significantly enhanced spectrin signal corresponding to the pronounced RNA localization pattern seen by in situ RNA hybridization. These data demonstrated that alpha-spectrin from the sea urchin is a highly conserved member of the spectrin superfamily of proteins and that its elevated expression in specific cell types anticipates overt morphogenesis.

Animals↗

PubMed1991

Structure and expression of the polyubiquitin gene in sea urchin embryos.

A cloned Lytechinus pictus cDNA has been identified, which includes seven direct repeats of a 228 bp sequence encoding ubiquitin and about 450 bp of 3' noncoding sequence. The deduced amino acid sequence is identical to that of ubiquitins of other animals (though repeats 3 and 5 each have single amino acid substitutions at different positions). Southern blot analysis revealed that the sea urchin genome contains a single copy of the polyubiquitin gene, and the number of 228 bp repeat units appears to vary from seven to ten among different alleles; no other ubiquitin coding sequences were detected. The size distribution of polyubiquitin mRNA is polymorphic among different individuals, probably corresponding to the differences in copy number of the repetitive coding sequence. The abundance of cytoplasmic polyubiquitin RNA is constant throughout embryogenesis and is similar in ectoderm, endoderm, and mesoderm cells. The constant prevalence of polyubiquitin mRNA apparently results from a balance between ontogenetic changes in its rate of synthesis and its stability in the presence of actinomycin D. Accumulation of polyubiquitin RNA was not heat shock-inducible during embryogenesis.

Amino Acid Sequence↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1990

Myosin heavy chain accumulates in dissimilar cell types of the macromere lineage in the sea urchin embryo.

In this study, myosin heavy chain from sea urchin pluteus larvae was characterized by analysis of a 2.5-kb cDNA clone. DNA sequence of 1465 bp demonstrated a 71% similarity in the deduced amino acid sequence to the embryonic rat skeletal muscle sequence. Antibodies generated against a polypeptide encoded by the open reading frame of the cDNA clone specifically identified a 210-kDa myosin protein which accumulated in 8-12 muscle cells differentiating bilateral to the esophagus beginning at early larval stages. This same myosin also accumulated in cells of the endodermal epithelium that comprise the three sphincters of the larval gut. Thus, a gene encoding myosin heavy chain is expressed in dissimilar cell types of the macromere lineage, and the pattern of accumulation in the gut identifies functionally distinct cells of the endodermal epithelium.

Amino Acid Sequence↗

PubMed1990

Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.

The Spec1 and Spec2 genes of Strongylocentrotus purpuratus are closely associated with the differentiation of aboral ectoderm. To examine cis-regulatory elements involved in the spatial expression of the Spec genes, we fused the Escherichia coli lacZ gene containing a nuclear targeting signal to 5'flanking DNA plus 5' untranslated leader sequences from Spec1, Spec2a, and Spec2c. All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions. The Spec-lacZ reporter gene plasmids were microinjected into eggs of S. purpuratus, Lytechinus variegatus, and L. pictus, and beta-galactosidase activity was determined in situ by X-gal staining. The Spec2a-lacZ fusion gene, which contained 1516 bp of 5' flanking DNA and 18 bp of 5' untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species. The Spec1-lacZ fusion gene was expressed in a strikingly different fashion--preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells. The staining pattern was the same in either homologous or heterologous embryos. The Spec2c-lacZ fusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed. To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5' flanking DNA from the Spec2a-lacZ fusion gene, resulting in a delta Spec2a-lacZ fusion gene that contained only the conserved DNA region. This gene fusion showed preferential expression in aboral ectoderm cells. However, the cell type specificity was not as great as with the parental Spec2a-lacZ plasmid. These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between -1516 and -697 bp in the 5' flanking DNA of Spec2a.

Animals↗

PubMed1989

Gastrulation in the sea urchin is accompanied by the accumulation of an endoderm-specific mRNA.

Spatial diversification of the endoderm during gastrulation in the sea urchin Lytechinus variegatus was examined with an endoderm-specific cDNA clone. This cDNA clone, LvN1.2, was identified by a differential cDNA screen between the ectoderm and endoderm/mesoderm fractions from prism stage embryos. The LvN 1.2-kb mRNA was first detectable by Northern blots at the mesenchyme blastula stage just prior to gastrulation and then accumulated approximately 15-fold from gastrulation to the pluteus stage. In situ hybridization analysis demonstrated that the mRNA accumulated specifically in endoderm and was restricted to the hindgut-midgut regions. This restricted localization was apparent during gastrulation and predicted the morphological distinction between foregut and midgut eventually seen at prism and pluteus stages. Sequence analysis showed that the 189-amino acid open reading frame represented a novel protein. In vitro translation of synthetically produced LvN1.2 mRNA and Western blot analysis with antibodies to the protein sequence yielded the same 25-kDa polypeptide on SDS-PAGE. The LvN1.2 protein resided within discrete granules of the hindgut and midgut cells. These particles were concentrated to the luminal aspect of the cells, suggesting the LvN1.2 protein participates in the digestive function of this region of the gut.

Amino Acid Sequence↗

PubMed1989

Cortical granule-specific components are present within oocytes and accessory cells during sea urchin oogenesis.

The coordinate expression of cortical granule-specific components in sea urchin oogenesis was studied using antibody probes. The components used to generate the organelle-specific antibodies included the whole cortical granule exudate, fertilization envelopes, hyalin, beta, 1-3,glucanase, and Ig8. Using immunolocalization techniques at both the light and electron microscopic levels, these molecules were found to be specific to cortical granules in three distinct cell types: developing oocytes, eggs, and accessory cells. In early oocytes, each of the cortical granule components are coordinately accumulated in the developing cortical granules dispersed throughout the cytoplasm. No other organelle within the developing oocytes or eggs contained detectable levels of any of these epitopes. In the somatic interstitial tissue of the ovary, cortical granule components also were accumulated specifically within cortical granule structures. Found only in select accessory cells, these cortical granules were indistinguishable in morphology and epitope composition from eggs and were contained within cytoplasmic aggregates termed mosaic globules. The mechanism of cortical granule concentration into mosaic globules is unknown, but this demonstration of common organelle constituents between oocytes and accessory cells may provide insight for such an understanding.

Animals↗

PubMed1989

Transcription of the Spec 1-like gene of Lytechinus is selectively inhibited in response to disruption of the extracellular matrix.

The influence of the extracellular matrix (ECM) on differential gene expression during sea urchin development was explored using cell-type-specific cDNA probes. The ECM of three species of sea urchin, Strongylocentrotus purpuratus, Lytechinus variegatus and Lytechinus pictus, was disrupted with the lathrytic agent beta-aminopropionitrile (BAPN), which inhibits collagen deposition in the ECM and arrests gastrulation (Wessel & McClay, Devl Biol. 121: 149, 1987). The levels of several mRNAs (Spec 1, Spec 2, CyIIa actin, CyIIIa actin and collagen in S. purpuratus, and metallothionine, ubiquitin and LpS3 in L. pictus and L. variegatus) were compared in BAPN-treated and control embryos. These mRNAs accumulated normally during BAPN treatment, even though the embryos did not gastrulate. To determine if the expression of any gene product is sensitive to ECM disruption, a differential cDNA screen compared poly (A+) RNA from BAPN-arrested and control embryos in Lytechinus. A cDNA clone was isolated from this screen that represented a 2.1 kb mRNA that did not accumulate during BAPN treatment. Removal of BAPN resulted in the accumulation of this transcript coincident with the onset of gastrulation. This cDNA clone encodes a L. variegatus homologue of LpS1, recently demonstrated to be an ancestral homologue of the aboral ectoderm-specific Spec 1-Spec 2 gene family in S. purpuratus. Nuclear run-on assays in L. pictus suggested that transcriptional activity of LpS1 was selectively inhibited by BAPN treatment. Thus, although the accumulation of many gene products occurred independently of the embryonic collagenous matrix, the accumulation of LpS1 and LvS1 appeared to be mediated by the ECM.

Actins↗

PubMed1988

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.

Animals↗

PubMed1987

A cortical granule-specific enzyme, B-1,3-glucanase, in sea urchin eggs.

The ultrastructural localization of B-1,3-glucanase in three species of sea urchin eggs was determined using a monospecific antibody in an electronmicroscopic immunogold procedure. In all three species, Lytechinus variegatus, Strongylocentrotus purpuratus, and Arbacia punctulata, B-1,3-glucanase was localized specifically to the cortical granules. No other organelle within the egg contained significant label. During the fertilization reaction, B-1,3-glucanase was released from cortical granules into the perivitelline space and became associated with the hyaline layer. No significant label was found in association with the fertilization envelope.

Animals↗

PubMed1987

Gastrulation in the sea urchin embryo requires the deposition of crosslinked collagen within the extracellular matrix.

This study demonstrates that a collagenous extracellular matrix (ECM) is necessary for gastrulation in the sea urchin embryo. The approach taken was to disrupt collagen processing with two types of agents (a lathyritic agent, beta-aminopropionitrile (BAPN), and three types of proline analogs: dehydroproline, cis-OH-proline, and azetidine carboxylic acid) and to assess the effect on embryogenesis by morphological, immunological, and biochemical criteria. Embryos chronically exposed to either of the agents following fertilization displayed no detectable developmental abnormalities before the mesenchyme blastula stage. These embryos, however, did not gastrulate nor differentiate any further and remained at the mesenchyme blastula stage for at least 36 hr. Upon removal of the agents, the embryos resumed a normal developmental schedule and formed pluteus larvae that were indistinguishable from control embryos. By immunofluorescence studies with monospecific antibodies to type I and type IV collagens it is seen that the lathyritic agent BAPN reduces the accumulation of collagens within the ECM. This effect is confirmed and quantitated by use of an ELISA and by a biochemical determination of OH-proline. When the agents are removed from the inhibited embryos, collagen deposition returns to normal, coincident with gastrulation. Western-blot analysis, using monospecific antibodies to collagen, demonstrates that the effect of the lathyritic agent is to reduce the stability of the extracellular collagen by inhibiting the intra- and intermolecular crosslinking of collagen molecules. BAPN exhibits a dose-dependent effect on morphogenesis, but has no effect on respiration nor on protein synthesis of the embryos throughout development. Although the lathyritic agent affects collagen deposition, it is shown to not affect the expression of other molecules of the ECM, nor that of several cell surface molecules. However, a cell surface molecule that is expressed specifically in the endoderm, termed Endo 1, is not expressed in the inhibited embryos. Endo 1 is expressed after removal of the lathyritic agent and its appearance is coincident with gastrulation in the recovered embryos. These results suggest that a collagenous ECM is important for gastrulation and subsequent differentiation in the sea urchin, but not for earlier developmental processes. In addition, the dependence of Endo 1 expression on the collagenous ECM raises the possibility that this cell surface molecule is in some way regulated by interactions of the presumptive endodermal cells with the ECM.

Aminopropionitrile↗