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.     

A new extracellular matrix protein of the sea urchin embryo with properties of a substrate adhesion molecule

Summary A new embryonic extracellular matrix protein has been purified from eggs of the sea urchin Paracentrotus lividus. The molecule is a 210 kD dimer consisting of two 105 kD subunits that are held together by S-S bridges. In the unfertilized egg, the protein is found within granules uniformly distributed throughout the cytoplasm. After the egg is fertilized, the antigen is polarized to the apical surface of ectodermal and endodermal cells during all of the developmental stages examined, until the pluteus larva is formed. The protein promotes the adhesion of blastula cells to the substrate and is antigenically distinct from echinonectin, a well characterized substrate adhesion molecule. This report adds a new candidate to the list of known extracellular matrix molecules for the regulation of differentiation and morphogenesis in the sea urchin embryo. Offprint requests to: V. Matranga     

Constraint, flexibility, and phylogenetic history in the evolution of direct development in sea urchins

Development in sea urchins typically involves the production of an elaborate feeding larva, the pluteus, within which the juvenile sea urchin grows. However, a significant fraction of sea urchins have completely or partially eliminated the pluteus, and instead undergo direct development from a large egg. Direct development is achieved primarily by heterochrony, that is, by the abbreviation or elimination of larval developmental processes and the acceleration of processes involved in development of adult features. Direct development has evolved independently several times, and in several ways. These radically altered ontogenies offer remarkable opportunities for the study of the mechanisms by which early development undergoes evolutionary modification. The recent availability of monoclonal antibody and cDNA probes that recognize homologous cells in embryos of closely related typical and direct developing species makes possible an experimental analysis of the cellular and molecular bases for heterochronic changes in development.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase in the sea urchin embryo is developmentally regulated

The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, an enzyme which plays a regulatory role in the synthesis of cholesterol, dolichol, and coenzyme Q, has been measured in the developing embryo of the sea urchin. Enzyme activity increased at least 200-fold during development from the unfertilized egg to the pluteus stage embryo. Mixing experiments suggested that the low level of enzyme activity found at early stages was not due to the presence of inhibitor(s) in the egg or zygote. The enzyme in the sea urchin embryo exhibited properties different from that found in mammals: only a fraction of the activity could be solubilized from microsomes, and mild trypsinization inactivated the enzyme without releasing any of it from the microsomes in soluble form. To further study the sea urchin HMG-CoA reductase, a genomic clone was identified by hybridization to a cDNA encoding hamster HMG-CoA reductase. Sequence analysis of this clone revealed a coding region that shares a high degree of homology with the carboxyl-terminal domain of hamster HMG-CoA reductase. Analysis of sea urchin embryo HMG-CoA reductase mRNA levels using a restriction fragment derived from the genomic clone revealed a 5.5-kilobase poly(A)+ mRNA that increased 15-fold during development from the egg to the gastrula stage and then decreased 1.5-fold at the pluteus stage. Since the relative increase in HMG-CoA reductase mRNA was less than the increase in enzyme activity (15-fold versus 200-fold) factors in addition to the level of mRNA may control the activity of this enzyme during embryogenesis.     

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.     

Isolation and characterization of cDNA encoding a spicule matrix protein in Hemicentrotus pulcherrimus micromeres.

A cDNA clone, termed pHPSMC, was obtained from the Japanese sea urchin Hemicentrotus pulcherrimus, and it was found to be highly homologous in sequence to the spicule matrix protein cDNA of Strongylocentrotus purpuratus (Sucov et al., Dev. Biol. 120: 507-519, 1987). During early embryogenesis, mRNA complementary to pHPSMC appeared in gastrulae and remained at a similar level until the pluteus stage. In situ hybridization revealed that the mRNA was localized exclusively in primary mesenchyme cells in gastrulae. pHPSMC mRNA was detected in micromeres in vitro after 48 h of culture, but it was not found in blastomeres immediately after isolation. These features suggested that pHPSMC represents the spicule matrix protein cDNA cognate in Hemicentrotus pulcherrimus. In the derived polypeptide, we detected a domain containing a tandemly repeated 13-amino acid sequence as did Sucov et al. (1987). Unexpectedly, the sequence of the repeated element was completely different from that originally reported for Strongylocentrotus purpuratus, but it was very similar to the corrected sequence that appeared recently.     

A large calcium-binding protein associated with the larval spicules of the sea urchin embryo

A tissue-specific, high molecular weight, calcium-binding protein from the sea urchin embryo is described. This protein, designated as CBP 180, has a molecular weight of 180,000 under reducing conditions, and is extractable with 1% Triton X-100. It accumulates rapidly during development, starting roughly at the onset of spiculogenesis. When embryos are cultured in the presence of inhibitors of spicule formation, such as tunicamycin and zinc ions, accumulation of CBP180 is depressed or stopped. By immunofluorescence technique and by using an antibody specifically generated against this protein, CBP180 is mainly localized in primary mesenchyme cells and spicular syncytium of the pluteus larva. Little or none is detectable in ectoderm, endoderm or blastocoelar extracellular matrix. These results suggest that the protein is involved in calcium sequestration in the differentiation of larval spicules.     

Insulin-related molecules and insulin effects in the sea urchin embryo

Insulin, the polypeptide hormone secreted by the differentiated pancreas, may play a role in vertebrate development at prepancreatic stages. In an invertebrate embryo, the sea urchin Strongylocentrotus purpuratus, we now find that insulin modulates the levels of developmentally regulated mRNAs of different lineages (one ectoderm-specific, one mesoderm-specific, and one found in all cell types). Using indirect immunofluorescence, we have localized a molecule which shares antigenic determinants with mammalian insulin in the unfertilized egg as well as in the gut of pluteus larva sea urchins. In addition, Southern hybridization reveals high similarity between sea urchin DNA sequences and the human insulin receptor gene. Our results suggest the presence of an insulin/insulin receptor-related system in sea urchin development.     

Multiple levels of regulation of protein synthesis at fertilization in sea urchin eggs

Fertilization of sea urchin eggs results in a large stimulation of protein synthesis. This increase in protein synthesis is mediated by the mobilization of stored maternal mRNA (mRNPs) into polysomes, but the details of the molecular mechanisms which regulate this process are not well understood. Using a sea urchin egg cell-free translation system, evidence has been obtained which indicates that the capacity to initiate protein synthesis on new mRNAs is limited. Addition of exogenous mRNAs failed to stimulate overall protein synthesis, whereas supplementing the system with a nuclease-treated reticulocyte lysate, an S-100 supernatant fraction, or purified eIF-2 stimulated nearly twofold. In addition, the levels of 43 S preinitiation complexes containing a 40 S ribosomal subunit and methionyl-tRNA were increased at pH 7.4 compared to pH 6.9, or when reticulocyte S-100 was added. However, other experiments showed clearly that mRNA availability may also regulate translation in the sea urchin egg. Sea urchin lysates only stimulated poorly the nuclease-treated reticulocyte lysate system, and the mRNPs in the sea urchin lysate did not bind to reticulocyte 43 S preinitiation complexes. Since purified sea urchin egg mRNA was active in both assays, the bulk of sea urchin mRNA must be masked in the egg, and remain masked in the in vitro assays. Thus, protein synthesis appears to be regulated at both the level of mRNA availability and the activity of components of the translational machinery.     

Sea urchin TgBMP2/4 gene encoding a bone morphogenetic protein closely related to vertebrate BMP2 and BMP4 with maximal expression at the later stages of embryonic development.

We have cloned a gene fragment (named TgBMP2/4) that encodes a protein homologous to vertebrate bone morphogenetic protein (BMP) 2 and BMP4 in the sea urchin Tripneustes gratilla. This peptide sequence contains 204 amino acids with 7 conserved cysteine residues at the C-terminus of the coding region and a cluster of basic amino acids that may serve as a signal for proteolytic cleavage. Sequence comparison and phylogenetic analyses reveal that TgBMP2/4 is closely related to vertebrate BMP2 and BMP4 as well as to amphioxus BMP2/4, with similarity levels ranging from 90% to 94% at the mature C-terminal domain. Northern blot analyses show that a 6.3-kb TgBMP2/4 mRNA appears first at the mesenchyme blastula stage and increases to a maximal level at the gastrula and pluteus stages. This expression pattern is different from that of a BMP2/4-related gene previously found in sea urchin.     

Developmental regulation,induction, and embryonic tissue specificity of sea urchin metallothionein gene expression

Metallothionein (MT) is shown to be present in sea urchin embryos on the basis of its characteristic properties as a small protein (6–7 Da) of extraordinarily high cysteine content, whose biosynthesis is readily induced by heavy metals. Induction by Zn²⁺ results in the accumulation of the cysteine-rich MT protein, a 0.8 kb MT mRNA and a 2.9 kb nuclear RNA. The amount of MT mRNA is regulated intrinsically through the course of embryogenesis to the pluteus stage: A maternal MT mRNA is poly(A)-deficient and is polyadenylated after fertilization. New MT mRNA begins to accumulate between the seventh and eighth cell cleavage, reaches a maximum at the mesenchyme blastula stage, decreases during gastrulation, and rises again in the early pluteus stage. “Animalizing” embryos with Zn²⁺ during early embryogenesis causes a sustained accumulation of MT mRNA to levels greater than 25 times the normal amount. MT mRNA is present in high amount in the ectoderm of the pluteus, but is barely detectable in the mesoderm-endoderm tissue fraction. Treatment of either the pluteus or its isolated tissue fractions with Zn²⁺ results in the induction of MT mRNA accumulation in the mesoderm-endoderm but not in the already MT mRNA-enriched ectoderm. Furthermore, differences in Zn²⁺ induction of the MT gene in the blastula and gastrula are consistent with a developmental pattern in which MT gene expression is maintained constitutively at a high level in the ectoderm and at a low level in the mesoderm-endoderm tissues, which are, however, preferentially inducible by Zn²⁺.     

Territorial expression of three different trans-genes in early sea urchin embryos detected by a whole-mount fluorescence procedure.

We have developed a new procedure for detection of the protein product of chloramphenicol acetyltransferase (CAT) reporter genes in whole mounted sea urchin embryos. The position of a commercially available anti-CAT antibody is visualized by video or confocal microscopy, and thus the spatial domains of exogenous reporter gene expression can be determined with regard to the intact three-dimensional structures of the embryo. We show that in pluteus stage embryos CAT protein expression patterns for SM50 . CAT or CyIIIa . CAT reporter genes are similar to those previously obtained by in situ hybridizations with radioactive probes. Taking advantage of the superior resolution of cellular CAT expression patterns using the antibody visualization method, we found for the first time that, in addition to the expression in aboral ectoderm, some cells in the ciliated band of the pluteus express CyIIIa . CAT. The expression of a new fusion construct, CyIIa . CAT, was also examined. As expected from the localization of endogenous CyIIa mRNA, CAT protein was expressed under control of the CyIIa promoter in gut and skeletogenic mesenchyme cells.     

Structure and tissue-specific developmental expression of a sea urchin arylsulfatase gene

Arylsulfatases are a group of enzymes that remove sulfate moieties from a diverse set of substrates including glycoproteins, steroids, and cerebrosides. We have isolated recombinant cDNA clones corresponding to an arylsulfatase (SpARS) message that encodes an abundant protein of pluteus larvae of the sea urchin Strongylocentrotus purpuratus. Although vertebrate arylsulfatases have broad tissue distributions, in situ hybridization with a probe for SpARS shows that the sea urchin message accumulates in the embryo only in the single cell type of aboral ectoderm and its precursors. The message is first detectable by RNase protection assays around hatching blastula stage and accumulates through pluteus larva stage. The open reading frame of cDNA clones is 1701 nt long and encodes a deduced protein with a predicted molecular mass of 61 kDa. Analysis of corresponding genomic DNA clones reveals that the pre-mRNA contains six exons. Consistent with the fact that arylsulfatase enzyme activity is extracellular, this polypeptide has a hydrophobic leader sequence and three potential glycosylation sites. Furthermore, hybridization in situ shows that in blastulae arylsulfatase message is preferentially concentrated around nuclei at the basal sides of cells. The S. purpuratus sequence is very similar to that recently reported for the same enzyme from Hemicentrotus pulcherrimus and 30% of the amino acid residues are also identical to those of both human arylsulfatase C (steroid sulfatase) and arylsulfatase A. Sequence relationships among these four mRNAs suggest that, assuming equal rates of evolution, the duplication separating the human genes occurred at about the time of separation of the echinoderm and vertebrate lineages.