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.     

Inhibition of mitogen activated protein kinase signaling affects gastrulation and spiculogenesis in the sea urchin embryo

The mitogen activated protein (MAP) kinase signaling cascade has been implicated in a wide variety of events during early embryonic development. We investigated the profile of MAP kinase activity during early development in the sea urchin, Strongylocentrotus purpuratus, and tested if disruption of the MAP kinase signaling cascade has any effect on developmental events. MAP kinase undergoes a rapid, transient activation at the early blastula stage. After returning to basal levels, the activity again peaks at early gastrula stage and remains high through the pluteus stage. Immunostaining of early blastula stage embryos using antibodies revealed that a small subset of cells forming a ring at the vegetal plate exhibited active MAP kinase. In gastrula stage embryos, no specific subset of cells expressed enhanced levels of active enzyme. If the signaling cascade was inhibited at any time between the one cell and early blastula stage, gastrulation was delayed, and a significant percentage of embryos underwent exogastrulation. In embryos treated with MAP kinase signaling inhibitors after the blastula stage, gastrulation was normal but spiculogenesis was affected. The data suggest that MAP kinase signaling plays a role in gastrulation and spiculogenesis in sea urchin embryos.     

Biosynthesis of N-glycosidically linked glycoproteins during gastrulation of sea urchin embryos.

Embryos of the sea urchin, Stronglyocentrotus purpuratus, synthesize several classes of sulfated and non-sulfated glycoproteins during gastrulation. The antibiotic tunicamycin, which is a specific inhibitor of the N-glycosylation of proteins, inhibits the synthesis of lipid-linked oligosaccharides in these embryos at concentrations which have little effect on the biosynthesis of other classes of glycolipids or on protein synthesis. As a consequence of this inhibition, glycoproteins with oligosaccharide side chains of the general type (Man)5-7-(GlcNAc)2 are not synthesized. In addition, the biosynthesis of a novel class of sulfated glycoproteins is inhibited. In contrast, no effect upon the synthesis of sulfated glycosaminoglycans is seen. The morphogenetic consequence of tunicamycin treatment is that development of embryos from the mesenchyme blastula to the gastrula stage is arrested. The results provide evidence that during development glycoproteins containing both unsulfated and sulfated N-glycosidically linked oligosaccharide chains are synthesized via the lipid-linked pathway. The biosynthesis of these molecules appears to be a prerequisite to the differentiation and morphogenesis that occurs during gastrulation.     

Mitochondrial profile densities and areas in different developmental stages of the sea urchin Sphaerechinus granularis

Mitochondrial profile densities in electronmicrographs were counted in the swimming blastula, mesenchyme blastula, gastrula and prism stages of the sea urchin embryos Sphaerechinus granularis. No numerical changes were statistically apparent. When profile areas were investigated, the mean values of the swimming blastula, the gastrula and the prism stage showed no statistical differences. However, increased areas were measured in the mesenchyme blastula stage. This increase might be related to an increase of the embryonic volumina in the mesenchyme blastula stage. In contrast to earlier reported data, the results indicate that the mitochondrial density in S. granularis embryos does not alter during development in these stages.     

Developmental regulation of glycosyltransferases involved in synthesis of N-linked glycoproteins in sea urchin embryos

Previous in vivo studies using drugs that inhibit the N-glycosylation of proteins have demonstrated that newly synthesized N-linked glycoproteins are required for gastrulation in embryos of two species of sea urchins, Strongylocentrotus purpuratus and Arbacia punctulata. To understand the biochemical events regulating glycoprotein synthesis during gastrulation in S. purpuratus embryos, we examined the in vitro activities of enzymes catalyzing several of the early steps in N-linked glycoprotein synthesis. The activities of glycosyl transferases responsible for production of N,N-diacetylchitobiosylpyrophosphoryldolichol and glucosylphosphoryldolichol, two intermediates in the formation of oligosaccharylpyrophosphoryldolichol (the carbohydrate donor for N-glycosylation), were low but detectable in membranes from eggs. After fertilization these activities remained constant or increased slowly up to the blastula stage and thereafter increased rapidly at gastrulation. In agreement with these in vitro findings, in vivo labeling experiments revealed that the rate of incorporation of [3H]Man into oligosaccharylpyrophosphoryldolichol and into protein increased three- to fourfold prior to gastrulation and then slightly more at the prism stage. In contrast, in vitro activity of mannosylphosphoryldolichol synthase, another enzyme in the pathway of N-linked glycosylation, was maximal in membranes from egg and embryos in the early stages of development and declined prior to gastrulation. Furthermore, the level of this activity was at least 100-fold greater than that for enzymes involved in the formation of the chitobiosyl and glucosyl lipids. With the exception of mannosylphosphoryldolichol synthase activity, these data indicate that there is a general activation of the glycosylation apparatus before gastrulation in sea urchin embryos. Possible explanations for the decrease in mannosylphosphoryldolichol synthase activity are discussed.     

Prolyl hydroxylase activity during sea urchin development

Prolyl hydroxylase activity appears at the blastula stage of development in the sea urchin Strongylocentrotus purpuratus and increases over 7-fold by the prism larva stage. The enzyme requires ascorbate, ferrous ions, and α-ketoglutarate for maximum activity and is inhibited by α,α′-dipyridyl. The significance of prolyl hydroxylase activity in embryonic collagen metabolism and morphogenesis is discussed.     

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.     

Characterization and expression of two matrix metalloproteinase genes during sea urchin development

Matrix metalloproteinases (MMPs) play an essential role in a variety of processes in development that require extracellular matrix remodeling and degradation. In this study, we characterize two MMPs from the sea urchin Strongylocentrotus purpuratus. These clones can both be identified as MMPs based on the presence of conserved domains such as the cysteine switch, zinc-binding, and hemopexin domains. In addition, both of these genes contain consensus furin cleavage sites and putative transmembrane domains, classifying them as membrane-type MMPs. We have named these clones SpMMP14 and SpMMP16 based on the vertebrate MMPs with which they share the greatest similarity. SpMMP14 is expressed in all cells from the egg to mesenchyme blastula stage embryo. Expression of this gene is strongest in the animal and vegetal poles early in gastrulation and in the animal pole only later in gastrulation. SpMMP16 is expressed at low levels in eggs. Expression of SpMMP16 becomes more pronounced in the vegetal pole region at the blastula and mesenchyme blastula stages and becomes confined to vegetal pole descendants, such as pigment cells, later in development. In the future, we hope to learn more about the possible functions of these genes in sea urchin development.     

Stimulation of Protein Synthesis in the Mitochondria of Sea Urchin Embryos before Gastrulation

A transient increase in protein synthesis was observed in mitochondria at the mesenchyme blastula stage of sea urchin ( Hemicentrotus pulcherrimus ) embryos. This stimulated activity was inhibited by chloramphenicol but not by cycloheximide. Reconstituting experiments in which poly U-dependent protein synthesis was carried out showed the mitochondrial peptide elongation factor to be essential for increasing the protein synthetic activity in mesenchyme blastula, but aminoacyl tRNA synthetase and ribosome fraction containing initiation factor not to be involved in this increase. These findings are discussed in relation to the differentiation of embryos at the gastrulation stage.     

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.     

Change in phosvitin kinase activity during early development of the sea urchin

Protein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE-cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE-cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X-100 but was extracted by 1 M NaCl.     

Effect of actinomycin and puromycin on the deoxyribonuclease activity in P. Lividus embryos at various stages of development

The pattern of DNAse activity in sea urchin Paracentrotus lividus during early embryonic development is altered by actinomycin.When the drug is added to the embryos soon after fertilization, the decrease of DNAse activity that normally occurs before the onset of gastrulation is prevented. If actinomycin is added when DNAse activity starts to decrease, the enzyme pattern remains the same as in the control. Addition of the drug at late gastrula stage, on the other hand, brings about a transient increase of activity with respect to that of untreated embryos.Puromycin has no effect on DNAse activity during the period from fertilization to the blastula stage, whereas it inhibits the increase of activity which occurs after gastrulation. The type of regulatory mechanism involved is discussed.     

Expression of type IV collagen-degrading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis

Type IV collagen-degrading activity was expressed in homogenates of Lytechinus pictus embryos during embryogenesis. Activity was concentrated 1,600-fold by ammonium sulfate fractionation, ion exchange, and gel chromatography and could not be activated further upon trypsin or organomercurial treatment. This enzyme activity could also degrade gelatin but had no affinity for type I, III, and V collagens. Activity was inhibited by addition of excess type IV collagen or gelatin, but was unaffected by addition of excess amounts of non-collagenous proteins of the extracellular matrix. Chelators such as 1,10-phenanthroline or Na₂EDTA reduced activity to control levels. Inhibitors of plasmin and of serine and thiol proteases were without effect. Type IV collagen-degrading activity first became apparent at the stage of early mesenchyme blastula. It then increased by a small increment and remained stable up to the stage of late mesenchyme blastula, coinciding with first detection of collagen synthesis and the appearance of the archenteron. Thereafter, a sharp increase in activity was observed, concurrently with remodelling of the archenteron. Maximum activity was attained at prism stage and was retained throughout to pluteus-larva stage. The specific inhibitors of collagen biosynthesis 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide and tricyclodecane-9-yl xanthate arrested sea urchin embryo development at early blastula, prevented the invagination of the archenteron, and reverted the expression of type IV collagen-degrading activity to non-detectable levels. Removal of the inhibitors allowed embryos to gastrulate and express type IV collagen-degrading activity.