In vitro methylation of histones in sea urchin nuclei during early embryogenesis

Nuclei isolated from sea urchin embryos incubated in vitro in the presence of S-adenosyl-[methyl-3H]methionine, methylate their own basic proteins. The protein methylase activity varies during the embryonic development with two peaks of activity at mesenchymal blastula and at young gastrula. Histones H3 and H4 are the main substrates of the reaction. The extent of methylation of the two histones depends on the S-adenosylmethionine concentration. At low S-adenosylmethionine concentrations, the in vitro methyl-accepting ability of H3 is 10-times that of H4, while at high concentrations it is 3-times that of H4. This finding is clearly evident in the equilibrium saturation experiments with blastula and gastrula nuclei, which both show two distinct Km values for S-adenosylmethionine. The major and perhaps only product of methylation is epsilon-N-methyl-lysine. Enzyme activity is clearly correlated with specific embryonic stages, while no correlation is apparent between enzyme activity and the amount of DNA in the embryos.     

Spermidine labels proteins during sea urchin embryogenesis

We have previously described the presence of a protein containing intact, covalently bound spermidine during very early embryogenesis of the sea urchin (Strongylocentrotus purpuratus). Proteins containing other polyamine metabolites also appear as embryogenesis proceeds. These proteins which contain label derived from exogenous radioactive spermidine show a characteristic pattern which changes during the course of embryonic development. We document for the first time that hypusine, the polyamine metabolite which is a characteristic component of the eukaryotic protein translation initiation factor eIF-4D, is present in more than one species of macromolecule. In addition, N1-acetylspermidine has also been identified as a significant intracellular metabolite of spermidine during embryogenesis.     

Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis

We have determined spatial patterns of expression of individual actin genes in embryos of the sea urchin Strongylocentrotus purpuratus. Radioactively labeled probes specific for each of five cytoplasmic-type (Cy) and the single muscle-type (M) mRNAs were hybridized in situ to sections of fixed embryos. M actin mRNA appears only late in development and is confined to a few cells associated with the coelomic rudiments. The five Cy mRNAs fall into three sets, whose times and sites of expression during development are highly distinctive. Different cell lineages express messages of one or more of these sets, but never all three. Although all Cy actin mRNAs exhibit monophasic accumulation in the RNA of whole embryos during the course of development, such accumulation in many cases results from the summation of both increases and decreases in abundance within individual sets of cells. Within the genomic linkage group CyI-CyIIa-CyIIb, expression of CyI and CyIIb appears to be co-ordinate, and quite distinct from that of CyIIa. CyI and CyIIb are expressed in all lineages at some point in embryogenesis, but confined mainly to oral ectoderm and portions of the gut of the pluteus larva. CyIIa mRNAs are restricted to mesenchyme lineages throughout late gastrula stage, and subsequently accumulate in parts of the gut. The CyIIIa and CyIIIb genes, which form a separate linkage group, are expressed only in aboral ectoderm and its precursors. Furthermore, CyIII messages are the only detectable actin mRNAs in this cell lineage after late blastula stage.     

Spatio-temporal expression of pamlin during early embryogenesis in sea urchin and importance of N-linked glycosylation for the glycoprotein function

Expression of pamlin, a heterotrimeric primary mesenchyme cell (PMC) adhesion glycoprotein, and its role during early embryogenesis were examined using immunochemistry and microinjection of pamlin to tunicamycin-treated embryos of the sea urchin, Hemicentrotus pulcherrimus. Pamlin faintly detected in egg cortex before fertilization was strongly expressed in the hyaline layer after fertilization. The embryonic apical surface retained pamlin throughout early embryogenesis, whereas pamlin on the basal surface showed a dynamic change of spatio-temporal distribution from morula to gastrula stage. Pamlin distributed on the entire basal surface of the ectoderm before onset of invagination gradually disappeared from the presumptive archenteron during gastrulation, and then was restricted to the apical tuft region and the PMC sessile sites in early gastrulae. Tunicamycin, an inhibitor of N-glycosydically linked carbohydrate formation, inhibited PMC migration and gastrulation. Tunicamycin also inhibited the assembly of mannose moieties of 180 and 52 kDa subunits of pamlin. Pamlin microinjection to the tunicamycin-treated embryos rescued them from this morphogenetic disturbance. PMCs did not bind to pamlin isolated from the tunicamycin-treated embryos. The present study indicated that pamlin plays an essential role in PMC migration, its termination and gastrulation, and the presence of N-glycosydically linked carbohydrate moieties that contain mannose are necessary to preserve the biological function of pamlin.     

Patterns of protein synthesis and metabolism during sea urchin embryogenesis

We have analyzed the patterns of protein synthesis in developing embryos of the sea urchin Strongylocentrotus purpuratus by two-dimensional gel electrophoresis. There was an increase in the number of proteins detectably synthesized during development, as well as significant changes in relative rates of synthesis involving approximately 20% of the nearly 900 newly synthesized polypeptides. The majority of these changes were increases rather than decreases in synthesis; about half were of at least 10-fold, while a few were of more than 100-fold. Very few changes were detected upon fertilization and during the first several hours of development, while about 60% of the changes detected occurred between the hatching and the beginning of invagination. An analysis of proteins detected by silver staining indicated that most remained nearly constant in mass during embryonic development, but several increased or declined substantially. Many proteins present in eggs were not detectably synthesized in either eggs or embryos.     

dCMP-aminohydrolase activity during early sea urchin development. An example of negative enzyme control during embryogenesis

In sea urchin, unfertilized eggs have a very high level of dCMP-aminohydrolase (dCMPase) activity, which decreases gradually and at the pluteus stage it is only about a quarter of that found in the unfertilized egg. But in abnormal embryos and in disaggregated cells from embryos, no decrease in the dCMPase activity takes place. To understand the control mechanism involved in this enzyme activity during development, we have analyzed the effect of various drugs which interfere with information transfer, such as actinomycin C, puromycin, 5-azacytidine, 2-thio-uracil and p-fluoro-DL-phenylalanine on dCMPase activity in embryos of Paracentrotus lividus and Sphaerechinus granularis. Among these drugs only actinomycin induces a remarkable increase of the dCMPase activity in embryos with respect to unfertilized eggs. Puromycin has a differential and dose-dependent effect. Other drugs, although they affect normal development and macromolecular synthesis, do not significantly alter the dCMPase activity. On the basis of these results we suggest the presence of a repressor mechanism in the control of dCMP-aminohydrolase level during early embryogenesis of sea urchin.     

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.