Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton

Spicule matrix proteins are the products of primary mesenchyme cells, and are present in calcite spicules of the sea urchin embryo. To study their possible roles in skeletal morphogenesis, monoclonal antibodies against SM50, SM30 and another spicule matrix protein (29 kDa) were obtained. The distribution of these proteins in the embryo skeleton was observed by immunofluorescent staining. In addition, their distribution inside the spicules was examined by a 'spicule blot' procedure, direct immunoblotting of proteins embedded in crystallized spicules. Our observations showed that SM50 and 29 kDa proteins were enriched both outside and inside the triradiate spicules of the gastrulae, and also existed in the corresponding portions of growing spicules in later embryos and micromere cultures. The straight extensions of the triradiate spicules and thickened portions of body rods in pluteus spicules were also rich in these proteins. The SM30 protein was only faintly detected along the surface of spicules. By examination using the spicule blot procedure, however, SM30 was clearly detectable inside the body rods and postoral rods. These results indicate that SM50 and 29 kDa proteins are concentrated in radially growing portions of the spicules (normal to the c-axis of calcite), while SM30 protein is in the longitudinally growing portions (parallel to the c-axis). Such differential distribution suggests the involvement of these proteins in calcite growth during the formation of three-dimensionally branched spicules.     

Ultrastructure of collagen in sea urchin embryos

Summary Collagen fibrils with a main period banding of 610 Å and 220 Å in width were observed in the blastocoel of 72-h embryos of the sea urchin,Strongylocentrotus purpuratus. Non-striated fibrils of 50 Å diameter were also observed. The collagen is seen in highest concentration in the vicinity of mesenchyme cells which are richly endowed with endoplasmic reticulum and secretory vesicles. A role for collagen in cell attachment, orientation and spicule formation is discussed.     

Inhibition of translation and modification of translation factors during apoptosis induced by the DNA-damaging agent MMS in sea urchin embryos

Translational control was investigated in sea urchin eggs and embryos in response to the DNA-damaging agent methyl methanesulfonate (MMS). We have shown in this report that exposure of sea urchin embryos to MMS induces drastic effects on protein synthesis activity, and on translation factors level, integrity and post-translational modifications. In response to the treatment of embryos by the DNA-damaging agent MMS, protein synthesis is inhibited independently of the translation inhibitor 4E-BP and in correlation with phosphorylation of the translation factor eIF2alpha subunit. Furthermore, a low molecular weight form of translation initiation factor eIF4G is detected correlatively with MMS-induced apoptosis. We propose that modifications of translation factors play an important role in protein synthesis modulation that occurs during DNA-damage induced apoptosis.     

Electron microscope visualization of giant polysomes in sea urchin embryos

Chromatin spreading techniques have been applied to the electron microscopic visualization of polysomes in sea urchin (Strongylocentrotus purpuratus) eggs and embryos. Polysomes of giant size are commonly found after the 8-cell stage. The largest seen, from an early gastrula, was 13.6 m in length, carried 277 ribosomes, with a message calculated to contain 6.49×10⁴ nucleotides and potentially to encoded 2.38×10⁶ daltons of peptide. Polysomes are rare and very large ones absent from lysates of unfertilized eggs. Giant polysomes appear in 4- to 8-cell stages and are common in 16-cell stages and thereafter. They are of two forms: a compact form with no spacing between ribosomes characteristic of stages through early mesenchyme blastulae, and an extended form found only after late mesenchyme blastulae. Both have potential for massive informational content. Some of each type have ribosome-free tails at one end, as long as 733 Å in the compact forms, and 7,890 Å in the extended ones. Occasionally they have a single array of fibrous material increasing from one end of a polysome to the other, interpreted to be nascent peptide chains. Polysomes are not found after brief, mild exposure of lysates to RNase A, or from embryos treated with puromycin. Very large polysomes are present in lysates of blastulae exposed since fertilization to actinomycin D, cycloheximide, or cordycepin. They appear in parthenogenetically activated or fertilized enucleate merogones, but are absent from unactivated merogones, demonstrating that egg masked messages can generate them. A potential embryological significance of giant, potentially polycistronic polysomes is suggested.     

The contractility of elongated microvilli in early sea urchin embryos

Summary Elongated microvilli attach the early sea urchin embryo to the fertilization envelope and support it in a concentric position within the perivitelline space. The contractility of the elongated microvilli was demonstrated in several ways. (1) During normal cleavage, these microvilli change their length to adapt to the change in shape and numbers of blastomeres. (2) When treated with calcium-free sea water, embryos become eccentrically located and the microvilli extend further than normal on one side; when returned to normal sea water, the embryos become centered again. (3) Several agents cause the fertilization envelope to become higher and thinner than normal and the elongated microvilli to extend correspondingly if treated within ten min after fertilization. In some cases, both elongated microvilli and fertilization envelope return to normal size when returned to normal sea water. (4) Fertilization in a papain solution causes the elongated microvilli and the fertilization envelope to contract to the surface of the embryo. (5) Refertilization after the papain-induced contraction can bring about the elongation of these microvilli and the elevation of the fertilization envelope a second time. It was also shown that elongated microvilli are extended immediately upon fertilization, at the same time as the short microvilli. The firm adherence of the tips of elongated microvilli to the fertilization envelope by means of extracellular matrix fibers is shown in a high voltage electron microscope stereoimage. This allows us to understand why it is that when the elongated microvilli extend or contract, the fertilization envelope also extends and contracts accordingly.     

Relative importance of parental and larval nutrition on larval development and metamorphosis of the sea urchin Strongylocentrotus droebachiensis

We examined the relative importance of parental nutritional condition and larval food ration on the rates of development, growth and metamorphosis of larvae of Strongylocentrotus droebachiensis (Müller) in a laboratory experiment. Parents were reared for 22 months on either a high ration of kelp (Laminaria spp., 6 days week⁻¹) supplemented with mussel flesh (Mytilus spp., 1 day week⁻¹) (KM), or a low ration of kelp (1 day week⁻¹) (KL). Larvae were fed either a high ration (5000 cells ml⁻¹) or a low ration (500 cells ml⁻¹) of microalgae (Dunaliella tertiolecta). Larval food ration had a strong effect on the rates of development, growth, and metamorphosis, which were all significantly greater in larvae fed the high ration. Test diameter of settlers also was significantly greater in the high than the low ration. Parental nutritional condition had little or no effect on the rates of development and growth, and no effect on settler size. The rate of metamorphosis was significantly higher in larvae from the KM than the KL treatment in the high but not the low ration (where rates of metamorphosis were similar). Although parental condition generally had a small effect on larval development, our results suggest that when planktonic food is abundant, larvae of adults from nutritionally rich habitats (such as kelp beds) may metamorphose sooner than those of adults from nutritionally poor habitats (such as barrens).     

Distribution and redistribution of pigment granules in the development of sea urchin embryos

Summary Pigment granules (PGs) are embeded in the cortex of embryos of the Japanese sea urchins,Hemicentrotus pulcherrimus and Anthocidaris crassispina. PGs in the cortex actively retreated from the vegetal pole area at the 4-cell stage and then a notable PG-distribution gradient formed along the egg axis (the polar redistribution of PGs). The polar redistribution of PGs in the cortex occurred at the same time after fertilization even in solutions of microtubule disrupting reagents such as Colcemid, vinblastine sulfate or griseofulvin. Consequently, the polar redistribution of PGs was not associated with the microtubules. However, the polar redistribution of PGs was interrupted in seawater containing cytochalasin B (CB), dithiothreitol (DTT) or tetracaine, and the distribution pattern of PGs in the cortex was definitely disturbed. Moreover, CB, DTT and tetracaine altered the division pattern of vegetal blastomeres at the 4th cleavage which is normally unequal so that all the blastomeres divided equally. Microtubule disrupting reagents did not have such an effect on the cleavage pattern. Thus the cortical movement along the egg axis reflected by the polar redistribution of PGs seems to correlate with the micromere formation.     

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.     

DNA amplification in sea urchin oocytes

Summary In situ hybridization of ribosomal RNA withParacentrotus lividus ovaries suggests that ribosomal DNA undergoes amplification in the mononucleolate oocytes of this sea urchin.     

Cyclic appearance of cytoplasmic NOR-silver-stained particles in sea urchin embryos.

When sea urchin embryos were subjected to nucleolar organizer region (NOR)-silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell-cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine-treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) showed that 95-kDa and 38-kDa proteins were the NOR-silver-staining proteins in sea urchin embryos. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95-kDa and 38-kDa proteins are the major NOR-silver-staining proteins in sea urchin embryos.     

Pulse treatment of sea urchin embryos with A23187 blocks their hatching out

Summary Pulse treatment of sea urchin embryos with 3 µM A23187 for 2 h at 20° C, starting from 3 to 6 h of development, prevented the embryos from hatching. Many embryos thus treated with A23187 produced mesenchyme cells and underwent gastrulation while still enclosed within the fertilization membrane. The pulse treatment in this pre-hatching period exerts markedly stronger inhibitory effects on hatching than on other events in early development. Treatment beginning at times earlier than 2 h and later than 8 h of development caused only a slight delay of hatching. The activity of hatching enzyme, known to increase between 6 and 8 h after fertilization, was quite low, if present at all, in embryos in which hatching was blocked by A23187. Hatching enzyme synthesis is probably blocked by the preceding pulse treatment. However, overall protein synthesis, estimated with methionine S 35 incorporation, was somewhat augmented in embryos by the pulse treatment. The blockage of hatching and the augmentation of overall protein synthesis by A23187 were appreciably reversed by procaine, tetracaine, ruthenium red or verapamil. Probably, an artificial Ca²⁺ signal induced by A23187 activates protein synthesis but blocks the induction of hatching enzyme synthesis.     

Single-channel activity in sea urchin sperm revealed by the patch-clamp technique

Ionic fluxes are deeply involved in the response of spermatozoa to the egg. Using the patch-clamp technique, we show for the first time single ion channel activity in sea urchin spermatozoa and spermatozoa heads. Due to their small size gigaseals were obtained in suspended cells by applying suction through the pipette. The rate of gigaseal formation was very low and improved to 6% (n = 1145) when flagella were detached from sperm. Current-voltage curves created from single-channel events showed conductances of approx. 65 and 170 pS, suggesting the presence of two types of channels. At least one appears to be a K⁺ channel.     

cAMP-dependent protein kinase in sea urchin embryos

cAMP-dependent protein kinase in the supernatant fraction of the homogenate of sea urchin eggs and embryos obtained by centrifugation at 105,000g was investigated in the present study. In the previous report, the dissociation constant between cAMP-binding proteins and cAMP changed during the development. This suggests that the nature of cAMP-dependent protein kinase, which has been well established to be the major cAMP receptor, changes during the development. In the present study, four protein kinases were separated through DEAE-cellulose column from the supernatant of unfertilized egg homogenate. One of them was cAMP-dependent protein kinase. The others were cAMP-independent ones. One among them was phosvitin kinase, and the others were not identified at present. The activity of cAMP-dependent protein kinase gradually increased during a period from fertilization to the swimming blastula stage. During this period, cleavages occurred at a high rate, and the rate decreased after hatching out. Thus, it is supposed that cAMP-dependent protein kinase in the supernatant may take a part in the mechanism of cleavage. The activity, however, became very low at the mesenchyme blastula, the gastrula, and the pluteus stages. cAMP-binding capacity was observed in the sedimentable fraction and the supernatant fraction, respectively, obtained by 105,000g centrifugation at all stages examined. If the structure-bound cAMP-binding protein is also cAMP-dependent protein kinase, it may play different roles in the mechanism of development.     

The isolation of isohistones by preparative gel electrophoresis from embryos of the sea urchin Parechinus angulosus

Ten isohistones from the embryo of the sea urchin Parechinus angulosus have been isolated by preparative gel electrophoresis and characterised by electrophoretic mobility in two detergent systems, amino acid composition and partial sequences. This brings the total number of different histones identified which are synthesized at one or the other time during the life cycle of the sea urchin to a minimum of 24 structurally characterised polypeptides.     

The organic matrix of the skeletal spicule of sea urchin embryos

The micromeres that arise at the fourth cell division in developing sea urchin embryos give rise to primary mesenchyme, which in turn differentiates and produces calcareous endoskeletal spicules. These spicules have been isolated and purified from pluteus larvae by washing in combinations of ionic and nonionic detergents followed by brief exposure to sodium hypochlorite. The spicules may be demineralized and the integral matrix dissolves. The matrix is composed of a limited number of glycoproteins rich in aspx, glux, gly, ser, and ala, a composition not unlike that found in matrix proteins of biomineralized tissues of molluscs, sponges, and arthropods. There is no evidence for collagen as a component of the matrix. The matrix contains N-linked glycoproteins of the complex type. The matrix arises primarily from proteins synthesized from late gastrulation onward, during the time that spicule deposition occurs. The mixture of proteins binds calcium and is an effective immunogen. Electrophoresis of the glycoproteins on SDS-containing acrylamide gels, followed by blotting and immunocytochemical detection, reveals major components of approximately 47, 50, 57, and 64 kD, and several minor components. These same components may be detected with silver staining or fluorography of amino acid-labeled proteins. In addition to providing convenient molecular marker for the study of the development of the micromere lineage, the spicule matrix glycoproteins provide an interesting system for investigations in biomineralization.     

Functional organization of DNA elements regulating SM30α, a spicule matrix gene of sea urchin embryos

The SM30a gene encodes a protein in the embryonic endoskeleton of the sea urchin Strongylocentrotus purpuratus, and is specifically expressed in the skeletogenic primary mesenchyme cell lineage. To clarify the mechanism for the differentiation of this cell lineage, which proceeds rather autonomously in the embryo, regulation of the SM30alpha gene was investigated previously and it was shown that the distal DNA region upstream of this gene from - 1.6 to - 1.0 kb contained numerous negative regulatory elements that suppressed the ectopic expression of the gene in the gut. Here we study the influence of the proximal region from - 303 to + 104 bp. Analysis of the expression of reporter constructs indicated that a strong positive enhancer element existed in the region from -142 to -105bp. This element worked both in forward and reverse orientations and additively when placed tandemly upstream to the reporter gene. In addition, other weaker positive and negative regulatory sites were also detected throughout the proximal region. Electrophoretic gel mobility shift analyses showed that multiple nuclear proteins were bound to the putative strong enhancer region. One of the proteins binding to this region was present in ear y blastulae, a time when the SM30 gene was still silent, but it was not in prism embryos actively expressing the gene. The binding region for this blastula-specific protein was narrowed down to the region from - 132 to -122 bp, which included the consensus binding site for the mammalian proto-oncogene product, Ets. Two possible SpGCF1 binding sites were identified in the vicinity of the enhancer region. This information was used to make a comparison of the general regulatory architecture of genes that contribute to the formation of the skeletal spicule.     

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5′-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.     

Development of the esophageal muscles in embryos of the sea urchin Strongylocentrotus purpuratus

Summary Development of the esophageal muscles in embryonic sea urchins is described using light- and electron microscopy. The muscles develop from processes of about 14 cells of the coelomic epithelium that become immunore-active to anti-actin at about 60 h (12–14° C). Initially, eachmyoblast extends a single process with numerous fine filopodia around the esophagus. By 72 h the processes have reached the midline and fused with those from cells of the contralateral coelomic sac. Myoblasts begin to migrate out of the coelomic epithelium between 72 and 84 h. By 72 h the processes stain with the F-actin specific probe NBD-phallacidin. The contractile apparatus is not evident in transmission electron-microscopic preparations of embryos at 70 h, but by 84 h the contractile apparatus is present and the muscle cells are capable of contraction. Because the myoblasts migrate free of the coelomic epithelium and are situated on the blastocoelar side of the basal lamina, it is suggested that that they should be considered as a class of mesenchymal cells.     

Midbody sealing after cytokinesis in embryos of the sea urchin Arabacia punctulata

Summary Cytokinesis consists of a contractile phase followed by sealing of the connecting midbody to form two separated cells. To determine how soon the midbody sealed after cleavage furrow contraction, the fluorescent dye Lucifer Yellow CH(457.3 M.W.) was microinjected into cells at various intervals after cleavage had begun. Mitotic PtK₂ cells were recorded with video-microscopy so that daughter cells in the epithelial sheet could be identified for several hours after cell division. One daughter cell of each pair followed was microinjected to determine whether the dye diffused into the other daughter cell. For intervals up to four hours after the beginning of cytokinesis, diffusion took place between daughter cells. After this time the dye did not spread between daughter cells. In sea urchin blastomeres of the first, second and third divisions, Lucifer Yellow passed between daughter blastomeres only during the first 15 min after cytokinesis. If one cell of a two-cell, four-cell or eight-cell embryo was microinjected more than 15 min after the last cleavage, the dye remained in the injected cell and was distributed to all progeny of that cell, resulting in blastulae that were either one-half, one-quarter or one-eighth fluorescent, respectively. Thus, although cleavage furrow contraction takes approximately the same amount of time in sea urchin blastomeres and PtK₂ cells, the time of midbody sealing differs dramatically in the two cell types. Our results also indicate the importance of knowing the mitotic history of cells when injecting dyes into interphase cells for the purpose of detecting gap junctions.     

Spicule matrix protein LSM34 is essential for biomineralization of the sea urchin spicule.

Biomineralized skeletal structures are composite materials containing mineral and matrix protein(s). The cell biological mechanisms that underlie the formation, secretion, and organization of the biomineralized materials are not well understood. Although the matrix proteins influence physical properties of the structures, little is known of the role of these matrix proteins in the actual formation of the biomineralized structure. We present here results using an antisense oligonucleotide directed against a spicule matrix protein, LSM34, present in spicules of embryos of Lytechinus pictus. After injection of anti-LSM34 into the blastocoel of a sea urchin embryo, LSM34 protein in the primary mesenchyme cells decreases and biomineralization ceases, demonstrating that LSM34 function is essential for the formation of the calcareous endoskeletal spicule of the embryo. Since LSM34 is found primarily in a specialized extracellular matrix surrounding the spicule, it is probable that this matrix is important for the biomineralization process.