Gastrulation In Sea Urchin Embryos Research Articles

Shared regulatory function of non-genomic thyroid hormone signaling in echinoderm skeletogenesis

Thyroid hormones are crucial regulators of metamorphosis and development in bilaterians, particularly in chordate deuterostomes. Recent evidence suggests a role for thyroid hormone signaling, principally via 3,5,3′,5′-Tetraiodo-L-thyronine (T4), in the regulation of metamorphosis, programmed cell death and skeletogenesis in echinoids (sea urchins and sand dollars) and sea stars. Here, we test whether TH signaling in skeletogenesis is a shared trait of Echinozoa (Echinoida and Holothouroida) and Asterozoa (Ophiourida and Asteroida). We demonstrate dramatic acceleration of skeletogenesis after TH treatment in three classes of echinoderms: sea urchins, sea stars, and brittle stars (echinoids, asteroids, and ophiuroids). Fluorescently labeled thyroid hormone analogues reveal thyroid hormone binding to cells proximal to regions of skeletogenesis in the gut and juvenile rudiment. We also identify, for the first time, a potential source of thyroxine during gastrulation in sea urchin embryos. Thyroxine-positive cells are present in tip of the archenteron. In addition, we detect thyroid hormone binding to the cell membrane and nucleus during metamorphic development in echinoderms. Immunohistochemistry of phosphorylated MAPK in the presence and absence of TH-binding inhibitors suggests that THs may act via phosphorylation of MAPK (ERK1/2) to accelerate initiation of skeletogenesis in the three echinoderm groups. Together, these results indicate that TH regulation of mesenchyme cell activity via integrin-mediated MAPK signaling may be a conserved mechanism for the regulation of skeletogenesis in echinoderm development. In addition, TH action via a nuclear thyroid hormone receptor may regulate metamorphic development. Our findings shed light on potentially ancient pathways of thyroid hormone activity in echinoids, ophiuroids, and asteroids, or on a signaling system that has been repeatedly co-opted to coordinate metamorphic development in bilaterians.

A Report on the Fertilisation and Early Development in the Sea Urchin

The primary objective of this study was to investigate fertilisation, cleavage and gastrulation in sea urchin embryos. It was found that a difference between room and 4°C stored samples were obtained, with only 55% of the embryos reaching the plateus larvae stage. After 72h, cell death occurred in the 4°C stored samples. The parthenogenic embryos died, and didn't pass the blastula stage of early gastrulation. This paper addresses 4 main topics of sea urchin embryo development, including parthenogenesis and the interference of fertilisation.

Behavior of Pigment Cells Closely Correlates the Manner of Gastrulation in Sea Urchin Embryos

To know whether behavior of pigment cells correlates the process of gastrulation or not, gastrulating embryos of several species of regular echinoids (Anthocidaris crassispina, Mespilia globulus and Toxopneustes pileolus) and irregular echinoids (Clypeaster japonicus and Astriclypeus manni) were examined. In M. globulus and A. crassispina, the archenteron elongated stepwise like in well-known sea urchins. In the embryos of both species, fluorescent pigment cells left the archenteron tip and migrated into the blastocoel during gastrulation. In T. pileolus, C. japonicus and A. manni, on the other hand, the archenteron elongated at a constant rate throughout gastrulation. In these species, no pigment cell was observed at the archenteron tip during invagination processes; pigment cells began to migrate in the ectoderm from the vegetal pole side toward the apical plate without entering the blastocoel. These results clearly indicate that the behavior of pigment cells closely correlated the manner of gastrulation. Further, it was examined whether the archenteron cells are rearranged during invagination, by comparing the number of cells observed on cross sections of the archenteron at the early and late gastrula stages. The rearrangement was not conspicuous in A. crassispina and M. globulus, in which archenteron elongated stepwise. In contrast, the archenteron cells were remarkably rearranged in C. japonicus, alothough the archenteron elongated continuously. Thus, neither the behavior of pigment cells nor the manner of gastrulation matches the current taxonomic classification of echinoids.

Genomic organization of the gene that encodes the precursor to EGF-related peptides, exogastrula-inducing peptides, of the sea urchin Anthocidaris crassispina

Exogastrula-inducing peptides (EGIPs) were identified in embryos of the sea urchin Anthocidaris crassispina as polypeptides with structural similarity to epidermal growth factor (EGF) that severely affect gastrulation of sea urchin embryos to induce exogastrulation. Here we have obtained genomic clones for the EGIP precursor gene ( EGIP) and determined its genomic organization. The EGIP gene spans the length of 9 kb in the genome and is composed of seven exons and six introns. Each of the four EGF motifs in the precursor protein is encoded by a single exon, and all the exon boundaries are in phase 1, suggesting that EGIP have been generated during evolution by duplication of an exon encoding a single ancient EGIP sequence. The 5′-flanking sequence of EGIP from −4372 to +194 revealed the presence of multiple repeat sequences including direct and inverted repeats as well as two clusters of GGGG/CCCC elements. The function of the upstream flanking region of EGIP was examined by introducing the gene constructs into embryos in which different regions from the flanking DNA were placed upstream to the GFP reporter gene. Systematic deletion of the upstream DNA revealed the presence of potent enhancer activity between −372 and −210.

Potential of veg2 blastomeres to induce endoderm differentiation in sea urchin embryos.

Two different modes of gastrulation in sea urchin embryos have been reported. The first mode, reported in Hemicentrotus pulcherrimus and some other species, consists of two phases: a primary and a secondary invagination. The second mode involves gastrulation with a continuous convolution of cells near the blastopore; this mode has been reported to occur in the embryos of the sand dollar, Scaphechinus mirabilis. The rudimentary gut is comprised of fewer cells in the embryos of the former species than in the latter. We assumed that the differences in gastrulation modes could be related to the different potentials of the veg2 layer to induce endoderm differentiation in the upper layer. In the present study, we produced chimeric embryos consisting of an animal cap recombined with veg2 layer blastomere(s) to compare the inductive effect of the veg2 layer and/or the blastomere(s) in H. pulcherrimus and S. mirabilis embryos. Our results showed that the inductive effect of the veg2 layer is stronger in S. mirabilis embryos than in H. pulcherrimus embryos. Moreover, it was suggested that the difference in the strength of inductive effects of veg2 layers is related to the difference in gastrulation modes.

Cell movements during the initial phase of gastrulation in the sea urchin embryo

The morphogenetic processes responsible for the initial phase of gastrulation in sea urchin embryos are not known. Here we report observations of the size and position of clones of cells derived from horseradish peroxidase (HRP)-injected mesomeres and macromeres. The displacement of these clones during the initial phase of gastrulation suggests that involution is a mechanism involved in primary invagination. Experiments with embryos marked with vital dyes indicate that movements occur only during a brief phase coincident with the invagination of the vegetal plate. Counts of cells derived from HRP-injected mesomeres and macromeres suggest it unlikely that localized growth in the vegetal plate is involved in gastrulation. An analysis of changes in cell shape during the initial phase of gastrulation indicates that there is a stage-dependent shift from cells being columnar to having their apices skewed toward the vegetal plate and an increase in the proportion of cells having basal processes during gastrulation. When embryos are grown in the presence of monoclonal antibodies to the apical lamina or monovalent fragments of these antibodies, the initial phase of gastrulation is delayed and they form partial exogastrulae. Analysis of embryos marked with HRP indicate that the antibody treatments interfere with the cellular movements observed in untreated embryos. We conclude that directed movements of cells within the blastoderm, probably employing tractoring on components of the hyaline layer, cause the buckling of the vegetal plate and displacement of presumptive endoderm cells seen during the initial phase of gastrulation.

Chlorpromazine-sensitive developmental processes in the sea urchin, Lytechinus pictus—II. Effects of neuroactive agents on the susceptibility of the gastrulation process to chlorpromazine

1. 1. The complete inhibition of gastrulation in sea urchin embryos continuously incubated in 1 μM chlorpromazine beginning at the hatched blastula stage was partially reversed by serotonin (10 μM), lanthanum ions (1 μM), dibutyrl cAMP (1 μM), or reserpine (10 μM) but not by dopamine, norepinephrine, epinephrine, acetylcholine, dibutyrl cGMP, or excess calcium ions. 2. 2. The specific inhibition of the secondary phase of gastrulation by 0.5 μM chlorpromazine was not reversed by any of the above agents. 3. 3. Hatched blastula embryos cultured in 1 μM chlorpromazine sulfoxide or 1 μM haliperidol, a dopamine antagonist, developed to normal pluteus larvae. 4. 4. A serotonergic morphogenetic mechanism is proposed.

Biphasic stage sensitivity to UV suppression of gastrulation in sea urchin embryos

The effects of ultraviolet light (UV) on the gastrulation of sea urchin embryos were examined. The results suggest that gastrulation is inhibited by UV irradiation and that stage sensitivity to UV suppression of gastrulation changes biphasically: higher sensitivity at early and late blastula, and lower sensitivity at the mid-blastula stages. The UV-induced inhibition of gastrulation was completely reversible by subsequent exposure to visible light.

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.

Regulation of sea urchin glycoprotein mRNAs during embryonic development.

Gastrulation in sea urchin embryos is accompanied by a striking increase in the synthesis of N-linked glycoproteins, and inhibitors of this process block gastrulation. In this report, the messages coding for N-glycosylatable proteins in the developing embryo of the sea urchin, Strongylocentrotus purpuratus, were examined. Total mRNA and mRNA isolated from membranes of the embryos at various stages of development were used to program a cell-free translation/glycosylation system prepared from rabbit reticulocyte lysate supplemented with dog pancreas microsomes. The glycosylated translation products were separated from the nonglycosylated products by concanavalin A-agarose and analyzed by gel electrophoresis. The results indicate that although the RNA derived from the membranes of gastrula-stage embryos contains messages coding for numerous glycoproteins, only trace amounts of glycoprotein messages are associated with membranes at earlier stages of development. mRNAs coding for four glycoproteins of M(r)s 70,000, 65,000, 51,000, and 30,000 were examined further in total RNA preparations from the developing embryo. The data indicate that the messages coding for the glycoproteins of M(r)s 65,000 and 51,000 are present also in the unfertilized egg and in the pregastrulation embryo. Because these two messages are not found associated with the membranes until gastrula stage, it is likely that the synthesis of these glycoproteins during gastrulation is regulated at the translational level. The messages coding for glycoproteins of M(r)s 70,000 and 30,000, on the other hand, are not detectable in the unfertilized egg and may be synthesized de novo by the embryos. Thus, the expression of these two glycoproteins during gastrulation is regulated at least in part on the transcriptional level. On the basis of these findings, it appears that different modes of regulation are used for different glycoproteins that are synthesized during gastrulation.

Induction of N-linked glycoprotein synthesis during gastrulation of sea urchin embryos

Induction of N-linked glycoprotein synthesis during gastrulation of 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.

Effect of 5-Bromodeoxyuridine on Differentiation: II. The Effect of Early BUdR Treatment on Gastrulation of Sea Urchin Embryos

BUdR treatment of early cleavage stages is more effective on gastrulation the earlier the drug is administered before the 16-cell stage. The distribution of percentages of embryos gastrulating is in good agreement with the theoretical calculated values presented in part I and permit the assumption that BUdR may act by preventing normal transcription at, or shortly after, the 16-cell stage.

Identification of four classes of cell surface antigens appearing at gastrulation in sea urchin embryos

An antiserum to isolated membranes of gastrula-stage embryos of the sea urchin Lytechinus variegatus was characterized by absorption and cell agglutination specificities. The antiserum was found to recognize four distinct classes of antigens on the embryonic cell surface: (1) an early embryonic class or “maternal” class present from the earliest stages of development, (2) an embryonic class of antigens which appeared on all cells beginning at gastrulation, (3) a class of antigens present on ectoderm cells, and (4) a class of antigens present on endoderm cells. All four classes of antigens were shown indirectly to be synthesized on embryonic mRNA since a hybrid embryo of the cross Tripneustes ♀ × Lytechinus ♂ expressed all four classes of Lytechinus-specific antigens beginning at gastrulation. Each class was Lytechinus specific in that hybrid cells were agglutinated if beyond the beginning of gastrulation, while normal Tripneustes ♀ × Tripneustes ♂ cells were not agglutinated.

OCCURRENCE OF MUCOPOLYSACCHARIDES IN THE EARLY DEVELOPMENT OF THE SEA URCHIN EMBRYO AND ITS ROLE IN GASTRULATION.

The occurrence of acid mucopolysaccharides in the early development of the sea urchin embryo was studied by histochemical stainings as well as by autoradiographic methods. By histochemical methods acid niucopolysacchdride was demonstrated at the vegetal region in the early stage of gastrulation as a globular structure. Experiments with 35 S-labeled sulfate which was incorporated into acid mucopolysaccharides confirmed the result obtained by histochemical observation. It was revealed that sulfate polysaccharide in the vegetal region moved toward the blastocoel in parallel with the shedding of the primary mesenchyme cells. When the incorporation of sulfate into the acid mucopolysaccharides was inhibited by selenate, the primitive gut development was remarkably repressed. The substance seems to be indispensable for smooth cell movements essential for the gastrulation of sea urchin embryo.