Polysaccharides sulfated at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus

Based on the fact that the development of sea urchin embryos is arrested at the blastula stage in sulfate-free sea water (SFSW), we attempted in the present study to elucidate the nature of sulfated polysaccharides (PSs) which appear at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus. Electrophoretic analysis of PSs prepared from embryos at different developmental stages revealed that three kinds of PSs (3A, 3B, 3C) appear de novo at the gastrula stage, and that these PSs are not found in embryos at the hatching blastula stage, nor are they found in permanent blastula reared in SFSW. These, three PSs were mostly of extracellular matrix origin. Among them, 3C was identified as dermatan sulfate on the basis of its electrophoretic mobility and sensitivity to enzymatic digestion. 3A and 3B remained to be identified. Further, a plausible precursor of 3C, which was sulfated under normal conditions, was detected as 6D in the embryos reared in SFSW. Autoradiographic analysis using [35S]sulfate revealed that these three PSs, accounted for more than 90% of [35S]sulfate incorporated into the acid PS fraction during gastrulation.     

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.     

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.     

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.     

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.     

Specificity of cell-cell interactions in sea urchin embryos: Appearance of new cell-surface determinants at gastrulation

Studies on normal and hybrid sea urchin embryos show that, beginning at gastrulation, hybrid cells express cell-surface antigens specific to both species. The appearance of these antigens is shown to be correlated with a change in the adhesive specificity of hybrid cells: Beginning at gastrulation, hybrid cells recognize and adhere to embryonic cells of both normal genotypes. Prior to gastrulation, hybrid cells adhere to cells of the maternal genotype only. Two adhesion assays demonstrate these adhesive preferences. (i) When cell aggregates are placed together in a dish, Lytechnius aggregates fuse together, and Tripneustes aggregates fuse together, but aggregates of the two species do not fuse with each other. Hybrid cell aggregates, if they are past the beginning of gastrulation, fuse to both Tripneustes and Lytechinus aggregates. (ii) In a collection assay, midgastrula cells of the hybrid embryos are collected at a high rate to aggregates of either species. Pregastrula hybrid cells collect at a high rate to aggregates of the maternal species only. This change in adhesive preference is temporally correlated with the appearance of new cell surface antigens. Antiserum was prepared in rabbits against membranes from Lytechinus gastrulae. Indirect immunofluorescence tests show that hybrid cells of the cross (T♀ × L♂) express Lytechinus-specific antigens at the cell surface beginning at gastrulation. Furthermore, an apparent relationship between the new cell-surface antigens and adhesion exists in that Lytechinus cell adhesion is inhibited specifically after binding Fab fragments of the Lytechinus antiserum. The antiserum has no effect on Tripneustes adhesion. The Lytechinus adhesion-inhibiting activity can be removed by absorption of the antiserum with Lytechinus cells.     

p38 MAPK is essential for secondary axis specification and patterning in sea urchin embryos

Most eggs in the animal kingdom establish a primary, animal-vegetal axis maternally, and specify the remaining two axes during development. In sea urchin embryos, the expression of Nodal on the oral (ventral) side of the embryo is the first known molecular determinant of the oral-aboral axis (the embryonic dorsoventral axis), and is crucial for specification of the oral territory. We show that p38 MAPK acts upstream of Nodal and is required for Nodal expression in the oral territory. p38 is uniformly activated early in development, but, for a short interval at late blastula stage, is asymmetrically inactivated in future aboral nuclei. Experiments show that this transient asymmetry of p38 activation corresponds temporally to both oral specification and the onset of oral Nodal expression. Uniform inhibition of p38 prevents Nodal expression and axis specification, resulting in aboralized embryos. Nodal and its target Gsc each rescue oral-aboral specification and patterning when expressed asymmetrically in p38-inhibited embryos. Thus, our results indicate that p38 is required for oral specification through its promotion of Nodal expression in the oral territory.     

Agrobacterium-mediated transformation of sea urchin embryos

Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. In the present work, we studied whether Agrobacterium can transfer genetic information to animal (sea urchin) embryos. Sea urchin embryos were co-cultivated with A. tumefaciens strains carrying binary vectors containing the nptII marker gene and agrobacterial rolC and rolB oncogenes. Bacterial plasmid T-DNA-sea urchin DNA junction sites were identified in the genome of these embryos, thus indicating successful transformation. The nptII and both rol genes were expressed in the transformed embryos. The processes of transgene integration and transgene expression were suppressed when Agrobacteria contained mutated virA, virB or virG genes, suggesting that Agrobacterium transforms sea urchin cells by a mechanism similar to that which mediates T-DNA transfer to plants. Some of the embryos co-cultivated with Agrobacterium developed teratoma-like structures. The ability of Agrobacterium strains to trigger formation of teratoma-like structures was diminished when they contained the mutated vir genes. In summary, our results demonstrate that Agrobacterium is able to transform animal (sea urchin) embryonic cells, thus indicating a potential of this natural system for gene delivery to animal hosts. We also discuss the possibility of horizontal gene transfer from Agrobacterium to marine invertebrates.     

Target recognition by the archenteron during sea urchin gastrulation

During sea urchin gastrulation filopodia are sent out by secondary mesenchyme cells (SMCs) at the tip of the archenteron in continual cycles of extension, attachment, and retraction. Eventually the archenteron ceases its elongation and its tip localizes to the animal pole region of the embryo (Gustafson and Kinnander, 1956, Exp. Cell Res. 11, 36-57; Dan and Okazaki, 1956, Biol. Bull. 110, 29-42). We have investigated the mechanisms and specificity of this localization by analyzing filopodial behavior and by experimental manipulation of the interaction of the archenteron with the animal pole region. When the tip of the archenteron nears the animal pole, some filopodia make contact with a well-defined locus within this region. Filopodia that make contact with the locus remain attached 20-50 times longer than attachments observed at any other site along the blastocoel wall. The SMCs bearing the long-lived filopodia eventually change their phenotype by flattening and spreading onto this region. Several lines of experimental evidence indicate that contact with the animal pole locus, or "target" region, is crucial for the change in phenotype of the SMCs: (1) the phenotypic change can be induced precociously by bringing the animal pole region within reach of the tip of the archenteron early in gastrulation. Precocious contact with other regions of the blastocoel wall does not induce a similar change. (2) The phenotypic change can be delayed by placing the animal pole out of reach late in gastrulation, resulting in artificial prolongation of exploratory behavior by filopodia. (3) Ectopic combinations of animal pole ectoderm and archenterons in fused multiple embryos and chimaeras result in attachment of archenterons to the nearest available target, and (4) freely migrating SMCs are observed to migrate randomly within the blastocoel, then stop at the animal pole and undergo the change in phenotype. Filopodia rapidly attach to the animal pole when the shape of early gastrulae is altered such that the animal pole is less than 35 microns from the tip of the archenteron, even though such attachments only occur in normal embryos at the 2/3-3/4 gastrula stage. Since it has previously been shown that the archenteron elongates autonomously to 2/3 of its final length (Hardin, 1988, Development 103, 317-324), it appears that autonomous extension of the archenteron is required to place filopodia close enough to the animal pole to allow them to interact with it.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rho-kinase in sea urchin eggs and embryos

The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.     

Exogenous hyalin and sea urchin gastrulation. Part III: biological activity of hyalin isolated from Lytechinus pictus embryos

Hyalin is a large glycoprotein, consisting of the hyalin repeat domain and non-repeated regions, and is the major component of the hyaline layer in the early sea urchin embryo of Strongylocentrotus purpuratus. The hyalin repeat domain has been identified in proteins from organisms as diverse as bacteria, sea urchins, worms, flies, mice and humans. While the specific function of hyalin and the hyalin repeat domain is incompletely understood, many studies suggest that it has a functional role in adhesive interactions. In part I of this series, we showed that hyalin isolated from the sea urchin S. purpuratus blocked archenteron elongation and attachment to the blastocoel roof occurring during gastrulation in S. purpuratus embryos, (Razinia et al., 2007). The cellular interactions that occur in the sea urchin, recognized by the U.S. National Institutes of Health as a model system, may provide insights into adhesive interactions that occur in human health and disease. In part II of this series, we showed that S. purpuratus hyalin heterospecifically blocked archenteron-ectoderm interaction in Lytechinus pictus embryos (Alvarez et al., 2007). In the current study, we have isolated hyalin from the sea urchin L. pictus and demonstrated that L. pictus hyalin homospecifically blocks archenteron-ectoderm interaction, suggesting a general role for this glycoprotein in mediating a specific set of adhesive interactions. We also found one major difference in hyalin activity in the two sea urchin species involving hyalin influence on gastrulation invagination.     

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.     

Electron microscopic studies on primary mesenchyme cell ingression and gastrulation in relation to vegetal pole cell behavior in sea urchin embryos

Early morphogenetic events of primary mesenchyme cell (PMC) ingression and gastrulation were examined by scanning and transmission electron microscopy, with special attention directed to changes in the shape of vegetal pole cells, the length of their microvilli, and interactions between microvilli and the hyaline layer (HL). Eight cells (vegetal pole cells) with elongated microvilli remained in the vegetal pole region while surrounding cells ingressed into the blastocoel to form the primary mesenchyme. These vegetal pole cells indented with the surrounding cells at the stage of gastrulation. The outer surface area with elongated microvilli of vegetal pole cells expanded at the stage of PMC ingression, but was considerably reduced at gastrulation. Microvilli on vegetal pole cells continued to adhere to the HL up to the stage of PMC ingression, but ceased to do so at the time of gastrulation. Thus, the area with separated HL, which is restricted to the region of the PMC released at the stage of PMC ingression, spreads almost entirely throughout the area of the indenting vegetal plate at gastrulation. The apical lamina, apparently consisting of fibrous material intertwinning the stalks of the microvilli, filled the space between the HL and ectodermal cells. The cells surrounding those of the vegetal pole and indenting with those at the stage of gastrulation appeared to behave in the same way as ingressing PMCs in both cell-shape and loss of adhesion of microvilli to HL. The role of vegetal pole cells in early morphogenetic events is discussed.     

Glycoprotein synthesis in developing sea urchin embryos

Surface membrane glycoproteins have been postulated in many mammalian cells to be involved in external surface membrane functions such as cell adhesion, cell-cell recognition, and cell movement. In developing echinoderm embryos, cell adhesion, recognition, and movement of individual cell types have been attributed to differences in the external surface membranes of these cells. Results reported here suggest that the three cell types of 16-cell sea urchin embryos have a mechanism that could establish differences in the carbohydrate portion of glycoproteins located in the external surface membrane. The results demonstrate 1) that glycoproteins are synthesized during early sea urchin development and 2) that slightly different rates of glycoprotein synthesis exist for the three types of blastomeres from 16-cell sea urchin embryos.     

Thymidine uptake by developing sea urchin embryos

Unfertilized Paracentrotus lividus eggs accumulate very little thymidine. Upon fertilization, however, uptake increases sharply. The pool for thymidine and/or its metabolic products is saturated after 40 min of exposure. Its size is expandable and proportional to the initial concentration of thymidine in the medium. The uptake rate is low shortly after fertilization, increases until 40 min after fertilization and remains constant thereafter. Of the radioactivity taken up in the form of [³H]thymidine during a 30 min exposure beginning at 60 min after fertilization, about 1% is associated with the acid-insoluble fraction and 99% with the acid-soluble fraction.