Elongated Microvilli on Vegetal Pole Cells in Sea Urchin Embryos

The ultrastructure of cells in the vegetal pole region of sea urchin embryos during early development to the mesenchyme blastula stage was examined by scanning electron microscopy. Vegetal pole cells in the ectoderm with longer microvilli than those of neighboring cells were first detectable at the early blastula stage just before hatching. These cells with elongated microvilli remained in the central region of the vegetal plate when most vegetal plate cells ingressed into the blastocoel to form primary mesenchyme. When first detectable in the sea urchin, Anthocidaris crassispina , four vegetal pole cells had elongated microvilli, but at the time of primary mesenchyme cell ingression, the number of cells with elongated microvilli had increased to eight, apparently by cell division. These vegetal pole cells were wedge-shaped with a broad surface adhering to the hyaline layer at the time of primary mesenchyme cell ingression. SEM observation of the outer surface of embryos showed that the microvilli extended into the hyaline layer. The reinforced attachment of vegetal pole cells to the hyaline layer through their elongated microvilli may explain why these cells could remain at the vegetal pole when the surrounding cells ingressed into the blastocoel as primary mesenchyme cells.     

Endo16, a lineage-specific protein of the sea urchin embryo, is first expressed just prior to gastrulation

We have isolated and characterized a new endoderm-specific gene, designated Endo16, from a sea urchin gastrula stage cDNA library. Northern blot analysis and in situ hybridization experiments indicate that this gene is first expressed in the vegetal plate, a group of endodermal and mesenchymal precursor cells that are poised to invaginate in the first movement of gastrulation. Expression becomes progressively restricted to a subset of endodermal cells as development proceeds. To study the Endo16 gene product, a polyclonal antiserum was raised against bacterially expressed Endo16 protein. Indirect immunofluorescence experiments in midgastrula stage embryos reveal that the Endo16 protein is localized to the surface of endoderm and secondary mesenchyme cells. In Western blot experiments, the antiserum detects a small set of high molecular weight proteins ranging from 180 to greater than 300 kDa. Analysis of the nucleotide-derived amino acid sequence from a partial Endo16 cDNA clone reveals a highly repetitive, extremely acidic protein segment that includes the Arg-Gly-Asp (RGD) tripeptide known to be important in cell binding domains of a number of extracellular proteins. Taken together, these data suggest that the Endo16 protein may be an adhesion molecule involved in gastrulation of the sea urchin embryo.     

Jun N‐terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N‐terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125‐treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation. genesis 53:762–769, 2015. © 2015 Wiley Periodicals, Inc.     

Expression of Cyclophilin during the Embryonic Development of the Sea Urchin

The spatial and temporal expression pattern of cyclophilin (Cyp) was examined during the embryonic development of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus using Western blot analysis and indirect immunofluorescence microscopy. In this study, affinity-purified anti-human Cyp A antibody was used as the primary antibody. Western blot analysis revealed that a single 17.5 kDa immunoreactive band of Cyp was present in unfertilized eggs, in embryos during several stages of development, and in ovaries and testes of adult sea urchins. Cyp was also recognized in unfertilized eggs and embryonic sea urchin cells by indirect immunofluorescence microscopy, but its concentrations within the embryonic tissues varied significantly during embryogenesis. Expression of Cyp during the cleavage stage was thought to be attributable to maternal Cyp products, with zygotic expression appearing after gastrulation. Cyp expression appears to increase depending on the Cyp concentration in the vegetal and apical plates and primary mesenchyme cells in mesenchyme blastulae, and in the oral ectodermal ridge, gut and skeletogenetic mesenchyme cells in pluteus larvae. These results suggest that widespread embryonic distribution and an increased Cyp content occur during the gastrulation in sea urchin development.     

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.     

Brachyury homolog (HpTa) is involved in the formation of archenteron and secondary mesenchyme cell differentiation in the sea urchin embryo

Sea urchin Brachyury homolog (HpTa) is expressed exclusively in the vegetal plate and secondary mesenchyme cells in the embryos of sea urchin Hemicentrotus pulcherrimus. In order to gain insights into the role of HpTa during sea urchin development, we designed experiments to perturb the embryo by inducing ectopic overexpression of HpTa by injecting fertilized eggs with HpTa mRNA. The overexpression of HpTa resulted in suppression of the formation of vegetal plate and secondary mesenchyme cells. We assume that the interaction of HpTa with unknown factors is required for the activation of the HpTa target genes, and that the excess amount of HpTa proteins produced from injected HpTa mRNA depletes the co-factors. In consequence, the target genes of HpTa would be repressed by the overexpression of HpTa. We suggest that HpTa is involved in the formation of the vegetal plate and the differentiation of secondary mesenchyme cells.     

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 ³⁵S-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.     

The role of lysyl oxidase and collagen crosslinking during sea urchin development

Lysyl oxidase, the only enzyme involved in collagen crosslinking, is shown to be present in embryos of the sea urchin Strongylocentrotus purpuratus. The enzyme specific activity increases over six-fold during development, showing the greatest rise during gastrulation and prism larva formation. The enzyme is inhibited by the specific inhibitor, beta-aminoproprionitrile (BAPN). Continuous BAPN treatment of S. purpuratus and Lytechinus pictus embryos from late cleavage stages onward increases the amount of noncrosslinked collagen present in prism larvae. When BAPN is added at the 128- or 256-cell stage it causes developmental arrest at the mesenchyme blastula stage. Embryos can be maintained in the arrested state for at least 96 h and will resume normal development and morphogenesis following BAPN removal. If BAPN is added after the mesenchyme blastula stage, it has little adverse effect on development; consequently nonspecific toxic effects of the drug are unlikely. The results suggest that lysyl oxidase and collagen crosslinking play a vital role in primary mesenchyme migration, gastrulation, and morphogenesis during sea urchin development and indicate that BAPN may be very useful in studying the extracellular matrix-cell interactions at the cellular and molecular level.     

Specification process of animal plate in the sea urchin embryo

The most animal part of the ciliated band of sea urchin larvae, the animal plate, is a specialized region in which elongated cells form long and non-beating cilia. To learn how this region is specified, animal halves were isolated from the early cleavage to pregastrulation stages. As is well known, the animal half that is isolated at the eight-cell stage develops into a 'dauerblastula', which forms long and non-beating cilia all around the surface. The region with long cilia, however, became restricted toward the animal pole when separation was delayed. If separated before primary mesenchyme ingression, even a small animal-pole-side fragment formed a normal-sized animal plate. Thus, the prospective animal plate region is gradually restricted by some signal from the vegetal hemisphere, and the specification process terminates before the mesenchyme blastula stage. It was also known that a normal-sized animal plate was formed in micromere-less embryos, indicating that the signal does not depend on micromeres or their descendants. Further, the animal-pole-side fragments were isolated from embryos in which the third cleavage plane was shifted toward the vegetal pole. They formed a normal-sized animal plate, containing more than 75% of the egg volume from the animal pole. This indicates that the egg cytoplasm delivered to veg₁ -lineage blastomeres plays an important role in the animal plate specification. Interestingly, the an1-less embryo formed long and non-beating cilia at its top region, but thickening did not occur. The cytoplasm near the animal pole might contain some factors necessary for the animal plate to become thick.