Nuclear activity during oogenesis in sea urchins

Summary Early meiotic stages of Arbacia punctulata oocytes have revealed the presence of synaptinemal complexes in the chromosomes, which persist through zygotene-pachytene. The synaptinemal complexes conform broadly to the usual tripartite structures found in other higher forms. In addition, nuclei at these stages consist of a small nucleolus and dense bodies of varying sizes. The nucleolus is fibrillar in texture throughout and does not seem to incorporate Uridine-5-³H after pulse labeling, whereas the chromosomes are labeled. The nucleolar label is visualized at diplotene stages and onwards. The nuclear envelope differentiates by the appearance of numerous nuclear pore complexes with dense material in the annuli, and the chromosomes become markedly diffused. At vitellogenesis stage the nucleolus and chromatin become highly labeled after pulse incorporation of Uridine, indicating synthesis of ribosomal and chromosomal RNAs.This investigation was supported by grants No. A-5049, A-3624 and D-17 from National Research Council, Canada, grant No. DRB-9340-05 (U6) from Defense Research Board, Canada, and grant No. DRG-918 AT from Damon Runyon Memorial Fund for Cancer Research.     

Constraint, flexibility, and phylogenetic history in the evolution of direct development in sea urchins

Development in sea urchins typically involves the production of an elaborate feeding larva, the pluteus, within which the juvenile sea urchin grows. However, a significant fraction of sea urchins have completely or partially eliminated the pluteus, and instead undergo direct development from a large egg. Direct development is achieved primarily by heterochrony, that is, by the abbreviation or elimination of larval developmental processes and the acceleration of processes involved in development of adult features. Direct development has evolved independently several times, and in several ways. These radically altered ontogenies offer remarkable opportunities for the study of the mechanisms by which early development undergoes evolutionary modification. The recent availability of monoclonal antibody and cDNA probes that recognize homologous cells in embryos of closely related typical and direct developing species makes possible an experimental analysis of the cellular and molecular bases for heterochronic changes in development.     

Spine skeleton morphogenesis during regeneration in clypeasteroid and camarodont sea urchins

The process of skeleton morphogenesis is described for broken and totally removed spines in clypeasteroid (hollow spine) and camarodont (solid spine) sea urchins. Spine regeneration after total spine removal is completed in 40–45 days in clypeasteroids and in 60–70 days in camarodont sea urchins. Along with common stages of formation of longitudinal ribs in both hollow and solid spines, fundamental differences were found between the initial stages of reparative growth of the spine shaft. The spine shaft is formed from a single median process in clypeasteroids and from many simultaneously growing processes in camarodont sea urchins. Reparative morphogenesis of totally removed and partly broken spines in clypeasteroid sea urchins and totally removed spines in camarodont sea urchins leads to the formation of a skeletal structure identical to the intact spine. However, during the regeneration of broken camarodont spines, lateral growth is markedly retarded. As a result, the regenerated part of the spine shaft has a smaller diameter when the initial spine length is achieved. A hypothesis is proposed on a paedomorphic origin of spines in the clypeasteroid sea urchins on the basis of the juvenile stage of definitive spines in the camarodont sea urchins.     

Evolutionary crossroads in developmental biology: sea urchins

Embryos of the echinoderms, especially those of sea urchins and sea stars, have been studied as model organisms for over 100 years. The simplicity of their early development, and the ease of experimentally perturbing this development, provides an excellent platform for mechanistic studies of cell specification and morphogenesis. As a result, echinoderms have contributed significantly to our understanding of many developmental mechanisms, including those that govern the structure and design of gene regulatory networks, those that direct cell lineage specification, and those that regulate the dynamic morphogenetic events that shape the early embryo.     

Functional studies of MP62 during male chromatin decondensation in sea urchins

In amphibians, sperm histone transition post‐fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin‐like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin‐like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post‐fertilization and this phosphorylation is dependent on CDK‐cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin‐like activity in sea urchins participating during male pronucleus formation post‐fecundation. J. Cell. Biochem. 114: 1779–1788, 2013. © 2013 Wiley Periodicals, Inc.     

Embryonic expression of engrailed in sea urchins

Neural patterning genes that are expressed along the anterior-posterior axis of deuterostomes are expressed late in larval development in echinoderms and are thought to function in establishing the highly-derived, adult body plan. We have used genomic resources to clone an engrailed gene (SpEn) from Strongylocentrotus purpuratus, and with this we have developed an antibody specific for SpEn. SpEn is expressed late in embryogenesis in the developing larval nervous system. At the prism stage, a small number of neuroblasts in the oral ectoderm on the edge of the larval mouth begin expressing SpEn. The cells are in bilaterally symmetric positions. The expression of SpEn precedes the expression of the neural markers, synaptotagmin and serotonin in the SpEn immunoreactive cells. The SpEn cells are located on the margin of the domain of cells expressing SpNK2.1, but they do not have nuclear SpNK2.1. Expression of engrailed in a pair of bilateral neural structures in early development appears to be a shared feature of bilaterians.     

Localization of fodrin during fertilization and early development of sea urchins and mice

Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian alpha-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian alpha-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrin-specific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.     

Evolutionary reorganizations of ontogenesis in sea urchins

The data published during recent 15-20 years on comparative, experimental and molecular embryology of unusually developing sea urchins have been reviewed. These animals are characterized by large lipidrich eggs, highly modified embryogenesis, and the absence of a planktotrophic larva. Such a type of development is evolutionary advanced and arose independently in various phylogenetic lineages of the sea urchins.     

The study of proteins during early stages of embryogenesis of sea urchins

Summary The distribution of the preexisting and of thede novo synthesized proteins among soluble, and insoluble fractions, as well as between immunoprecipitable and non-immunoprecipitable soluble proteins has been studied in sea urchin embryos at different stages of development.In the S-100 fraction, which represented about 20% of total proteins, only a minor part of radioactivity was found. The majority of newly synthesized proteins was insoluble at neutral pH. Such distribution was practically invariant for all investigated stages of development, and was not markedly affected by Dactinomycin nor by 5-azacytidine.Only a small percentage of the total radioactivity of the S-100 fraction was found in the antigen-antibody complexes of soluble proteins. No shift of newly synthesized proteins towards the type of old, preexisting antigenic proteins was detected, and the majority of soluble newly synthesized proteins was found to be related to the non-immunoprecipitable soluble proteins.The authors gratefully acknowledge a generous gift of 5-azacytidine from Drs. Doskoil and ponar. We are also indebted to Dr. Miroslav Simi for kind interest and discussion.This work was supported in part by grants No 3111/1 from Federal Research Fund of Yugoslavia and No 02-020-1 from the National Institute of Health, U. S. Department of Health, Education and Welfare (PL-480 programme).     

Heterochrony and heterotopy in the phylogeny of sea urchins

The role of heterochrony is evident in ontogeny and phylogeny of irregular (exocyclic) sea urchins. After metamorphosis, a juvenile passes the stage of regular (endocyclic) sea urchin, in which the periproct is surrounded by plates of the apical system. A shift of the periproct in the area of the fifth interambulacrum occurs in extant taxa at early stages of postlarval development and is accompanied by the reduction of genital plate 5. In some ancient (Jurassic) adult irregular sea urchins, the endocyclic state of the apical system is retained for a long time and the derivative of the fifth genital plate is sometimes observed even in Early Cretaceous species. Considerable transformations in the structure of the lower test surface in members of the order Spatangoida are manifested in changes in the relative positions of plastron plates and ambulacral areas I and V, separation of sternal plates from the labrum, etc. The mechanism of these changes is connected with translocation or “sliding” of sutures of particular plates as a result of nonuniform growth and partial resorption. The study of evolutionary lineages of Cretaceous and Cenozoic sea urchins has shown that the evolution was connected with the directional changes in some morphological characters at late ontogenetic stages. The process was accompanied by either extension, peramorphosis (lineages of the genera Micraster, Infulaster–Hagenowia in the European Province), or the loss of these stages, paedomorphosis (Hemiaster (Bolbaster) lineage, Late Eocene–Middle Miocene of Australia). The phenomena of heterochrony and heterotopy in the development of peripetal, marginal, and lateroanal fascioles in the Late Cretaceous and Paleocene families Hemiasteridae, Schizasteridae, and Paleopneustidae are described. The heterotopy is also illustrated by the example of the development of additional genital pores on ocular plates II and IV of the Middle Jurassic species Pygomalus analis (Disasteroida); its apical system has five pores instead of four. In the Late Cretaceous species Guettaria roccardi (Holasteroida), ocular plates II and IV have two pores each; in the apical system, there are eight genital pores instead of four. In some members of the order Holectypoida, the place of lost genital plate 5 is occupied by a new plate sometimes pierced by a pore, but judging from crystallographic data, it is not homologous to other genital plates. The order Clypeasteroida is characterized by the development of very small pores in both ambulacral and interambulacral fields; they provide passage for numerous accessory tube feet.     

Variability of sperm specific histones in sea urchins

The variability of sperm histones was compared in two species of sea urchin. Whole sperm specific histones (SpH), were isolated from Tetrapygus niger (Arbacoida) and Parechinus angulosus (Echinoida). Individual histones were purified by chromatography on BioGel P-60 followed by reverse high pressure liquid chromatography (HPLC). The heterogeneity of each major histone type from T. niger was established from their HPLC elution patterns and further confirmed by electrophoresis in polyacrylamide gels containing 6 mM Triton X-100 combined with a transverse urea gradient (0--8 M). In T. niger, as well as in P. angulosus, a single form of SpH1 and SpH2A were found. In contrast, SpH2B was found to be heterogeneous, but represented by one major form in both species. The relatedness between both sets of histones was determined by establishing their immunological cross-reactivity. In this context, polyclonal antibodies elicited against T. niger sperm histones were assayed against individual histones from P. angulosus. From the results obtained, it emerged that histone SpH2A was the more closely related protein between these two species, followed by histone SpH1. In contrast, histone SpH2B was found to be only moderately related. These results confirm that SpH2A did not co-evolve with SpH2B, as was predicted for most species.     

Phylogeny and development of marine model species: strongylocentrotid sea urchins

The phylogenetic relationships of ten strongy-locentrotid sea urchin species were determined using mitochondrial DNA sequences. This phylogeny provides a backdrop for the evolutionary history of one of the most studied groups of sea urchins. Our phylogeny indicates that a major revision of this group is in order. All else remaining unchanged, it supports the inclusion of three additional species into the genus Strongylocentrotus (Hemicentrotus pulcherrimus, Allocentrotus fragilis, and Pseudocentrotus depressus). All were once thought to be closely related to this genus, but subsequent revisions separated them into other taxonomic groupings. Most strongylocentrotid species are the result of a recent burst of speciation in the North Pacific that resulted in an ecological diversification. There has been a steady reduction in the complexity of larval skeletons during the expansion of this group. Gamete attributes like egg size, on the other hand, are not correlated with phylogenetic position. In addition, our results indicate that the rate of replacement substitutions is highly variable among phylogenetic lineages. The branches leading to S. purpuratus and S. franciscanus were three to six times longer than those leading to closely related species.     

Phagocytosis and resorption during the reassembly of dissociated embryonic cells of sea urchins.

Time-lapse and electron microscopic observations were made on both epithelial and mesenchymal cells during the reassembly of embryos from dissociated cells of Strongylocentrotus purpuratus. In epithelial cells, where lysosomes are produced through the fusion of saccules formed from Golgi bodies, both phagocytosis of cell debris and resorption of differentiated cell structures were observed. In these cells, the lysosomes migrate and fuse with both autosomes and phagosomes. On the other hand, in the mesenchyme cells, where lysosomes are produced through the direct enlargement of the Golgi body's cisterna, neither phagocytosis nor resorption was observed. The migration of the lysosomes to the epithelial cell margins is the first indication of a re-establishment of cellular polarity after dissociation.