Endoderm Differentiationin VitroIdentifies a Transitional Period for Endoderm Ontogeny in the Sea Urchin Embryo

AbstractThe vegetal plate of the sea urchin embryo is specified during early cleavage divisions of the embryo as shown by the classical experiments of Horstadius (reviewed in “Experimental Embryology of Echinoderms,” 1973, Clarendon, Oxford). Not until gastrulation, though, do the cells within this territory differentiate into their characteristic cell types. Vegetal plate descendents comprise the coelomic epithelium, circumesophageal muscle, basal cells, pigment cells, and endodermal epithelium. We report here that cells of the endodermal lineage acquire the ability to differentiate autonomously several hours prior to gastrulation, between the late blastula and early mesenchyme blastula stages. Cells dissociated from whole embryos after the late blastula stage have the ability to differentiatein vitro,independent of cell contacts and of the embryonic environment. In contrast, preendoderm cells removed from the embryo prior to the late blastula stage show no ability to differentiate when culturedin vitroeven though cells of other lineages, e.g., ectoderm and skeletogenic mesenchyme, show morphological and molecular differentiation in these same cultures. We have used the expression of the endoderm-specific gene products Endo 1 and LvN1.2, detected by RNase protection assays and byin situimmunolabeling, to quantify endoderm differentiation independent of embryonic or cellular morphology. These studies define a transitional period in the ontogeny of the endoderm, from cells reliant on interactions to promote fate specification and organization of territories to later events involved in morphogenesis that result from cell-type-specific gene expression

Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin

AbstractSea urchin eggs secrete a serine protease activity, CGSP1, at fertilization that is essential for the block to polyspermy. Several targets of this proteolytic activity on the plasma membrane were identified here using a cell surface biotinylation approach. Amino acid microsequencing of one of these proteins led to the identification of a 4.75-kb cDNA clone from a Strongylocentrotus purpuratus ovary cDNA library that encodes a 160-kDa protein called p160. This protein contains five CUB domains and a putative transmembrane domain suggesting that p160 is an integral membrane protein with protein–protein interaction motifs facing the extracellular matrix of the egg. Whole-mount immunolocalization studies demonstrate that p160 is on the surface of the egg, enriched at the tips of microvilli. The protein is removed at fertilization in a protease-dependent manner, and functional assays suggest that p160 serves to link the plasma membrane to the vitelline layer until fertilization. Thus, p160 is a key candidate for a vitelline-layer linker protein, the selective proteolysis of which functions in the block to polyspermy in the sea urchin egg

Cyclin B synthesis is required for sea urchin oocyte maturation

Sea urchins are members of a limited group of animals in which meiotic maturation of oocytes is completed prior to fertilization. This is different from oocytes of most animals such as mammals and amphibians in which fertilization reactivates an arrested meiotic cycle. Using a recently developed technique for in vitro maturation of sea urchin oocytes, we analyzed the role of cyclin B, the regulatory component of maturation-promoting factor, in the control of sea urchin oocyte meiotic induction and progression. Oocytes of the sea urchin Lytechinus variegatus accumulate significant amounts of cyclin B mRNA and protein during oogenesis. We analyzed cyclin B synthetic requirements in oocytes and early embryos by inhibiting cyclin B synthesis with DNA and morpholino antisense oligonucleotides. Cyclin B synthesis is not necessary for the entry of G2-arrested oocytes into meiosis; however, it is required for the proper progression through meiotic divisions. Surprisingly, mature sea urchin eggs contain significant cyclin B protein following meiosis that serves as a maternal store for early cleavage divisions. We also find that cyclin A can functionally substitute for cyclin B in early embryos but not in oocytes. These studies provide a foundation for understanding the mechanism of meiotic maturation independent of the zygotic cell cycle

Oogenesis: Single cell development and differentiation

AbstractOocytes express a unique set of genes that are essential for their growth, for meiotic recombination and division, for storage of nutrients, and for fertilization. We have utilized the newly sequenced genome of Strongylocentrotus purpuratus to identify genes that help the oocyte accomplish each of these tasks. This study emphasizes four classes of genes that are specialized for oocyte function: (1) Transcription factors: many of these factors are not significantly expressed in embryos, but are shared by other adult tissues, namely the ovary, testis, and gut. (2) Meiosis: A full set of meiotic genes is present in the sea urchin, including those involved in cohesion, in synaptonemal complex formation, and in meiotic recombination. (3) Yolk uptake and storage: Nutrient storage for use during early embryogenesis is essential to oocyte function in most animals; the sea urchin accomplishes this task by using the major yolk protein and a family of accessory proteins called YP30. Comparison of the YP30 family members across their conserved, tandem fasciclin domains with their intervening introns reveals an incongruence in the evolution of its major clades. (4) Fertilization: This set of genes includes many of the cell surface proteins involved in sperm interaction and in the physical block to polyspermy. The majority of these genes are active only in oocytes, and in many cases, their anatomy reflects the tandem repeating interaction domains essential for the function of these proteins. Together, the expression profile of these four gene classes highlights the transitions of the oocyte from a stem cell precursor, through stages of development, to the clearing and re-programming of gene expression necessary to transition from oocyte, to egg, to embryo

The TATA Binding Protein in the Sea Urchin Embryo Is Maternally Derived

AbstractThe cDNA encoding the TATA binding protein was isolated from 8- to 16-cell and morula-stage embryonic libraries of two distantly related species of sea urchin,Strongylocentrotus purpuratusandLytechinus variegatus,respectively. The two proteins are 96% identical over both the N- and C-terminal domains, suggesting a conservation of transcriptional processes between the two species. The prevalence of SpTBP transcripts at several developmental time points was determined using the tracer excess titration method, and the corresponding number of TBP protein molecules was determined by quantitative Western blot analysis. Our results indicate that the amount of TBP mRNA and protein per embryo remains relatively constant throughout development. An initial large pool of TBP protein (>109) molecules in the egg becomes diluted as a consequence of cell division and decreases to about 2 × 106molecules per cell by the gastrula stage. We found byin situRNA hybridization that the oocyte contains a large amount of TBP mRNA which is depleted late in oogenesis so that the eggs and early embryos have extremely low levels of TBP mRNA. We conclude that the oocyte manufactures nearly all of the TBP protein necessary for embryogenesis

Cyclin E and Its Associated cdk Activity Do Not Cycle during Early Embryogenesis of the Sea Urchin

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity

Sea Star Wasting Disease in \u3cem\u3eAsterias forbesi\u3c/em\u3e along the Atlantic Coast of North America

As keystone species, sea stars serve to maintain biodiversity and species distribution through trophic level interactions in marine ecosystems. Recently, Sea Star Wasting Disease (SSWD) has caused widespread mass mortality in several sea star species from the Pacific Coast of the United States of America (USA) and Asterias forbesi on the Atlantic Coast. A densovirus, named Sea Star associated Densovirus (SSaDV), has been associated with the wasting disease in Pacific Coast sea stars, and limited samples of A. forbesi. The goal of this research is to examine the pathogenesis of SSWD in A. forbesi on the Atlantic Coast of the USA and to determine if SSaDV is associated with the wasting disease in this species. Histological examination of A. forbesi tissues affected with SSWD showed cuticle loss, vacuolation and necrosis of epidermal cells, and oedema of the dermis, but no consistent evidence indicating the cause of the lesions. Challenge experiments by cohabitation and immersion in infected water suggest that the cause of SSWD is viral in nature, as filtration (0.22 μm) of water from tanks with sea stars exhibiting SSWD did not prevent the transmission and progression of the disease. Death of challenged sea stars occurred 7–10 d after exposure to infected water or sea stars, and the infectivity crossed species (A. forbesi and Pateria miniata) with equal penetrance. Of the 48 stars tested by quantitative real time PCR, 29 (60%) were positive for the SSaDV VP1 gene. These stars represent field-collected sea stars from all geographical regions (South Carolina to Maine) in 2012–2015, as well as stars exposed to infected stars or water from affected tanks. However, a clear association between the presence of SSaDV and SSWD signs in experimental and field-collected A. forbesi was not found in this study

Germline factor DDX4 functions in blood‐derived cancer cell phenotypes

DDX4 (the human ortholog of Drosophila Vasa) is an RNA helicase and is present in the germ lines of all metazoans tested. It was historically thought to be expressed specifically in germline, but with additional organisms studied, it is now clear that in some animals DDX4/Vasa functions outside of the germline, in a variety of somatic cells in the embryo and in the adult. In this report, we document that DDX4 is widely expressed in soma‐derived cancer cell lines, including myeloma (IM‐9) and leukemia (THP‐1) cells. In these cells, the DDX4 protein localized to the mitotic spindle, consistent with findings in other somatic cell functions, and its knockout in IM‐9 cells compromised cell proliferation and migration activities, and downregulated several cell cycle/oncogene factors such as CyclinB and the transcription factor E2F1. These results suggest that DDX4 positively regulates cell cycle progression of diverse somatic‐derived blood cancer cells, implying its broad contributions to the cancer cell phenotype and serves as a potential new target for chemotherapy

Vasa Protein Expression is Restricted to the Small Micromeres of the Sea Urchin, but is Inducible in Other Lineages Early in Development

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.Organismic and Evolutionary Biolog

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation

AbstractThe sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm–egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca2+ signaling and homeostasis modules present in the egg and zygote. The sea urchin “calcium toolkit” as predicted by the genome is described. Emphasis is on the Ca2+ signaling modules operating during egg activation, but the Ca2+ signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca2+ at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca2+ serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies