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Gary M. Wessel - One of the best experts on this subject based on the ideXlab platform.
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Post-translational regulation by gustavus contributes to selective Vasa protein accumulation in multipotent cells during embryogenesis
Developmental biology, 2010Co-Authors: Eric A. Gustafson, Mamiko Yajima, Celina E. Juliano, Gary M. WesselAbstract:Vasa is a broadly conserved DEAD-box RNA helicase associated with germ line development and is expressed in multipotent cells in many animals. During embryonic development of the sea urchin Strongylocentrotus purpuratus, Vasa protein is enriched in the small micromeres despite a uniform distribution of Vasa transcript. Here we show that the Vasa coding region is sufficient for its selective enrichment and find that gustavus, the B30.2/SPRY and SOCS box domain gene, contributes to this phenomenon. In vitro binding analyses show that Gustavus binds the N-terminal and DEAD-box portions of Vasa protein independently. A knockdown of Gustavus protein reduces both Vasa protein abundance and its propensity for accumulation in the small micromeres, whereas overexpression of the Vasa-interacting domain of Gustavus (GusΔSOCS) results in Vasa protein accumulation throughout the embryo. We propose that Gustavus has a conserved, positive regulatory role in Vasa protein accumulation during embryonic development.
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Vasa protein expression is restricted to the small micromeres of the sea urchin but is inducible in other lineages early in development
Developmental Biology, 2008Co-Authors: Ekaterina Voronina, Eric A. Gustafson, Celina E. Juliano, Manuel Lopez, Jia L Song, Cassandra G Extavour, Sophie B George, Paola Oliveri, David R Mcclay, Gary M. WesselAbstract: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.
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Vasa protein expression is restricted to the small micromeres of the sea urchin but is inducible in other lineages early in development
Developmental Biology, 2008Co-Authors: Ekaterina Voronina, Eric A. Gustafson, Celina E. Juliano, Jia L Song, Cassandra G Extavour, Sophie B George, Paola Oliveri, David R Mcclay, Manuel E Lopez, Gary M. WesselAbstract: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.
Yunhan Hong - One of the best experts on this subject based on the ideXlab platform.
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light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka
Gene, 2014Co-Authors: Yongming Yuan, Yunhan HongAbstract:Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as Vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development.
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Medaka Vasa is required for migration but not survival of primordial germ cells
Mechanisms of Development, 2009Co-Authors: Ni Hong, Jianfang Gui, Yunhan HongAbstract:Vasa is essential for germline development. However, the precise processes in which Vasa involves vary considerably in diverse animal phyla. Here we show that Vasa is required for primordial germ cell (PGC) migration in the medakafish. Vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of Vasa RNA. Interestingly, Vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. importantly, Vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcT4b in migratory tissues remained unchanged by Vasa knockdown. By chimera formation we show that PGCs from Vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in Vasa-disrupted embryos. Furthermore, ectopic PGCs in Vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka Vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival. (C) 2009 Elsevier Ireland Ltd. All rights reserved.
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Medaka Vasa is required for migration but not survival of primordial germ cells.
Mechanisms of development, 2009Co-Authors: Ni Hong, Jianfang Gui, Yunhan HongAbstract:Vasa is essential for germline development. However, the precise processes in which Vasa involves vary considerably in diverse animal phyla. Here we show that Vasa is required for primordial germ cell (PGC) migration in the medakafish. Vasa knockdown by two morpholinos led to the PGC migration defect that was rescued by coinjection of Vasa RNA. Interestingly, Vasa knockdown did not alter the PGC number, identity, proliferation and motility even at ectopic locations. We established a cell culture system for tracing PGCs at the single cell level in vitro. In this culture system, control and morpholino-injected gastrulae produced the same PGC number and the same time course of PGC survival. Importantly, Vasa-depleted PGCs in culture had similar motility and locomotion to normal PGCs. Expression patterns of wt1a, sdf1b and cxcr4b in migratory tissues remained unchanged by Vasa knockdown. By chimera formation we show that PGCs from Vasa-depleted blastulae failed to migrate properly in the normal environment, whereas control PGCs migrated normally in Vasa-disrupted embryos. Furthermore, ectopic PGCs in Vasa-depleted embryos also retained all the PGC properties examined. Taken together, medaka Vasa is cell-autonomously required for PGC migration, but dispensable to PGC proliferation, motility, identity and survival.
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differential expression of Vasa rna and protein during spermatogenesis and oogenesis in the gibel carp carassius auratus gibelio a bisexually and gynogenetically reproducing vertebrate
Developmental Dynamics, 2005Co-Authors: Jianfang Gui, Yunhan HongAbstract:The RNA helicase Vasa is a germ cell marker in animals, and its homolog in vertebrates to date has been limited to bisexual reproduction. We cloned and characterized CagVasa, a Vasa homolog from the gibel carp, a fish that reproduces bisexually or gynogenetically. CagVasa possesses 14 RGG repeats and eight conserved motifs of Vasa proteins. In bisexually reproducing gibel carp, Vasa is maternally supplied and its zygotic expression is restricted to gonads. By in situ hybridization on testicular sections, Vasa is low in spermatogonia, high in primary spermatocytes, reduced in secondary spermatocytes, but disappears in spermatids and sperm. In contrast, Vasa persists throughout oogenesis, displaying low-high-low levels from oogonia over vitellogenic oocytes to maturing oocytes. A rabbit anti-Vasa antibody (alpha Vasa) was raised against the N-terminal CagVasa for fluorescent immunohistochemistry. On testicular sections, Vasa is the highest in spermatogonia, reduced in spermatocytes, low in spermatids, and absent in sperm. In the ovary, Vasa is the highest in oogonia but persists throughout oogenesis. Subcellular localization of Vasa and its protein changes dynamically during oogenesis. The aVasa stains putative primordial germ cells in gibel carp fry. It detects gonadal germ cells also in several other teleosts. Therefore, CagVasa encodes a Vasa ortholog that is differentially expressed in the testis and ovary. Interestingly, the alpha Vasa in combination with a nuclear dye can differentiate critical stages of spermatogenesis and oogenesis in fish. The cross-reactivity and the ability to stain stage-specific germ cells make this antibody a useful tool to identify fish germ cell development and differentiation. (c) 2005 Wiley-Liss, Inc.
Claire M Peppiattwildman - One of the best experts on this subject based on the ideXlab platform.
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supplementary material for an intact kidney slice model to investigate Vasa recta properties and function in situ
2017Co-Authors: C Crawford, C Sprott, L Sawbridge, T M Kennedylydon, R J Unwin, Scott S P Wildman, Jgl Munday, Tejal A Desai, Claire M PeppiattwildmanAbstract:Background: Medullary blood flow is via Vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending Vasa recta show that pericytes can constrict/dilate descending Vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated Vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in ‘live’ kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in Vasa recta diameter. Results: Pericytes were identified on Vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in Vasa recta diameter (10–30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E 2 ) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L -NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating Vasa recta function in situ and the role of pericytes as regulators of Vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.
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nonsteroidal anti inflammatory drugs alter Vasa recta diameter via pericytes
American Journal of Physiology-renal Physiology, 2015Co-Authors: C Crawford, Scott S P Wildman, Claire M PeppiattwildmanAbstract:We have previously shown that Vasa recta pericytes are known to dilate Vasa recta capillaries in the presence of PGE2 and contract Vasa recta capillaries when endogenous production of PGE2 is inhib...
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an intact kidney slice model to investigate Vasa recta properties and function in situ
Nephron Physiology, 2012Co-Authors: C Crawford, C Sprott, L Sawbridge, R J Unwin, Scott S P Wildman, Jgl Munday, Tejal A Desai, Claire M PeppiattwildmanAbstract:Background: Medullary blood flow is via Vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending Vasa recta show that pericytes can constrict/dilate descending Vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated Vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in ‘live’ kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in Vasa recta diameter. Results: Pericytes were identified on Vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in Vasa recta diameter (10–30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E2) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating Vasa recta function in situ and the role of pericytes as regulators of Vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.
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an intact kidney slice model to investigate Vasa recta properties and function in situ
Nephron Physiology, 2012Co-Authors: C Crawford, C Sprott, L Sawbridge, T M Kennedylydon, R J Unwin, Scott S P Wildman, Jgl Munday, Tejal A Desai, Claire M PeppiattwildmanAbstract:Background: Medullary blood flow is via Vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending Vasa recta show that pericytes can constrict/dilate descending Vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated Vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in ‘live’ kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in Vasa recta diameter. Results: Pericytes were identified on Vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in Vasa recta diameter (10–30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E2) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating Vasa recta function in situ and the role of pericytes as regulators of Vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.
Eric A. Gustafson - One of the best experts on this subject based on the ideXlab platform.
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Post-translational regulation by gustavus contributes to selective Vasa protein accumulation in multipotent cells during embryogenesis
Developmental biology, 2010Co-Authors: Eric A. Gustafson, Mamiko Yajima, Celina E. Juliano, Gary M. WesselAbstract:Vasa is a broadly conserved DEAD-box RNA helicase associated with germ line development and is expressed in multipotent cells in many animals. During embryonic development of the sea urchin Strongylocentrotus purpuratus, Vasa protein is enriched in the small micromeres despite a uniform distribution of Vasa transcript. Here we show that the Vasa coding region is sufficient for its selective enrichment and find that gustavus, the B30.2/SPRY and SOCS box domain gene, contributes to this phenomenon. In vitro binding analyses show that Gustavus binds the N-terminal and DEAD-box portions of Vasa protein independently. A knockdown of Gustavus protein reduces both Vasa protein abundance and its propensity for accumulation in the small micromeres, whereas overexpression of the Vasa-interacting domain of Gustavus (GusΔSOCS) results in Vasa protein accumulation throughout the embryo. We propose that Gustavus has a conserved, positive regulatory role in Vasa protein accumulation during embryonic development.
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Vasa protein expression is restricted to the small micromeres of the sea urchin but is inducible in other lineages early in development
Developmental Biology, 2008Co-Authors: Ekaterina Voronina, Eric A. Gustafson, Celina E. Juliano, Manuel Lopez, Jia L Song, Cassandra G Extavour, Sophie B George, Paola Oliveri, David R Mcclay, Gary M. WesselAbstract: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.
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Vasa protein expression is restricted to the small micromeres of the sea urchin but is inducible in other lineages early in development
Developmental Biology, 2008Co-Authors: Ekaterina Voronina, Eric A. Gustafson, Celina E. Juliano, Jia L Song, Cassandra G Extavour, Sophie B George, Paola Oliveri, David R Mcclay, Manuel E Lopez, Gary M. WesselAbstract: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.
Celina E. Juliano - One of the best experts on this subject based on the ideXlab platform.
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Post-translational regulation by gustavus contributes to selective Vasa protein accumulation in multipotent cells during embryogenesis
Developmental biology, 2010Co-Authors: Eric A. Gustafson, Mamiko Yajima, Celina E. Juliano, Gary M. WesselAbstract:Vasa is a broadly conserved DEAD-box RNA helicase associated with germ line development and is expressed in multipotent cells in many animals. During embryonic development of the sea urchin Strongylocentrotus purpuratus, Vasa protein is enriched in the small micromeres despite a uniform distribution of Vasa transcript. Here we show that the Vasa coding region is sufficient for its selective enrichment and find that gustavus, the B30.2/SPRY and SOCS box domain gene, contributes to this phenomenon. In vitro binding analyses show that Gustavus binds the N-terminal and DEAD-box portions of Vasa protein independently. A knockdown of Gustavus protein reduces both Vasa protein abundance and its propensity for accumulation in the small micromeres, whereas overexpression of the Vasa-interacting domain of Gustavus (GusΔSOCS) results in Vasa protein accumulation throughout the embryo. We propose that Gustavus has a conserved, positive regulatory role in Vasa protein accumulation during embryonic development.
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Vasa protein expression is restricted to the small micromeres of the sea urchin but is inducible in other lineages early in development
Developmental Biology, 2008Co-Authors: Ekaterina Voronina, Eric A. Gustafson, Celina E. Juliano, Manuel Lopez, Jia L Song, Cassandra G Extavour, Sophie B George, Paola Oliveri, David R Mcclay, Gary M. WesselAbstract: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.
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Vasa protein expression is restricted to the small micromeres of the sea urchin but is inducible in other lineages early in development
Developmental Biology, 2008Co-Authors: Ekaterina Voronina, Eric A. Gustafson, Celina E. Juliano, Jia L Song, Cassandra G Extavour, Sophie B George, Paola Oliveri, David R Mcclay, Manuel E Lopez, Gary M. WesselAbstract: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.
