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- Author: Mitinori Saitou x
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Department of Anatomy and Cell Biology, Laboratory for Mammalian Germ Cell Biology, JST, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Department of Anatomy and Cell Biology, Laboratory for Mammalian Germ Cell Biology, JST, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
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Department of Anatomy and Cell Biology, Laboratory for Mammalian Germ Cell Biology, JST, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Department of Anatomy and Cell Biology, Laboratory for Mammalian Germ Cell Biology, JST, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
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The specification of germ cell fate in development initiates mechanisms essential for the perpetuation of genetic information across the generations. Recent studies in mice have shown that germ cell specification requires at least three key molecular/cellular events: repression of the somatic program, re-acquisition of potential pluripotency, and an ensuing genome-wide epigenetic reprogramming. Moreover, a signaling and transcriptional principle governing these processes has been identified, raising the possibility of inducing the germ cell fate precisely from pluripotent stem cells in culture. These advances will in turn serve as a basis to explore the mechanism of germ cell specification in other mammals, including humans. The recapitulation of germ cell development in humans in culture will provide unprecedented opportunities to understand the basis of the propagation of our genome, both under normal and diseased conditions.
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Laboratory for Mammalian Germ Cell Biology, Department of Biosystems Science, Laboratory of Molecular Cell Biology and Development, RIKEN Kobe Institute, Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
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Laboratory for Mammalian Germ Cell Biology, Department of Biosystems Science, Laboratory of Molecular Cell Biology and Development, RIKEN Kobe Institute, Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
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The ability to monitor the development of a given cell lineage in a non-invasive manner by fluorescent markers both in vivo and in vitro provides a great advantage for the analysis of the lineage of interest. To date, a number of transgenic or knock-in mouse strains, in which developing germ cells are marked with fluorescent reporters, have been generated. We here describe a novel double transgenic reporter mouse strain that expresses membrane-targeted Venus (mVenus), a brighter variant of yellow fluorescent protein (YFP), under the control of Prdm1 (Blimp1) regulatory elements and enhanced cyan fluorescent protein (ECFP) under the control of Dppa3 (Stella/Pgc7). The double transgenic strain unambiguously marked Prdm1 expression in the lineage-restricted precursors of primordial germ cells (PGCs) in the proximal epiblast at embryonic day (E) 6.25 and specifically illuminated Prdm1- and Dppa3-positive migrating PGCs after E8.5. The double transgenic reporter also precisely recapitulated dynamic embryonic expression of Prdm1 outside the germ cell lineage. Moreover, we derived ES cells that bore both transgenes. These cells made a robust contribution both to the germ and somatic cell lineages in chimeras with accurate Prdm1-mVenus and Dppa3-ECFP expression. The transgenic strain and the ES cells will serve as valuable experimental materials not only for analyzing the origin and properties of the germ cell lineage in vivo, but also for establishing a culture system to efficiently induce proper germ cells with temporally coordinated Prdm1 and Dppa3 expression in vitro.
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Laboratory for Mammalian Germ Cell Biology, Department of Development and Differentiation, Department of Genetics and Division of Mammalian Development, Department of Anatomy and Cell Biology, JST, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
Laboratory for Mammalian Germ Cell Biology, Department of Development and Differentiation, Department of Genetics and Division of Mammalian Development, Department of Anatomy and Cell Biology, JST, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
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Mutations of RNA-binding proteins such as NANOS3, TIAL1, and DND1 in mice have been known to result in the failure of survival and/or proliferation of primordial germ cells (PGCs) soon after their fate is specified (around embryonic day (E) 8.0), leading to the infertility of these animals. However, the mechanisms of actions of these RNA-binding proteins remain largely unresolved. As a foundation to explore the role of these RNA-binding proteins in germ cells, we established a novel transgenic reporter strain that expresses NANOS3 fused with EGFP under the control of Nanos3 regulatory elements. NANOS3–EGFP exhibited exclusive expression in PGCs as early as E7.25, and continued to be expressed in female germ cells until around E14.5 and in male germ cells throughout the fetal period with declining expression levels after E16.5. NANOS3–EGFP resumed strong expression in postnatal spermatogonia and continued to be expressed in undifferentiated spermatogonial cells in adults. Importantly, the Nanos3–EGFP transgene rescued the sterile phenotype of Nanos3 homozygous mutants, demonstrating the functional equivalency of NANOS3–EGFP with endogenous NANOS3. We found that throughout germ cell development, a predominant amount of NANOS3–EGFP co-localized with TIAL1 (also known as TIAR) and phosphorylated eukaryotic initiation factor 2α, markers for the stress granules, whereas a fraction of it showed co-localization with DCP1A, a marker for the processing bodies. On the other hand, NANOS3–EGFP did not co-localize with Tudor domain-containing protein 1, a marker for the intermitochondrial cements, in spermatogenic cells. These findings unveil the presence of distinct posttranscriptional regulations in PGCs soon after their specification, for which RNA-binding proteins such as NANOS3 and TIAL1 would play critical functions.