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  • Odie Beyer posted an update 3 months, 1 week ago

    A number of reports have demonstrated that pluripotent ESCs can form embryoid bodies (EBs) and spontaneously differentiate into PGCs and haploid spermatids in vitro (Geijsen et al., 2004; Hayashi et al., 2011; Kee et al., 2009; Toyooka et al., 2003), thereby providing an alternative model to investigate the signaling networks during early germ cell formation. Pluripotency related genes, Oct4 and Nanog, which are expressed at high levels in ESCs, are also enriched in PGCs, and therefore can serve as markers with SSEA1 surface antigen for ESC-derived PGCs (Geijsen et al., 2004; West et al., 2009). In addition, loss of genomic imprinting occurs solely in germ cell lineages, and thus DNA methylation erasure of imprinted genes has been a unique in vitro surrogate to confirm the identity of PGCs (Geijsen et al., 2004; West et al., 2009). In two recent papers, DAZ family members were shown to promote PGC formation from both mouse and human ESCs, but also to function in the later stages of meiosis and gamete formation in vitro (Kee et al., 2009; Yu et al., 2009). LIN28, a RNA binding protein, enhances BLIMP1 Ridaforolimus by blocking the maturation of let-7, a miRNA regulator of BLIMP1, and thus promotes PGC derivation from mouse ESCs indirectly (West et al., 2009). Therefore, the ESC in vitro differentiation model provides a useful system to dissect early germ cell development during embryogenesis.GASZ (also called ASZ1) is a recently identified Germ cell specific protein with four Ankyrin repeats, a Sterile alpha motif, and a putative leucine Zipper (Ma et al., 2009; Yan et al., 2004; Yan et al., 2002). In a previous report, we demonstrated that the loss of function of Gasz leads to male infertility due to defects in post-natal meiosis as well as abnormal piRNA biosynthesis and retrotransposon expression (Ma et al., 2009). However, the biological function of GASZ in early germ cell development has yet to be elucidated. In the present study, using in vitro differentiation models of human and mouse ESCs, we demonstrate that GASZ promotes PGC derivation and enhances the expression of genes involved in early germ cell development. In addition, we found that GASZ was highly enriched in post-migrating PGCs, and its deficiency led to defects in expression of early germ cell markers during embryogenesis in vivo. Further analyses reveal that GASZ interacts with DAZL to synergistically enhance PGC formation from ESCs. Therefore, our data demonstrate a novel role of GASZ in mammalian germ cell development both from ESCs and during early embryogenesis.Materials and methodsResultsDiscussionThe ESC differentiation system is a powerful, sensitive, and assessable in vitro model to explore the molecular pathways in lineage determination during early embryonic development. So far, several studies suggested that germ cells can be spontaneously developed from ESCs under appropriate conditions and can be further enriched by sorting them with SSEA1, CXCR4, c-kit or CD61 antigens (Bucay et al., 2009; Hayashi et al., 2011; Park et al., 2009; Tilgner et al., 2008) or by GFP under germ cell specific promoters (Hayashi et al., 2011; Kee et al., 2009). In this study, we utilized ESC differentiation systems to show that ectopic expression of Gasz, a gene known to be involved in post-natal spermatogenesis, could stimulate germ cell derivation from both human and mouse ESCs. Our study thus proposes a potential usage of ectopic gene manipulation in improving germ cell differentiation from ESCs. In addition, we revealed that Gasz deficiency impaired the germ cell derivation from ESCs and confirmed its requirement for expression of key PGC regulators, DAZL and MVH, with a Gasz knockout mouse model. We further demonstrated a functional interaction of GASZ and DAZL in promoting PGCs from ESCs. Therefore, our findings implicate a novel role of GASZ in early germ cell development in mammals, and also provide a useful system for investigating the molecular pathways in embryonic germ cell formation.

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