Conditional knockdown of Nanog induces apoptotic cell death in mouse migrating primordial germ cells

Abstract

The pluripotency factor Nanog is expressed in peri-implantation embryos and primordial germ cells (PGCs). Nanog-deficient mouse embryos die soon after implantation. To explore the function of Nanog in germ cells, Nanog RNA was conditionally knocked down in vivo by shRNA. Nanog shRNA transgenic (NRi-Tg) mice were generated through the formation of germline chimeras with NRi-Tg embryonic stem cells. In E12.5 Cre-induced ER-Cre/NRi-Tg and TNAP-Cre/NRi-Tg double-transgenic embryos, the number of alkaline phosphatase-positive and SSEA1-positive PGCs decreased significantly. In the E9.5 and E10.5 migrating Nanog-knockdown PGCs, TUNEL-positive apoptotic cell death became prominent in vivo and in vitro, despite Oct4 expression. Single-cell microarray analysis of E10.5 Nanog-knockdown PGCs revealed significant up-and downregulation of a substantial number of genes, including Tial1, Id1 and Suz12. These data suggest that Nanog plays a key role in the proliferation and survival of migrating PGCs as a safeguard of the PGC-specific molecular network.

Epigenetic reprogramming in the germline: towards the ground state of the epigenome

“…These experiments hence suggested a potential role of Nanog in the reprogramming process. However, an independent study using the in vivo knockdown approach of Nanog revealed a gradual loss of PGCs prior to the reprogramming step, supporting the notion that Nanog is involved in the maintenance of the germline [17].…”

Primordial Germ Cells in Mice

“…Conditional knockout of Oct4 or conditional knockdown of Nanog in migrating PGCs results in the death of PGCs by apoptosis, indicating the importance of these gene products in the maintenance of PGCs (Table 1) (Kehler et al 2004;Chambers et al 2007;Yamaguchi et al 2009). Hypoxia-inducible factor 2a (HIF2a), which has a function to directly up-regulate Oct4, is also critical in PGC specification and/or survival ( Fig.…”

Switching on pluripotency: a perspective on the biological requirement of Nanog

“…Single-cell expression analysis in Nanog-depleted PGCs at E10.5 indicated significant up-or downregulation of several genes, including the repression of Id1 and the PRC2 subunit Suz12. However, major transcriptional regulators of germ cell fate were similarly expressed between Nanog knockdown and control PGCs [25]. These data suggest that lack of Nanog expression causes the loss of PGCs by E12.5 as a result of progressive apoptotic cell death, rather than trans-differentiation to a somatic cell fate.…”

“…Deletion of Nanog in established ES cells however does not impair ES cell self-renewal, and upon re-introduction of these in development they are competent to efficiently contribute to all somatic lineages [8]. However, Nanog-deficient ES cells fail to contribute to the germ lineage beyond E11.5 [8,25].…”

“…What is the fate of presumptive PGCs that lack Nanog? Yamaguchi and co-workers [25] addressed this question by conditional shRNA-mediated knockdown of Nanog mRNA in PGCs. Knockdown of Nanog in migrating PGCs resulted in the appearance of apoptotic cell death as early as E10.5.…”