Primitive Endoderm Differentiates via a Three-Step Mechanism Involving Nanog and RTK Signaling

Abstract

During preimplantation mouse development, the inner cell mass (ICM) differentiates into two cell lineages--the epiblast and the primitive endoderm (PrE)--whose precursors are identifiable by reciprocal expression of Nanog and Gata6, respectively. PrE formation depends on Nanog by a non-cell-autonomous mechanism. To decipher early cell- and non-cell-autonomous effects, we performed a mosaic knockdown of Nanog and found that this is sufficient to induce a PrE fate cell autonomously. Strikingly, in Nanog null embryos, Gata6 expression is maintained, showing that initiation of the PrE program is Nanog independent. Treatment of Nanog null embryos with pharmacological inhibitors revealed that RTK dependency of Gata6 expression is initially direct but later indirect via Nanog repression. Moreover, we found that subsequent expression of Sox17 and Gata4--later markers of the PrE--depends on the presence of Fgf4 produced by Nanog-expressing cells. Thus, our results reveal three distinct phases in the PrE differentiation program.

Smad2 Is Essential for Maintenance of the Human and Mouse Primed Pluripotent Stem Cell State

“…Importantly, up-regulation of Cdx2 expression in these cells did not result from decreased Nanog expression but from activated BMP signaling. That we did not see cross-regulation between Nanog and Cdx2 expression differs from observations in mESCs, in which the role of Nanog is controlled by Oct4 (70,71). Additionally, the effect of Nanog down-regulation on lineage specification upon BMP stimulation still remains unclear.…”

Histone methyltransferase Smyd3 regulates early embryonic lineage commitment in mice

“…Additionally, Nanog is known to negatively interact with Gata6, and both genes are known to be key regulators in the establishment of EPI and PE fates respectively (Morris et al 2010, Frankenberg et al 2011, Kang et al 2013, Schrode et al 2014. Nanog-deficient mouse embryos are arrested during post-implantation development because of the widespread expression of Gata6 in the EPI (Mitsui et al 2003, Frankenberg et al 2011. By contrast, Gata6-deficient mouse embryos are arrested during postimplantation development because of the widespread expression of Nanog in the PE (Morris et al 2010, Schrode et al 2014.…”

“…In mouse preimplantation embryos, Oct4, Nanog, and Sox2 are required for the maintenance of ICM pluripotency (Nichols et al 1998, Avilion et al 2003, Mitsui et al 2003. Additionally, Nanog is known to negatively interact with Gata6, and both genes are known to be key regulators in the establishment of EPI and PE fates respectively (Morris et al 2010, Frankenberg et al 2011, Kang et al 2013, Schrode et al 2014. Nanog-deficient mouse embryos are arrested during post-implantation development because of the widespread expression of Gata6 in the EPI (Mitsui et al 2003, Frankenberg et al 2011.…”

From blastocyst to gastrula: gene regulatory networks of embryonic stem cells and early mouse embryogenesis

“…However, Sox17 and Gata4, which are markers of an established PrE identity, are also absent in Nanog mutant embryos [61,110], suggesting that Fgf4 expression is downstream of Nanog and is intrinsically linked to Epi identity during specification of the PrE lineage. Thus, Nanog is believed to have a non-cell-autonomous role in PrE formation through this circuitry [61,110].…”

“…Indeed, differential levels of FGF4 and FGFR2 between cells of the ICM are essential for the dynamics of PrE/Epi formation [46,[107][108][109], leading to the mutually exclusive expression of Nanog and Gata6 in the Epi and PrE, respectively [104]. In the absence of Nanog, Epi formation is specifically impaired [19] and Fgf4 expression is downregulated [110]. However, Sox17 and Gata4, which are markers of an established PrE identity, are also absent in Nanog mutant embryos [61,110], suggesting that Fgf4 expression is downstream of Nanog and is intrinsically linked to Epi identity during specification of the PrE lineage.…”