Differential repression of Otx2 underlies the capacity of NANOG and ESRRB to induce germline entry.

Matúš Vojtek,Ian Chambers,Juanjuan Sun,Man Zhang,Jingchao Zhang

doi:10.1016/j.stemcr.2021.11.013

Abstract

SummaryPrimordial germ cells (PGCs) arise from cells of the post-implantation epiblast in response to cytokine signaling. PGC development can be recapitulated in vitro by differentiating epiblast-like cells (EpiLCs) into PGC-like cells (PGCLCs) through cytokine exposure. Interestingly, the cytokine requirement for PGCLC induction can be bypassed by enforced expression of the transcription factor (TF) NANOG. However, the underlying mechanisms are not fully elucidated. Here, we show that NANOG mediates Otx2 downregulation in the absence of cytokines and that this is essential for PGCLC induction by NANOG. Moreover, the direct NANOG target gene Esrrb, which can substitute for several NANOG functions, does not downregulate Otx2 when overexpressed in EpiLCs and cannot promote PGCLC specification. However, expression of ESRRB in Otx2+/− EpiLCs rescues emergence of PGCLCs. This study illuminates the interplay of TFs occurring at the earliest stages of PGC specification.

Highlights

Germline development and sexual reproduction depend on the establishment of primordial germ cells (PGCs)
ESRRB cannot activate the PGC-like cells (PGCLCs) program To examine whether ESRRB can induce cytokine-free PGCLC differentiation to NANOG, we overexpressed NANOG or ESRRB during cytokine-free PGCLC differentiation
Cell lines carrying tetracycline-inducible Nanog (TgiN) or Esrrb (TgiE) transgenes were generated by integrating piggyBac transposons into E14Tg2a embryonic stem cells (ESCs) expressing the modified reverse tetracycline transactivator (Urlinger et al, 2000) from Rosa26 (Figure 1A)

Summary

Introduction

Germline development and sexual reproduction depend on the establishment of primordial germ cells (PGCs). Prospective PGCs downregulate the epiblast transcription factor (TF) OTX2 and subsequently activate the key PGC TFs BLIMP1 (PRDM1), PRDM14 and AP2g (Kurimoto et al, 2008; Ohinata et al, 2005; Vincent et al, 2005; Weber et al, 2010; Yamaji et al, 2008; Zhang and Chambers, 2019) These events can be recapitulated in vitro by differentiating naive embryonic stem cells (ESCs) into epiblast-like cells (EpiLCs), which are transiently competent to specify PGC-like cells (PGCLCs) in response to BMP4 and associated cytokines (Hayashi et al, 2011; Hayashi and Saitou, 2013). Even though Nanog is not essential for the emergence of PGCs, or for germline transmission (Carter et al, 2014; Zhang et al, 2018b), Nanog deletion results in a large decrease in PGC numbers both in vitro and in vivo (Chambers et al, 2007; Murakami et al, 2016; Yamaguchi et al, 2009; Zhang et al, 2018b)

Results

Discussion

Conclusion