Embryonic stem cells (ESCs) can differentiate into all cell types of the embryonic germ layers. ESCs can also generate totipotent 2C-like cells and trophectodermal cells. However, these latter transitions occur at low frequency due to epigenetic barriers, the nature of which is not fully understood. Here, we show that treating mouse ESCs with sodium butyrate (NaB) increases the population of 2C-like cells and enables direct reprogramming of ESCs into trophoblast stem cells (TSCs) without a transition through a 2C-like state. Mechanistically, NaB inhibits histone deacetylase activities in the LSD1-HDAC1/2 corepressor complex. This increases acetylation levels in the regulatory regions of both 2C- and TSC-specific genes, promoting their expression. In addition, NaB-treated cells acquire the capacity to generate blastocyst-like structures that can develop beyond the implantation stage in vitro and form deciduae in vivo. These results identify how epigenetics restrict the totipotent and trophectoderm fate in mouse ESCs.

Multiple pluripotent states have been described in mouse and human stem cells. Here, we apply single-cell RNA-seq to a newly established BMP4 induced mouse primed to naïve transition (BiPNT) system and show that the reset is not a direct reversal of cell fate but goes through a primordial germ cell-like cells (PGCLCs) state. We first show that epiblast stem cells bifurcate into c-Kit⁺ naïve and c-Kit⁻ trophoblast-like cells, among which, the naïve branch undergoes further transition through a PGCLCs intermediate capable of spermatogenesis in vivo. Mechanistically, we show that DOT1L inhibition permits the transition from primed pluripotency to PGCLCs in part by facilitating the loss of H3K79me2 from Gata3/6. In addition, Prdm1/Blimp1 is required for PGCLCs and naïve cells, while Gata2 inhibits PGC-like state by promoting trophoblast-like fate. Our work not only reveals an alternative route for primed to naïve transition, but also gains insight into germ cell development.

Mouse primordial germ cells (PGCs), originate from the early post-implantation epiblast in response to BMP4 secreted by the extraembryonic ectoderm. However, how BMP4 acts here has remained unclear. Recent work has identified the transcription factor (TF), OTX2 as a key determinant of the segregation of the germline from the soma. OTX2 is expressed ubiquitously in the early post-implantation epiblast, decreasing rapidly in cells that initiate the PGC programme. Otx2 mRNA is also rapidly repressed by BMP4 in vitro, in germline competent cells. Supporting a model in which BMP4 represses Otx2, enforcing sustained OTX2 expression in competent cells blocks germline entry. In contrast, Otx2-null epiblast cells enter the germline with increased efficiency in vitro and in vivo and can do so independently of BMP4. Also, Otx2-null cells can initiate germline entry even without the crucial PGC TF, BLIMP1. In this review, we survey recent advances and propose hypotheses concerning germline entry.

Primordial 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.