ABSTRACT
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.
Subject(s)
Gene Expression Regulation, Developmental , Germ Cells/metabolism , Nanog Homeobox Protein/genetics , Otx Transcription Factors/genetics , Receptors, Estrogen/genetics , Biomarkers , Cell Differentiation/genetics , Cells, Cultured , Gene Knockdown Techniques , Genetic Heterogeneity , Germ Cells/cytology , Immunophenotyping , Nanog Homeobox Protein/metabolism , Otx Transcription Factors/metabolism , Protein Binding , Receptors, Estrogen/metabolismABSTRACT
Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germline specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESC self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential, whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces the efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification.
Subject(s)
Cell Differentiation/genetics , Cell Self Renewal/genetics , Germ Cells/metabolism , Mouse Embryonic Stem Cells/metabolism , Repressor Proteins/metabolism , Signal Transduction/genetics , Animals , Cell Line , Cloning, Molecular/methods , Gene Knockout Techniques/methods , Leukemia Inhibitory Factor/metabolism , Mice , Nanog Homeobox Protein/genetics , Nanog Homeobox Protein/metabolism , Plasmids , Receptors, Estrogen/metabolism , Repressor Proteins/genetics , Transfection , TransgenesABSTRACT
The successful segregation of germ cells from somatic lineages is vital for sexual reproduction and species survival. In the mouse, primordial germ cells (PGCs), precursors of all germ cells, are induced from the post-implantation epiblast1. Induction requires BMP4 signalling to prospective PGCs2 and the intrinsic action of PGC transcription factors3-6. However, the molecular mechanisms that connect BMP4 to induction of the PGC transcription factors that are responsible for segregating PGCs from somatic lineages are unknown. Here we show that the transcription factor OTX2 is a key regulator of these processes. Downregulation of Otx2 precedes the initiation of the PGC programme both in vitro and in vivo. Deletion of Otx2 in vitro markedly increases the efficiency of PGC-like cell differentiation and prolongs the period of PGC competence. In the absence of Otx2 activity, differentiation of PGC-like cells becomes independent of the otherwise essential cytokine signals, with germline entry initiating even in the absence of the PGC transcription factor BLIMP1. Deletion of Otx2 in vivo increases PGC numbers. These data demonstrate that OTX2 functions repressively upstream of PGC transcription factors, acting as a roadblock to limit entry of epiblast cells to the germline to a small window in space and time, thereby ensuring correct numerical segregation of germline cells from the soma.
