A Nanog-dependent gene cluster initiates the specification of the pluripotent epiblast

The epiblast (Epi) is the source of embryonic stem (ES) cells and all embryonic tissues. It differentiates alongside the primitive endoderm (PrE) in a randomly distributed \"salt and pepper\" pattern from the inner cell mass (ICM) during preimplantation of the mammalian embryo. NANOG and GATA6 are key regulators of this binary differentiation event, which is further modulated by heterogeneous FGF signalling. When and how Epi and PrE lineage specification is initiated within the developing embryo is still unclear. Here we generated NANOG and GATA6 double KO (DKO) mouse embryos and performed single-cell expression analyses. We found that the ICM was unable to differentiate in the DKO mice, allowing us to characterize the ICM precursor state. The normally heterogeneous expression of Fgf4 between cells was significantly reduced in DKO ICMs, impairing FGF signalling. In contrast, several pluripotency markers did still display cell-to-cell expression variability in DKO ICMs. This revealed a primary heterogeneity independent of NANOG, GATA6 and FGF signalling that may also be conserved in humans. We found that NANOG is key in the initiation of epiblast specification already between the 16- and 32-cell stages, enabling the cell-clustered expression of many pluripotency genes. Our data uncover previously unknown biology in the early mouse embryo with potential implications for the field of pluripotent stem cells in human and other mammals.