Establishing competence for trophectoderm specification in mouse embryos before the first lineage bifurcation

How embryonic blastomeres transition from totipotency to differentiation remains a fundamental question in developmental biology. Here, we uncover the transcription factor CEBPa as an orchestrator of this process. CEBPa expression is first activated by NR5A2 at the 2-cell to 4-cell transition and in a second wave peaks in the trophectoderm of blastocysts. Loss of CEBPa disrupts timely blastocyst formation and impairs trophectoderm development, while its forced expression in embryonic stem cells drives differentiation into trophectoderm-like cells. Mechanistically, CEBPa sequentially activates key trophectoderm enhancers in a dose-dependent manner. At the 4-cell stage, some of these trophectoderm enhancers are already active while others are primed or still closed, placing blastomeres in a state of alert. These findings establish CEBPa as a molecular switch that equips 4-cell blastomeres with the competence for trophectoderm differentiation, defining the earliest steps in cell fate bifurcation and shedding new light on the mechanisms driving embryonic lineage specification.