Transcription factor co-expression mediates lineage priming for embryonic and extra-embryonic differentiation

In early mammalian development, cleavage stage blastomeres and inner cell mass (ICM) cells co-express embryonic and extra-embryonic transcriptional determinants. Using a protein-based double reporter we identify an embryonic stem cell (ESC) population that co-expresses the extra-embryonic factor GATA6 alongside the embryonic factor SOX2. Based on single cell transcriptomics, we find this population resembles the unsegregated ICM, exhibiting enhanced differentiation potential for endoderm while maintaining epiblast competence. To relate transcription factor binding in these cells to future fate, we describe a complete enhancer set in both ESCs and naive extra-embryonic endoderm stem cells and assess SOX2 and GATA6 binding at these elements in the ICM-like ESC sub-population. Both factors support cooperative recognition in these lineages, with GATA6 bound alongside SOX2 on a fraction of pluripotency enhancers and SOX2 alongside GATA6 more extensively on endoderm enhancers, suggesting that cooperative binding between these antagonistic factors both supports self-renewal and prepares progenitor cells for later differentiation.