Cooperative genetic networks drive embryonic stem cell transition from naïve to formative pluripotency

In the mammalian embryo, epiblast cells must exit the naïve state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene networks involved in the exit from naïve pluripotency remains fragmentary. Here, we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems biology to delineate the regulatory circuits governing naïve state exit. Transcriptome profiles for 73 ESC lines deficient for regulators of the exit from naïve pluripotency predominantly manifest delays on the trajectory from naïve to formative epiblast. We find that gene networks operative in ESCs are also active during transition from pre- to post-implantation epiblast in utero. We identified 496 naïve state-associated genes tightly connected to the in vivo epiblast state transition and largely conserved in primate embryos. Integrated analysis of mutant transcriptomes revealed funnelling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signalling pathways direct this pivotal mammalian cell state transition. A total number of 340 samples were analyzed as part of this project. 319 samples of 73 different KO cell lines and WT cell lines at two different conditions were used for the analysis. KOs were done in replicates (pairs) for each condition. The number of WT replicates is higher as WT samples were included in all batches. 21 additional samples were used in a WT differentiation time course. Time points from 0 to 32 hours are included (in steps of 2 hours). 0 hour and 32 hour samples were done in pairs and two different conditions at 0 hours were included.