A gene regulatory network controls the balance between mesendoderm and ectoderm at pluripotency exit

During embryogenesis, differentiation of pluripotent cells into somatic cell types depends both on signaling cues and intrinsic gene expression programs. While the molecular underpinnings of pluripotency are well mapped, much less is known on how mouse embryonic stem cells (mESCs) differentiate. Using RNA‐Seq profiling during specification to the three germ layers, we showed that mESCs switched on condition‐specific gene expression programs from the onset of the differentiation procedure and that primed pluripotency did not constitute an obligatory intermediate state. After inferring the gene network controlling mESC differentiation, we tested the role of the highly connected nodes by deleting them in a triple knock‐in Sox1‐Brachyury‐Eomes mESC line reporting on ectoderm, mesoderm, and endoderm fates. This led to the identification of regulators of mESC differentiation that acted at several levels: Sp1 as a global break on differentiation, Nr5a2 controlling ectoderm specification, and notably Fos:Jun and Zfp354c as opposite switches between ectoderm and mesendoderm fate.