During mammalian development, the challenge for the embryo is to override intrinsic cellular plasticity to drive cells to distinct fates. Here, we unveil novel roles for the HIPPO signaling pathway in controlling cell positioning and expression of Sox2, the first marker of pluripotency in the mouse early embryo. We show that maternal and zygotic YAP1 and WWTR1 repress Sox2 while promoting expression of the trophectoderm gene Cdx2 in parallel. Yet, Sox2 is more sensitive than Cdx2 to Yap1/Wwtr1 dosage, leading cells to a state of conflicted cell fate when YAP1/WWTR1 activity is moderate. Remarkably, HIPPO signaling activity resolves conflicted cell fate by repositioning cells to the interior of the embryo, independent of its role in regulating Sox2 expression. Rather, HIPPO antagonizes apical localization of Par complex components PARD6B and aPKC. Thus, negative feedback between HIPPO and Par complex components ensure robust lineage segregation.

In mammals, the first few days of development entail segregation of pluripotent and extraembryonic trophectoderm cells. The challenge for the embryo at this time is to override cell plasticity to ensure that cells adopt distinct fates. Here, we identify novel mechanisms restricting expression of Sox2 and Cdx2 to mutually exclusive domains.We show that Sox2 is repressed in the trophectoderm downstream of ROCK1/2 and HIPPO pathway LATS1/2 kinases. LATS1/2 kinases are thought to antagonize YAP1 and WWTR1, the transcriptional partners of TEAD4. However, the combined loss of Yap1; Wwtr1 has not been reported. Using female germ line deletion and null alleles, we show that YAP1/WWTR1/TEAD4 simultaneously antagonize Sox2 and promote Cdx2 expression in trophectoderm. However, Cdx2 is less sensitive to Yap1 and Wwtr1 dosage, which can lead cells to aberrantly coexpress CDX2 and SOX2, reminiscent of conflicted cell fate. We show that HIPPO resolves cell fate conflicts by elevating Sox2 expression and driving cells to the inner cell mass, but Sox2 is not required for HIPPOmediated cell repositioning. Rather, HIPPO signaling represses cell polarity components PAR and aPKC, facilitating cell internalization upstream of Sox2. We propose that HIPPO engages in negative feedback and PAR-aPKC providing a fail-safe to ensure lineage segregation.

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