The Role of Hippo-Yap1 Signaling in Germ-layer Specification

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ABSTRACT Understanding diagnose, on signaling well-known for its essential activities promoting cell proliferation during development. suggest important roles of YAP as a cell-fate determinant in early embryogenesis. Accordingly, our ongoing studies using human Embryonic Stem Cells (hESCs) 2D- models of gastrulation reveals that YAP is essential for the correct specification of the three germ-layers. The YAP KO-derived human 2D-gastruloids display expanded mesoderm and endoderm layers (ME) and reduced ectoderm layer, compared to WT. NODAL signaling homeostasis is essential for normal gastrulation; active NODAL cues are needed for ME differentiation. However, efficient NODAL inhibition is needed for the acquisition of an ectodermal potential. Our data suggest that the gastrulation phenotype in YAP KO cells is associated with an overly active NODAL signaling. The NODAL gene codifies for the ligand that activates the intracellular Smad2.3 signaling. Interestingly, our transcriptomic and epigenomic analysis suggest that YAP is needed to repress the well-characterized Proximal Epiblast Enhancer (PEE) of the NODAL gene during the exit of pluripotency toward an ectodermal fate. Our data further suggest that YAP restricts the chromatin development the human embryo will lead to discovery therapeutic tools to treat and prevent birth defects. The overarching goal of this proposal is to advance our knowledge the signaling and epigenetic mechanisms that regulate the process of human gastrulation. The Hippo- effector YAP1 (YAP) is However, increasing evidences the of the of accessibility of the NODAL locus, essential for gene repression. These general specification”. findings form the premise of our hypothesis: “Epigenetic regulation NODAL signaling by YAP is crucial for correct germ-layer We will address this idea in the following Aims. In Aim1 of , we will analyze the role of YAP in human 3D-gastruloids. We hypothesize that the ability of human epiblast cells to undergo three-dimensional gastrulation, and organization in an anteroposterior axis is regulated by YAP. To test this idea, we will apply a state-of-art synthetic human embryology model to study gastrulation in 3D. We also hypothesize that YAP- repression of the PEE of the NODAL gene is essential to balance pluripotency and ectoderm differentiation. To address this idea, we will investigate enhancer function and chromatin structure by combining cutting-edge CRISPR and genome-sequencing approaches (Aim2). Findings from Aim 1 and 2 will inform about human- specific regulatory roles of YAP that occur during axial organization. Finally, we will utilize a conditional YAP KO mouse to investigate the role of YAP in the differentiating epiblast, in vivo (Aim3). Our findings will inform on new mechanisms that regulate the activity of NODAL signaling and help to decipher the role of YAP during gastrulation.