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

摘要 理解 诊断， 对 信号传导以其促进细胞增殖的基本活性而闻名， 发展提示雅普作为细胞命运决定因子在 早期胚胎发生因此，我们正在进行的使用人类胚胎干细胞（hESC）2D- 原肠胚形成模型揭示了雅普对于三个胚层的正确特化是必不可少的。的 雅普KO衍生的人2D-类胃窦显示出扩张的中胚层和内胚层（ME）以及减少的中胚层和内胚层（ME）。 外胚层层，与WT相比。NODAL信号稳态对正常原肠胚形成至关重要;活性 ME分化需要结节线索。然而，需要有效的NODAL抑制， 获得外胚层潜能。我们的数据表明雅普KO细胞的原肠胚形成表型是 与过度活跃的NODAL信号有关。NODAL基因编码的配体，激活 细胞内Smad2.3信号转导。有趣的是，我们的转录组和表观基因组分析表明，雅普是 需要抑制NODAL基因的特征性近端外胚层增强子（PEE）， 多能性向外胚层命运的退出。我们的数据进一步表明，雅普限制染色质 人类胚胎的发展将导致发现治疗工具， 治疗和预防出生缺陷。这项提案的首要目标是增进我们的知识 调节人类原肠胚形成过程的信号和表观遗传机制。河马- 效应子YAP 1（雅普） 越来越多的证据表明， The of的 NODAL基因座的可及性，对基因抑制至关重要。这些 一般 规范”。 调查结果构成了我们的前提， 假说：“雅普表观遗传调控NODAL信号传导对于正确的胚层发育至关重要 我们将在以下目标中阐述这一想法。目标1 的 ，我们将分析雅普在 人类3D类胃体。我们假设人类上胚层细胞进行三维重组的能力， 原肠胚形成和前后轴的组织由雅普调节。为了验证这个想法，我们将应用一个 最先进的合成人类胚胎学模型，用于研究3D原肠胚形成。我们还假设雅普- 抑制NODAL基因的PEE对于平衡多能性和外胚层分化是必需的。 为了解决这一问题，我们将结合最新的 CRISPR和基因组测序方法（Aim 2）。目标1和2的发现将为人类提供信息- 雅普在轴向组织中的特殊调节作用。最后，我们将使用条件雅普 KO小鼠体内研究雅普在分化的上胚层中的作用（Aim 3）。我们的发现将告诉 调节NODAL信号传导活性的新机制，并有助于破译雅普在 原肠胚形成

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.