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

摘要 理解 诊断， 在……上面 以其促进细胞增殖的基本活动而闻名的信号转导 发展。提示YAP作为细胞命运决定因素在 早期胚胎发育。因此，我们正在进行的使用人类胚胎干细胞(HESCs)2D的研究- 原肠发育模型表明，YAP对于正确指定三种胚层是必不可少的。这个 YAPKO来源的人2D-胃肠显示中胚层和内胚层(ME)扩张和减少 外胚层，与WT相比。节间信号动态平衡对正常原肠发育至关重要；活跃的 ME的分化需要节点线索。然而，有效的节点抑制对于 获得外胚层潜力。我们的数据表明Yap KO细胞的原肠形成表型是 与过度活跃的节点信号有关。结节基因编码的配体激活了 胞内Smad2.3信号。有趣的是，我们的转录和表观基因组分析表明，YAP是 需要抑制结节基因的特征良好的近端上皮细胞增强子(PEE) 多能性的退出，走向外胚层的命运。我们的数据进一步表明，YAP限制了染色质 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。这项建议的首要目标是增进我们的知识 调节人类原肠形成过程的信号和表观遗传机制。河马- 效应器YAP1(YAP)是 然而，越来越多的证据表明 The of the of the of the 对基因抑制至关重要的节位基因的可及性。这些 一般信息 规格“。 这些发现形成了我们的前提 假说：YAP的表观遗传调控节点信号对正确的生殖层至关重要 我们将在以下目标中解决这一想法。在Aim1 的 ，我们将分析YAP在 人体3D-胃肠。我们假设人类上皮细胞经历三维 原肠形成，在前后轴上的组织由YAP调节。为了测试这个想法，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原肠形成。我们还假设YAP- 抑制节状基因的PIE对于平衡多能性和外胚层分化是必不可少的。 为了解决这个问题，我们将结合前沿技术研究增强子功能和染色质结构 CRISPR和基因组测序方法(AIM2)。来自Aim 1和Aim 2的发现将提供关于人类的信息- 在轴向组织过程中发生的YAP的特定调节作用。最后，我们将使用条件YAP KO小鼠体内研究YAP在表皮母细胞分化中的作用(Aim3)。我们的发现将会告诉我们 关于调节结节信号活动和帮助破译YAP在 原肠形成。

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.