Inductive and morphogenetic processes shaping the zebrafish embryonic axes

摘要 这个最大化研究者的研究奖续期申请的总体目标是推进 了解脊椎动物胚胎发生过程中的诱导和形态发生过程是如何协调的 确保正常发展。早期诱导过程受母体和合子基因产物控制 建立胚胎极性和胚层，而会聚和伸展（C&E）原肠胚形成运动 沿着前后轴拉长胚胎组织并使其内侧变窄。非经典 Wnt/Planar Cell Polarity（Wnt/PCP）信号通路介导间充质细胞的形态和行为发生极化 形成胚胎体的原肠胚细胞。我们先前提出Wnt/PCP信号传导作为细胞内的一个信号传导通路， 一种使细胞相对于前后胚胎轴定向的指南针，但是指南针是如何被调节的 不被理解。在上一个资助期间，我们惊讶地发现，有几种途径在 与Wnt/PCP平行，在C&E期间使细胞凋亡。我们还在C&E中涉及另一种保守的调节因子， 平面极性，Dachsous非典型钙粘蛋白，这是必不可少的许多过程，包括胚胎 裂解和轴规格，通过促进微管动力学。我们投入了大量的精力， 新的遗传工具来探测Wnt/PCP罗盘的机制，并启动了新的遗传筛选， 原肠胚形成调节因子 这些发现和工具激励我们在三个研究主题的未来研究。首先，我们将调查 我们认为Wnt/PCP中涉及的许多膜受体，包括Gpr 125粘附GPCR， 指南针，在C&E期间进行交互使用内源性gpr 125基因座中的精确突变和标签， 和遗传实验，我们将测试Gpr 125是否促进Wnt/PCP复合物的形成， 选择组件。在第二个主题中，我们将扩展我们的研究Dachsous，并调查它是否 通过促进微管动力学调节原肠胚形成运动。使用Dachsous内源标记 利用GFP，我们将进行蛋白质组学研究以分离内源性相互作用物，并进行基因组编辑以定义 关键区域。在一个平行的无偏见的遗传方法，我们将继续一个有前途的遗传筛选， 损害胚胎发生和原肠胚形成的母体和母体合子突变。总之，我们的 平面细胞极性的Wnt/PCP和Dachsous调节因子的机制研究和无偏遗传筛选， 将推进MIRA的使命，并了解诱导和形态发生过程是如何 在脊椎动物原肠胚形成期间协调。由于这些途径的组成部分发生突变， 流产，出生缺陷和疾病，我们的研究将提供深入了解他们的理解和诊断， 并促进治疗的发展。

ABSTRACT The overall goal of this Maximizing Investigator’s Research Award renewal application is to advance understanding of how inductive and morphogenetic processes during vertebrate embryogenesis are coordinated to ensure normal development. Early inductive processes controlled by maternal and zygotic gene products establish embryonic polarity and germ layers, while convergence and extension (C&E) gastrulation movements elongate embryonic tissues down the anteroposterior axis and narrow them mediolaterally. The noncanonical Wnt/Planar Cell Polarity (Wnt/PCP) signaling pathway polarizes morphologies and behaviors of mesenchymal gastrula cells that shape embryonic body. We previously proposed that Wnt/PCP signaling acts as a cellular compass that orients cells with respect to the anteroposterior embryonic axis, but how the compass is regulated is not understood. During the previous funding period, we surprisingly found that several pathways work in parallel to Wnt/PCP to polarize cells during C&E. We also implicated in C&E another conserved regulator of planar polarity, Dachsous atypical cadherin, which is essential for many processes, including embryonic cleavages and axis specification, by promoting microtubule dynamics. We invested significant effort in generating new genetic tools to probe the mechanisms of the Wnt/PCP compass and initiated a genetic screen for new gastrulation regulators. These findings and tools motivate our future studies in three research themes. In the first, we will investigate how numerous membrane receptors, including Gpr125 adhesion GPCR, which we implicated in the Wnt/PCP compass, interact during C&E. Using precise mutations and tags in the endogenous gpr125 locus, proteomic and genetic experiments, we will test whether Gpr125 promotes formation of Wnt/PCP complexes composed of select components. In the second theme, we will extend our studies of Dachsous and investigate whether it regulates gastrulation movements by promoting microtubule dynamics. Using Dachsous endogenously tagged with GFP, we will carry out proteomic studies to isolate endogenous interactors, and genome editing to define its critical regions. In a parallel unbiased genetic approach, we will continue a promising genetic screen for maternal and maternal-zygotic mutations that impair embryogenesis and gastrulation. Altogether, our mechanistic studies of Wnt/PCP and Dachsous regulators of planar cell polarity and unbiased genetic screens, will both advance the MIRA mission and understanding how inductive and morphogenetic processes are coordinated during vertebrate gastrulation. As mutations in the components of these pathways cause miscarriages, birth defects and diseases, our studies will provide insights into their understanding and diagnosis, and facilitate development of therapies.