Inductive and morphogenetic processes shaping the zebrafish embryonic axes

摘要 本次最大化研究奖续展申请的总体目标是推进 脊椎动物胚胎发生中的诱导和形态发生过程是如何协调的 确保正常发展。母体和合子基因产物控制的早期诱导过程 建立胚性极性和胚层，同时融合和延伸(C&E)原肠运动 将胚胎组织沿前后轴线拉长，并向内侧缩小。非规范的 Wnt/平面细胞极性(Wnt/PCP)信号通路极化间充质的形态和行为 形成胚体的原肠胚细胞。我们先前提出WNT/PCP信号作为细胞 指南针相对于胚胎前后轴定位细胞，但指南针是如何调节的 是不理解的。在上一次资助期间，我们意外地发现有几条途径在 与Wnt/PCP平行，在C&E过程中使细胞极化。我们还发现在C&E中还有另一个保守的调节因子 平面极性，Dachsous非典型钙粘附素，这是许多过程所必需的，包括胚胎 卵裂和轴规范，通过促进微管动力学。我们投入了大量的精力来产生 新的遗传学工具探索Wnt/PCP指南针的机制并启动新的遗传筛查 原肠调节物质。 这些发现和工具推动了我们未来在三个研究主题上的研究。首先，我们将调查 许多膜受体，包括我们涉及的Wnt/PCP中的Gpr125黏附GPCr Compass，在C&E过程中相互作用使用内源gpr125基因座的精确突变和标签，蛋白质组 和遗传学实验，我们将测试Gpr125是否促进由以下组成的Wnt/PCP复合体的形成 选择Components。在第二个主题中，我们将扩展我们对Dachsous的研究，并考察它是否 通过促进微管运动来调节原肠运动。使用内源标记的腊肠 通过GFP，我们将进行蛋白质组学研究，以分离内源相互作用因子，并进行基因组编辑，以确定 它的关键区域。在一个平行的无偏见的基因方法中，我们将继续进行有希望的基因筛查 母体和母体合子突变损害胚胎发育和原肠形成。总之，我们的 Wnt/PCP和Dachsous平面细胞极性调节剂和无偏遗传筛选的机制研究， 将既推进Mira任务，又理解诱导和形态发生过程是如何 在脊椎动物原肠发育过程中协调的。因为这些通路组件的突变会导致 流产、出生缺陷和疾病，我们的研究将提供对它们的理解和诊断的洞察力， 并促进治疗方法的发展。

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.