Control of Cell Movements During Gastrulation in Xenopus

原肠形成过程中形态发生运动的控制对生物体的生存至关重要，因此，确定在原肠形成过程中调节细胞活动的分子途径是至关重要的。非洲爪哇和斑马鱼最近的工作提供了证据，证明原肠形成过程中极化的细胞运动和形状变化受Wnt/PCP途径的调节。这些研究已经确定了信号蛋白杂乱(Dsh)的关键作用，并表明Dsh通过调节肌动蛋白细胞骨架来控制细胞极性。米勒博士实验室的初步研究支持了这一假设，即信号蛋白Eps8与Dsh相互作用，在原肠胚形成过程中，细胞外信号与肌动蛋白细胞骨架之间存在潜在的联系。本研究项目的目的是以爪蛙胚胎为模型系统，确定Eps8在原肠发育过程中控制形态发生运动的机制。这项提案将寻求回答以下问题：(1)在开发过程中对EPS8的要求是什么？将使用基因敲除策略和显性-负性形式的Eps8的表达来研究Eps8在非洲爪哇发育中的功能。所产生的表型将使用形态、组织学和基因表达分析进行检测。(2)Eps8在原肠胚发育过程中调控哪些形态发生事件？获得和丧失功能的方法将结合(I)细胞极性、形状和突起活动、(Ii)肌动蛋白组织和(Iii)细胞-细胞黏附的详细分析来研究Eps8调节形态发生的机制。他们还将描述原肠胚过程中Eps8的亚细胞分布。(3)原肠发育过程中有哪些信号调节Eps8的功能？他们将利用生化和细胞生物学分析来检查Eps8的功能和/或它与Dsh的相互作用是否受PDGF或Wnt信号的调节。拟议研究的更广泛影响：通过利用他们在使用经典胚胎学方法和现代成像工具分析细胞运动和体内蛋白质定位方面的专业知识，Miller实验室将获得对脊椎动物原肠形成过程中控制形态发生运动的机制的重要见解。由于会聚伸展运动是整个动物界使用的基本过程，他们在非洲爪哇的研究将有助于更好地理解细胞运动是如何协调以控制其他系统中的组织图案的。此外，一名研究生和一名本科生将在这个项目上进行广泛的工作。作为该项目的一部分，他们将接受显微镜、生物化学、实验胚胎学和细胞生物学方面的广泛培训。

0315767MillerThe control of morphogenetic movements during gastrulation is critical for the viability of the organism and, therefore, determining the molecular pathways that regulate cell activities during gastrulation is of fundamental importance. Recent work from Xenopus and zebrafish provide evidence that polarized cell motility and shape changes during gastrulation are regulated by the Wnt/PCP pathway. These studies have defined a critical role for the signaling protein Dishevelled (Dsh) and suggest that Dsh controls cell polarity through regulation of the actin cytoskeleton. Preliminary studies from Dr. Miller's laboratory provide support for the hypothesis that the signaling protein Eps8 interacts with Dsh providing a potential link between extracellular signals and the actin cytoskeleton during gastrulation. The objective of the current research project is to determine the mechanisms by which Eps8 controls morphogenetic movements during gastrulation, using embryos of the clawed frog, Xenopus laevis, as a model system. This proposal will seek to answer the following questions: (1) What is the requirement for Eps8 during development? The function of Eps8 in Xenopus development will be investigated using gene-knockdown strategies and expression of dominant-negative forms of Eps8. The resulting phenotypes will be examined using morphological, histological, and gene expression analyses. (2) What morphogenetic events does Eps8 regulate during gastrulation? Gain- and loss-of-function approaches will be combined with a detailed analysis of (i) cell polarity, shape, and protrusive activity, (ii) actin organization, and (iii) cell-cell adhesion to investigate the mechanism by which Eps8 regulates morphogenesis. They will also characterize the subcellular distribution of Eps8 during gastrulation. (3) What signals regulate Eps8 function during gastrulation? They will utilize biochemical and cell biological assays to examine whether Eps8 function and/or its interaction with Dsh is regulated by PDGF or Wnt signals. Broader impact of the proposed research: By exploiting their expertise in using classical embryological approaches and modern imaging tools for the analysis of cell movements and protein localization in vivo, the Miller laboratory will gain important insights into the mechanisms that control morphogenetic movements during gastrulation in vertebrates. Since convergent extension movements are a fundamental process employed throughout the animal kingdom, their studies in Xenopus will lead to a greater understanding of the how cell movements are coordinated to control tissue patterning in other systems. In addition, a graduate student, and an undergraduate student will work extensively on this project. They will receive extensive training in microscopy, biochemistry, experimental embryology, and cell biology as part of this project.