Cell biological mechanisms of gastrointestinal tract formation

描述（由申请方提供）：本研究的目的是确定调节早期内胚层细胞迁移的机制，并了解细胞运动如何促进胃肠道的整体形成。虽然在确定指定内胚层细胞的转录因子方面取得了很大进展，但对随后发生的细胞生物学过程（包括细胞运动的开始）知之甚少。在特化后不久，内胚层细胞在称为原肠胚形成的过程中内化以形成身体的内层。使用斑马鱼胚胎作为模型系统，我将研究内胚层细胞迁移的两个方面-随机方向性和接触依赖性排斥。在这些研究中，我将使用活胚胎的高分辨率荧光成像和我开发的一种新型转基因系，该转基因系用荧光标记肌动蛋白细胞骨架。初步证据表明，Nodal，一种已知诱导内胚层命运的信号蛋白，可以以定向随机的方式促进内胚层迁移。我将描述Nodal信号对内胚层细胞内肌动蛋白细胞骨架的影响，并鉴定出促进Nodal诱导运动的肌动蛋白调节蛋白。在第二个具体目标中，我将研究调节接触依赖性排斥的机制。这是一个迁移的内胚层细胞在短暂接触后相互排斥的过程。使用候选和基于微阵列的方法，我将尝试识别启动接触排斥的细胞表面受体以及在这些受体下游起作用的细胞质信号蛋白。同时，定向随机运动和接触排斥似乎驱动内胚层细胞分散在早期胚胎的表面，之后这些细胞合并成一个单一的肠管。为了理解这种初始分散的意义，我将使用功能获得和丧失技术来干扰正常的内胚层迁移行为，并评估对胃肠道后续发育的影响。 在这项研究的过程中，我将获得进一步的专业知识，在脊椎动物肠道发育，全胚胎延时成像，并定量方法检查体内细胞迁移。我的主要导师Didier Escherier博士和我的共同导师Orion Weiner博士将在这些领域提供额外的培训，以帮助我实现我的研究目标。这项研究将揭示胃肠道发育的一个不太受重视的方面，并将成为未来R级应用的基础，因为我将继续探索细胞迁移和器官发育之间的关系。

DESCRIPTION (provided by applicant): The goal of this study is to identify the mechanisms regulating early endodermal cell migration and understand how cell movements contribute to the overall formation of the gastrointestinal tract. Although much progress has been made in identifying the transcription factors that specify endodermal cells, much less is known about the cell biological processes that occur subsequently, including the onset of cell motility. Shortly after specification, endodermal cells internalize to form the inner layer of the body during a process known as gastrulation. Using the zebrafish embryo as a model system, I will examine two aspects of endodermal cell migration - random directionality and contact-dependent repulsion. For these studies, I will use high-resolution fluorescence imaging of live embryos and a novel transgenic line that I developed that fluorescently labels the actin cytoskeleton. Preliminary evidence suggests that Nodal, a signaling protein already known to induce endodermal fate, can promote endodermal migration in a directionally random manner. I will characterize the effects of Nodal signaling on the actin cytoskeleton within endodermal cells, and I will identify the actin regulatory proteins that function to promote Nodal-induced motility. In the second specific aim, I will examine the mechanisms regulating contact-dependent repulsion. This is a process whereby migrating endodermal cells are repelled away from each other after making brief contact. Using both candidate and microarray- based approaches, I will attempt to identify the cell surface receptors that initiate contact repulsion as well as the cytoplasmic signaling proteins that act downstream of these receptors. Together, directionally random motility and contact repulsion appear to drive the dispersal of endodermal cells across the surface of the early embryo, after which these cells coalesce into a single gut tube. To understand the significance of this initial dispersal, I will use gain and loss of function techniques to interfere with normal endodermal migratory behavior and assess the effects on subsequent development of the gastrointestinal tract. In the course of this study, I will gain further expertise in vertebrate gut development, whole-embryo time-lapse imaging, and quantitative methods for examining in vivo cell migration. My primary mentor, Dr. Didier Stainier, and my co-mentor, Dr. Orion Weiner, will provide additional training in these areas to help me achieve my research goals. This study will shed light on a poorly appreciated aspect of gastrointestinal development and will form the basis for a future R-level application as I continue to explore the relationship between cell migration and organ development.