Cell biological mechanisms of gastrointestinal tract formation

描述(申请人提供)：这项研究的目标是确定调节早期内皮细胞迁移的机制，并了解细胞运动如何有助于胃肠道的整体形成。尽管在识别内皮细胞的转录因子方面已经取得了很大的进展，但对随后发生的细胞生物学过程，包括细胞运动的开始，还知之甚少。规格后不久，内胚层细胞在一个称为原肠形成的过程中内化，形成体内的内层。以斑马鱼胚胎为模型系统，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，以及我开发的一种新型转基因品系，该品系可以荧光标记肌动蛋白细胞骨架。初步证据表明，Nodal是一种已知的诱导内皮细胞命运的信号蛋白，可以以定向随机的方式促进内皮细胞的迁移。我将描述Nodal信号对内皮细胞内肌动蛋白细胞骨架的影响，并将确定促进Nodal诱导运动的肌动蛋白调节蛋白。在第二个具体目标中，我将研究调节依赖接触的排斥力的机制。这是一个过程，在这个过程中，迁移的内皮细胞在短暂接触后相互排斥。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作用于这些受体下游的细胞质信号蛋白。在一起，方向随机的运动和接触排斥似乎驱动内胚层细胞在早期胚胎表面的扩散，之后这些细胞结合成一个单一的肠管。为了了解这种最初扩散的意义，我将使用获得和丧失功能的技术来干扰正常的内皮迁移行为，并评估其对胃肠道后续发育的影响。在这项研究的过程中，我将在脊椎动物肠道发育、全胚胎时间推移成像以及检查体内细胞迁移的定量方法方面获得进一步的专业知识。我的主要导师Didier Stainier博士和我的合作导师Orion Weiner博士将在这些领域提供额外的培训，以帮助我实现研究目标。随着我继续探索细胞迁移和器官发育之间的关系，这项研究将阐明胃肠道发育一个鲜为人知的方面，并将为未来R级应用奠定基础。 公共卫生相关性：细胞迁移对胚胎发育、免疫功能、组织修复、炎症和肿瘤转移等许多过程都很重要。尤其是在胃肠道系统中，胚胎发育早期内胚层细胞迁移的缺陷会导致胃肠道的各种畸形。这项研究将促进我们对内皮细胞迁移如何调节的了解，这将有助于更好地理解消化系统疾病，其中许多疾病是由先天性缺陷以及其他条件下的细胞迁移发挥突出作用。

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. PUBLIC HEALTH RELEVANCE: Cell migration is important for many processes such as embryonic development, immune function, tissue repair, inflammation, and tumor metastasis. In particular for the gastrointestinal system, defects in endodermal cell migration during early embryogenesis have been reported to cause various malformations of the gastrointestinal tract. This study will advance our knowledge of how endodermal cell migration is regulated, which will contribute to a better understanding of digestive diseases, many of which are precipitated by congenital defects, as well as other conditions in which cell migration plays a prominent role.