In vivo analysis of gastrointestinal epithelium morphogenesis

 描述（由申请方提供）：本研究的目的是研究驱动胃肠道上皮形成的细胞生物学机制。虽然在胃肠道发育和疾病的分子遗传学方面已经取得了很大进展，但对器官形状和功能的动态细胞行为的理解还不太清楚。在胚胎发育过程中，内胚层细胞最初经历高度动态的单细胞迁移阶段，但随后聚集并粘附在一起形成连贯的内胚层片，最终形成肠管的上皮衬里。在这项研究中，我将使用活体斑马鱼胚胎的高分辨率荧光成像来研究从单细胞迁移到上皮形成的转变。初步实验表明，内胚层细胞通过上调细胞连接分子以促进细胞之间的粘附，同时还在空间上调节肌动蛋白聚合和膜突起以闭合新形成的片材中的间隙来启动上皮形成。在本研究的第一个目标中，我将确定细胞识别细胞的机制， 自由区域并将它们的膜延伸穿过这些间隙。同时，我将通过监测荧光标记的细胞连接组分α-连环蛋白，E-钙粘蛋白和ZO- 1的动态来定义细胞间粘附的进展。最后，我将使用RNA-Seq转录组分析来识别内胚层片层形成中的新因素，并通过CRISPR/Cas9技术产生突变体来测试这些新候选基因的功能。在Aim 2中，我将探索细胞骨架基因septin 9a（sept 9a）在内胚层片形成中的作用。我之前已经确定sept 9a是一种在片层形成开始时内胚层特异性上调的基因。已知隔膜调节细胞形状和皮质张力的变化，这可以提供形成连贯上皮所必需的结构完整性。在这个目标中，我将描述sept 9a功能丧失突变对内胚层细胞运动性，细胞间相互作用，以及细胞间粘附的启动和维持的影响。胃肠道上皮的正确形成是组织执行所需的 它在屏障保护、消化和营养吸收方面的功能以及上皮结构的缺陷可能导致诸如炎症性肠病的疾病。然而，我们目前对上皮形成的理解大多来自体外细胞培养系统，其可能不能准确地代表发育中胚胎中发生的上皮分化。因此，早期斑马鱼内胚层可能是一个急需的从头上皮形成的体内模型。该模型可用于未来的研究，探索上皮生物学的基本方面，以及胃肠道疾病和疾病的细胞生物学机制。

 DESCRIPTION (provided by applicant): The goal of this study is to investigate the cell biological mechanisms that drive the formation of the gastrointestinal epithelium. Although much progress has been made in understanding in the molecular genetics of gastrointestinal tract development and disease, the dynamic cell behaviors that contribute to organ shape and function are less well understood. During embryonic development, endodermal cells initially undergo a phase of highly dynamic single-cell migration but then later converge and adhere together into a coherent endodermal sheet, which ultimately gives rise to the epithelial lining of the gut tube. For this study, I will use high-resolution fluorescence imaging of live zebrafish embryos to investigate the transition from single- cell migration to epithelium formation. Preliminary experiments suggest that endodermal cells initiate epithelium formation by upregulating cell junction molecules to facilitate adhesion between cells while also spatially regulating actin polymerization and membrane protrusion to close gaps in the newly forming sheet. In the first aim of this study, I will determine the mechanisms by which cells identify cell free areas and extend their membrane across these gaps. Concurrently, I will define the progression of cell-cell adhesion by monitoring the dynamics of fluorescently labeled cell junction components alpha-catenin, E-cadherin, and ZO- 1. Finally, I will use RNA-Seq transcriptome profiling to identify new factors in endodermal sheet formation and will test the function of these new candidate genes by generating mutants with CRISPR/Cas9 technology. In Aim2, I will explore the role of the cytoskeletal gene septin9a (sept9a) in endodermal sheet formation. I had previously identified sept9a as gene that is upregulated specifically in the endoderm at the onset of sheet formation. Septins are known to regulate changes in cell shape and cortex tension, which may provide the necessary structural integrity to form a coherent epithelium. In this aim, I will characterize the effects of sept9a loss-of-function mutations on endodermal cell motility, cell-cell interactions, and the initiation and maintenance of cell-cell adhesion. Proper formation of the gastrointestinal epithelium is required for the tissue to perform its functions in barrier protection, digestion, and nutrient absorption, and defects in epithelial structure may lead to diseases such as inflammatory bowel disease. However, much of our current understanding of epithelium formation has come from in vitro cell culture systems that may not accurately represent epithelial differentiation as occurs in a developing embryo. Thus, the early zebrafish endoderm may be a much-needed in vivo model of de novo epithelium formation. This model could be used in future studies exploring fundamental aspects of epithelial biology as well as the cell biological mechanisms underlying gastrointestinal disorders and diseases.