In vivo analysis of gastrointestinal epithelium morphogenesis

 描述(申请人提供)：这项研究的目标是研究驱动胃肠道上皮形成的细胞生物学机制。尽管在胃肠道发育和疾病的分子遗传学方面已经取得了很大的进展，但对影响器官形态和功能的动态细胞行为却知之甚少。在胚胎发育过程中，内皮细胞最初经历了一个高度动态的单细胞迁移阶段，但后来会聚在一起，粘在一起形成一个连贯的内胚层，这最终形成了肠管的上皮衬里。在这项研究中，我将使用活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，内皮细胞通过上调细胞连接分子来促进细胞之间的粘连，同时也在空间上调节肌动蛋白聚合和膜突出来弥合新形成的薄层中的缝隙，从而启动上皮的形成。在这项研究的第一个目标中，我将确定细胞识别细胞的机制 自由的区域，并将它们的膜延伸到这些缝隙中。同时，我将通过监测荧光标记的细胞连接成分α-连环蛋白、E-钙粘蛋白和ZO-1的动态变化来定义细胞-细胞黏附的进展。最后，我将使用RNA-Seq转录组谱来识别内皮片形成的新因素，并将通过CRISPR/Cas9技术产生突变来测试这些新候选基因的功能。在AIM2中，我将探索细胞骨架基因Septin9a(Sept9a)在内皮片形成中的作用。我之前已经确定Sept9a基因是在片状形成开始时在内胚层特异上调的基因。已知的是，间隔蛋白调节细胞形状和皮质张力的变化，这可能为形成连贯的上皮提供必要的结构完整性。在这个目标中，我将描述Sept9a功能缺失突变对内皮细胞运动性、细胞-细胞相互作用以及细胞-细胞黏附的启动和维持的影响。胃肠道上皮的正常形成是组织执行任务所必需的。 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会导致炎症性肠病等疾病。然而，我们目前对上皮形成的大部分了解来自于体外细胞培养系统，这些系统可能不能像发育中的胚胎那样准确地代表上皮分化。因此，早期斑马鱼内胚层可能是一种急需的新生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.