In vivo analysis of gastrointestinal epithelium morphogenesis

胃肠道上皮形态发生的体内分析

• 批准号: 9482489
• 负责人: Stephanie Woo
• 金额: $ 0.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9482489
• 关键词: vivo; analysis; gastrointestinal; epithelium; morphogenesis

 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.

 描述（由适用提供）：这项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。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步，但对器官形状和功能有助于器官的动态细胞行为知之甚少。在胚胎发育过程中，内胚层细胞最初是在高度动态的单细胞迁移阶段下的，然后随后汇聚并粘附成连贯的内胚片，最终导致了肠管的上皮衬里。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过渡。初步实验表明，内皮细胞通过上调细胞连接分子来启动上皮形成，以促进细胞之间的粘附性，同时还可以在空间控制肌动蛋白聚合和膜突出以在新形成的片片中的紧密间隙。在这项研究的第一个目的中，我将确定细胞识别细胞的机制 自由区域并将其膜扩展到这些间隙。同时，我将通过监视荧光标记的细胞连接组件α-蛋白，电子钙粘蛋白和ZO-1的动态来定义细胞细胞粘合剂的发展。在AIM2中，我将探讨细胞骨架基因septin9a（Sept9a）在内胚层形成中的作用。我以前曾将Sept9a确定为基因，该基因在床单开始时在内胚层中进行了专门更新。已知Septins可以调节细胞形状和皮层张力的变化，这可能提供必要的结构完整性以形成连贯的上皮。在此目标中，我将表征SEPT9A功能丧失突变对内胚层细胞运动，细胞 - 细胞相互作用以及细胞 - 细胞粘附的主动性和维持的影响。需要正确形成胃肠上皮的组织才能执行 它在屏障保护，消化和营养吸收以及上皮结构中的功能可能导致疾病，例如炎症性肠病。但是，我们目前对上皮形成的许多理解都是来自体外细胞培养系统，这些系统可能无法准确地代表上皮分化，就像发育中的胚胎中发生的那样。那就早期的斑马鱼内胚层可能是从头上皮形成的体内模型急需的。该模型可用于探索上皮生物学基本方面以及胃肠道疾病和疾病的细胞生物学机制的未来研究。