Paradigms in Branching Morphogenesis

分支形态发生的范式

• 批准号: RGPIN-2016-05084
• 负责人: Gupta, Indra
• 金额: $ 2.4万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=709583
• 关键词: Paradigms; Branching; Morphogenesis

Branching morphogenesis is a fundamental process in organ development that establishes a branched tubular epithelial network. In the lungs, the salivary glands, and the kidneys, epithelial cells adhere to one another, proliferate and undergo cell shape changes to create buds, tubules and branch points. During formation of the salivary glands, an epithelial bud emerges from the oral epithelium and then elongates and bifurcates to generate a multi-lobed gland that produces and transports saliva into the oral cavity. During lung development, a network of branching bronchial tubules arises to conduct air flow. Branching morphogenesis is critical during kidney development to establish the network of the nephrons, the filtering units of the kidney. While many signaling molecules and transcription factors are known to be important for branching morphogenesis in all three organs, we know little about the mechanisms by which changes in cell shape result in bud formation, tubule elongation, and the creation of branch points. To explore cell shape changes and their role in branching morphogenesis, we have begun to evaluate the effect of epithelial cell junctional complexes on cell morphology. We have established that many members of the tight junction family of proteins, the claudins, are expressed during renal and pulmonary branching morphogenesis. In a cell culture model, we demonstrated that overexpression of one claudin, claudin-3, enhanced tubule formation. The enterotoxin of the bacterium Clostridium perfringens (CPE) is able to bind to the second extracellular loop of a subset of claudin members (claudin-3,-4,-6,-7,-8, and -14) and this permits bacteria to invade epithelial barriers. A modified version of the enterotoxin containing only the C-terminal tail (C-CPE) also binds to these claudins: the C-CPE does not cause cell toxicity, but it removes claudins from tight junctions. We cultured embryonic mouse kidneys in the presence of C-CPE and observed a severe defect in branching morphogenesis. We therefore hypothesize that claudin-based junctional complexes are required for renal, pulmonary, and salivary gland branching morphogenesis: they change epithelial cells from a columnar to a wedge shape by apical constriction and this creates buds, tubules, and branch points. To address this hypothesis, we will: Aim 1. Determine the function of claudin-based junctions during the cellular events that mediate branching morphogenesis in cultured mouse embryonic lung and submandibular explants. Aim 2. Determine the spatial and temporal function of claudin-based junctions during the cellular events that mediate branching morphogenesis in vivo during mouse kidney and lung branching morphogenesis. This proposal will examine how claudin-based junctions influence cell shape changes during branching morphogenesis and will generate important insights about organ development.

分支形态发生是器官发育的一个基本过程，它建立了一个分支的管状上皮网络。在肺、唾液腺和肾中，上皮细胞彼此粘附、增殖并经历细胞形状变化以产生芽、小管和分支点。 在唾液腺的形成过程中，上皮芽从口腔上皮中出现，然后伸长并分叉以产生多叶腺，该多叶腺产生唾液并将唾液输送到口腔中。 在肺的发育过程中，支气管小管的分支网络产生以引导空气流动。 在肾脏发育过程中，分支形态发生对于建立肾单位（肾脏的过滤单位）的网络至关重要。虽然已知许多信号分子和转录因子对所有三个器官的分支形态发生都很重要，但我们对细胞形状变化导致芽形成、小管伸长和分支点产生的机制知之甚少。 为了探索细胞形状的变化及其在分支形态发生中的作用，我们已经开始评估上皮细胞连接复合物对细胞形态的影响。我们已经建立了紧密连接蛋白家族的许多成员，claudins，在肾和肺分支形态发生过程中表达。 在细胞培养模型中，我们证明了一种claudin，claudin-3的过表达增强了小管形成。细菌产气荚膜梭菌（CPE）的肠毒素能够结合到封闭蛋白成员（封闭蛋白-3、-4、-6、-7、-8和-14）的子集的第二细胞外环，并且这允许细菌侵入上皮屏障。一种只含有C-末端尾（C-CPE）的肠毒素的修饰版本也与这些claudin结合：C-CPE不会引起细胞毒性，但它会从紧密连接中去除claudin。我们培养的胚胎小鼠肾脏中存在的C-CPE，观察到严重的缺陷，在分支形态发生。因此，我们假设，基于密蛋白的连接复合物是肾，肺和唾液腺分支形态发生所必需的：它们通过顶端收缩将上皮细胞从柱状变为楔形，从而产生芽，小管和分支点。为了解决这个假设，我们将： 目标1.在培养的小鼠胚胎肺和下颌下外植体中，确定在介导分支形态发生的细胞事件中基于密蛋白的连接的功能。 目标二。确定在小鼠肾脏和肺分支形态发生期间介导体内分支形态发生的细胞事件期间基于密蛋白的连接的空间和时间功能。 这项提案将研究如何claudin为基础的连接影响细胞形状的变化，在分支形态发生，并将产生重要的见解器官发育。