Analysis of RhoGTPase function in neural crest EMT in vivo

描述(申请人提供)：神经脊细胞(NCC)是脊椎动物特有的细胞，从发育中的神经管迁移并分化为多种组织，包括颅面结构和周围神经系统的神经元和胶质细胞。神经干细胞的一个重要特征是上皮细胞向间充质细胞的转变(EMT)，从神经上皮细胞剥离并开始迁移。EMT是一个戏剧性的过程，在这个过程中，细胞失去了上皮结构，并经历了细胞形态和运动性的重大变化，从而允许细胞迁移和形成新组织。EMT在许多发育过程中起着关键作用，在病理事件中也起重要作用，尤其是在肿瘤的侵袭和转移过程中。然而，体内EMT过程中调节细胞变化的机制仍然知之甚少，这主要是因为缺乏能够在自然环境中研究EMT细胞的模型系统。我们正在开发斑马鱼NCC EMT作为研究体内EMT机制的模型。我们已经对完整胚胎的NCC行为进行了高分辨率的实时成像。我们现在建议开发工具来成像分子活性和分析RhoGTP酶在体内EMT过程中的功能。我们的具体目标是：1)在活体内对NCC EMT过程中活跃的Rho进行成像。我们将使用生物传感器对在完整斑马鱼后脑中进行EMT的NCC中活性Rho的时空动态进行成像。2)确定特定的下游Rho效应器通路，以控制体内细胞运动性和F-肌动蛋白的特定变化。我们将抑制ROCK和DIA信号，以测试这两个效应器对驱动EMT的细胞黏附和突起的变化进行差异调节的假设。3)我们将筛选Rho上游调控因子、GEF和GAP，以确定哪些在NCC中具有特异性的亚细胞定位，以及哪些功能控制Rho在细胞内的精确时空激活。我们能够成像RhoGTP酶的活性，操纵它们的功能，并检查对动态细胞行为和F-肌动蛋白的影响，这将阐明RhoGTP酶在体内EMT中的精确功能。我们研究特定下游效应器和上游GEF和GAP的实验将使我们能够开始定义在细胞不同部分以不同方式控制Rho及其功能的分子途径。了解EMT的调节机制具有很高的医学意义，因为EMT是多种病理过程的基础。因此，我们的实验有可能为旨在治疗涉及细胞异常迁移的疾病的疗法提供信息。 公共卫生相关性：EMT在胚胎发育过程中对组织重塑极其重要，也是几个病理过程的中心事件，如纤维化、慢性炎症和癌症进展和转移。阐明控制EMT的分子机制对于理解这些发育和病理事件至关重要。我们的实验有可能为旨在治疗涉及细胞异常迁移的疾病的疗法提供信息。

DESCRIPTION (provided by applicant): Neural crest cells (NCCs) are vertebrate-specific cells that migrate from the developing neural tube and differentiate into multiple tissues including craniofacial structures and neurons and glia of the peripheral nervous system. A defining feature of NCCs is the epithelial to mesenchymal transition (EMT) they undergo to delaminate from the neuroepithelium and begin migration. EMT is a dramatic process in which cells lose epithelial structure and undergo major changes in cell morphology and motility that allow cell migration and formation of new tissues. EMTs are critical for numerous developmental processes, and are also co-opted during pathological events, most notably carcinoma invasion and metastasis. However, the mechanisms regulating cellular changes during EMT in vivo remain poorly understood, largely because of a paucity of model systems in which cells undergoing EMT can be studied in their natural environment. We are developing zebrafish NCC EMT as a model to investigate EMT mechanisms in vivo. We have carried out high resolution live imaging of NCC behavior in intact embryos. We now propose to develop the tools to image the molecular activity and analyze the function of RhoGTPase during EMT in vivo. Our specific aims are: 1) To image active Rho during NCC EMT in vivo. We will use a biosensor to image the spatiotemporal dynamics of active Rho in NCCs undergoing EMT in the intact zebrafish hindbrain. 2) To define specific downstream Rho effector pathways that control particular changes in cell motility and F-actin in vivo. We will inhibit ROCK and Dia signaling to test the hypothesis that these effectors differentially regulate changes in cell adhesions and protrusions that drive EMT. 3) We will screen upstream Rho regulators, GEFs and GAPs, to determine which have specific subcellular localization in NCCs, and which function to control precise spatiotemporal activation of Rho within a cell. Our ability to image activity of RhoGTPases, manipulate their function and examine effects on dynamic cell behaviors and F-actin will elucidate precise functions of RhoGTPases during EMT in vivo. Our experiments to investigate the specific downstream effectors and upstream GEFs and GAPs will allow us to begin defining molecular pathways that differentially control Rho and its functions in different parts of the cell. Understanding EMT regulatory mechanisms has high medical relevance as EMTs underlie multiple pathological processes. Our experiments thus have potential to inform therapies designed to treat diseases involving abnormal cell migration. PUBLIC HEALTH RELEVANCE: EMTs are extremely important for tissue remodeling during embryonic development, and are also central events in several pathological processes, such as fibrosis, chronic inflammation and cancer progression and metastasis. Elucidation of the molecular mechanisms controlling EMT is critical for understanding these developmental and pathological events. Our experiments have potential to inform therapies designed to treat diseases involving abnormal cell migration.