Analysis of RhoGTPase function in neural crest EMT in vivo

描述（由申请人提供）：神经嵴细胞（NCC）是脊椎动物特异性细胞，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神经胶质。NCC的一个定义特征是上皮向间充质转化（EMT），它们经历从神经上皮分层并开始迁移。EMT是一个戏剧性的过程，其中细胞失去上皮结构并经历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对于许多发育过程是至关重要的，并且在病理事件期间也被增选，最显著的是癌侵袭和转移。然而，在体内EMT过程中调节细胞变化的机制仍然知之甚少，这主要是因为缺乏模型系统，其中经历EMT的细胞可以在其自然环境中进行研究。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RhoGTdR在体内EMT过程中的功能。我们的具体目标是：1）在体内NCC EMT期间成像活性Rho。我们将使用一个生物传感器图像的时空动态活跃Rho在NCC进行EMT在完整的斑马鱼后脑。2)确定特定的下游Rho效应通路，控制体内细胞运动和F-肌动蛋白的特定变化。我们将抑制ROCK和Dia信号传导，以检验这些效应物差异调节驱动EMT的细胞粘附和突起变化的假设。3)我们将筛选上游Rho调节因子，GEF和GAP，以确定哪些在NCC中具有特定的亚细胞定位，哪些功能控制细胞内Rho的精确时空激活。我们的能力，图像活性的RhoGTPases，操纵他们的功能和检查动态细胞行为和F-肌动蛋白的影响将阐明在EMT在体内RhoGTPases的精确功能。我们的实验，以调查特定的下游效应和上游GEF和GAP将使我们开始定义的分子途径，差异控制Rho和它的功能在不同的部分细胞。了解EMT调节机制具有高度的医学相关性，因为EMT是多种病理过程的基础。因此，我们的实验有可能为治疗涉及异常细胞迁移的疾病提供信息。 公共卫生相关性：EMT对于胚胎发育期间的组织重塑极其重要，并且也是若干病理过程（例如纤维化、慢性炎症和癌症进展和转移）中的中心事件。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治疗涉及异常细胞迁移的疾病提供信息。

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.