Analysis of RhoGTPase function in neural crest EMT in vivo

体内RhoGTPase在神经嵴EMT中的功能分析

• 批准号: 8200471
• 负责人: MARY C HALLORAN
• 金额: $ 18.17万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8200471
• 关键词: Abnormal Cell; Adhesions; Animals; Behavior; Binding; Biological Models; Biosensor; Carcinoma; Cell Adhesion; Cell physiology; Cell-Cell Adhesion; Cells; Cellular Morphology; Chimeric Proteins; Chronic; Complex; Conflict (Psychology); Cytoskeleton; Development; Developmental Process; Disease; Embryo; Embryonic Development; Environment; Epithelial; Event; F-Actin; Family; Fibrosis; GTP Binding; GTPase-Activating Proteins; Guanine Nucleotide Exchange Factors; Image; In Vitro; Individual; Inflammation; Life; Malignant Neoplasms; Mediating; Medical; Modeling; Molecular; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; Neural Crest; Neural Crest Cell; Neural tube; Neuroglia; Neurons; Pathologic Processes; Pathway interactions; Peripheral; Peripheral Nervous System; Preparation; Process; Resolution; Rho-associated kinase; Role; Signal Pathway; Signal Transduction; Structure; Testing; Tissues; Zebrafish; cell behavior; cell motility; craniofacial; epithelial to mesenchymal transition; hindbrain; in vivo; in vivo Model; migration; molecular imaging; neuroepithelium; research study; rho; spatiotemporal; therapy design; tool; tumor progression

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.

描述（由申请人提供）：神经rest细胞（NCC）是脊椎动物特异性细胞，它们从发育中的神经管迁移，并分化为多种组织，包括颅面结构，神经元和周围神经系统的神经胶质。 NCCS的一个定义特征是它们向间质转变（EMT）进行分层并开始迁移。 EMT是一个戏剧性的过程，其中细胞会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对于众多发展过程至关重要，并且在病理事件中也被选中，最著名的是癌侵袭和转移。然而，调节体内EMT过程中细胞变化的机制仍然很少了解，这主要是因为模型系统很少，在这种模型系统中，可以在其自然环境中研究经历EMT的细胞。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中的NCC行为进行了高分辨率的实时成像。现在，我们建议开发图像分子活性并分析体内EMT期间Rhogtpase的功能的工具。我们的具体目的是：1）在体内NCC EMT期间为主动Rho映像。我们将使用一个生物传感器来成像在完整的斑马鱼后脑中EMT中活性RHO的时空动力学。 2）定义特定的下游RHO效应器途径，该途径控制体内细胞运动和F-肌动蛋白的特定变化。我们将抑制岩石和DIA信号传导，以测试这些效应子差异地调节驱动EMT的细胞粘附和突起的变化的假设。 3）我们将筛选上游的RHO调节剂，GEF和间隙，以确定哪些在NCC中具有特定的亚细胞定位，以及哪些功能可以控制细胞内RHO的精确时空激活。我们对Rhogtpases的活性，操纵其功能并检查对动态细胞行为的影响和F-肌动蛋白的影响将阐明EMT在体内EMT期间的精确功能。我们的实验研究了特定的下游效应子，上游GEF和间隙将使我们能够开始定义分子途径，该途径差异地控制RHO及其在细胞的不同部分中的功能。由于EMT是多个病理过程的基础，因此了解EMT监管机制具有很高的医学相关性。因此，我们的实验有可能为治疗涉及异常细胞迁移的疾病的疗法提供信息。 公共卫生相关性：EMT对于胚胎发育过程中的组织重塑非常重要，并且在几种病理过程中也是核心事件，例如纤维化，慢性炎症，癌症的进展和转移。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异常细胞迁移的疾病的疗法提供信息。