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成多种组织，包括颅面结构以及周围神经系统的神经元和神经胶质细胞。 NCC 的一个决定性特征是它们经历上皮间质转化 (EMT)，从神经上皮脱落并开始迁移。 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 对许多发育过程至关重要，并且在病理事件（尤其是癌症侵袭和转移）过程中也有参与。然而，体内 EMT 过程中调节细胞变化的机制仍然知之甚少，很大程度上是因为缺乏可以在自然环境中研究经历 EMT 的细胞的模型系统。我们正在开发斑马鱼 NCC EMT 作为模型来研究体内 EMT 机制。我们对完整胚胎中的 NCC 行为进行了高分辨率实时成像。我们现在建议开发工具来成像分子活性并分析 RhoGTPase 在体内 EMT 过程中的功能。我们的具体目标是：1）在体内 NCC EMT 期间对活性 Rho 进行成像。我们将使用生物传感器对完整斑马鱼后脑中经历 EMT 的 NCC 中活性 Rho 的时空动态进行成像。 2) 定义控制体内细胞运动和 F-肌动蛋白特定变化的特定下游 Rho 效应通路。我们将抑制 ROCK 和 Dia 信号传导，以检验这些效应器差异调节驱动 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，以及哪些功能可以控制细胞内Rho的精确时空激活。我们能够对 RhoGTPase 的活性进行成像、操纵其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 RhoGTPase 在体内 EMT 过程中的精确功能。我们研究特定下游效应器以及上游 GEF 和 GAP 的实验将使我们能够开始定义差异控制 Rho 及其在细胞不同部分的功能的分子途径。了解 EMT 调节机制具有很高的医学相关性，因为 EMT 是多种病理过程的基础。因此，我们的实验有可能为旨在治疗涉及异常细胞迁移的疾病的疗法提供信息。 公共健康相关性：EMT 对于胚胎发育过程中的组织重塑极其重要，也是纤维化、慢性炎症以及癌症进展和转移等多种病理过程的中心事件。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的疗法提供信息。