Determining the role of Pcdh10a in zebrafish migratory neural crest cells

确定 Pcdh10a 在斑马鱼迁移神经嵴细胞中的作用

• 批准号: 9269183
• 负责人: Jason Williams
• 金额: $ 2.6万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269183
• 关键词: Actin-Binding Protein; Actins; Affect; Alpha Cell; Binding; Biological Assay; Cadherins; Cartilage; Cell Adhesion; Cell Adhesion Molecules; Cell membrane; Cell-Cell Adhesion; Cells; Characteristics; Complex; Congenital Abnormality; Data; Defect; Development; Disease; Dorsal; Ectoderm; Embryo; Epitopes; F-Actin; Family; Fluorescent in Situ Hybridization; Genes; Homologous Gene; Human; Image; Immunofluorescence Immunologic; Immunoprecipitation; Injectable; Lateral; Live Birth; Location; Mammals; Mandibulofacial Dysostosis; Mediating; Mus; Neural Crest; Neural Crest Cell; Neural tube; Neuroglia; Neurons; Pathway interactions; Peripheral Nervous System; Phenotype; Pigments; Play; Population; Proteins; Publishing; Regulation; Role; Route; Sampling; Signal Transduction; Somites; Surface Ectoderm; Syndrome; System; Testing; Tissues; Transgenic Organisms; Travel; United States; Western Blotting; Zebrafish; axon guidance; cell motility; cellular imaging; cleft lip and palate; craniofacial; craniofacial development; epithelial to mesenchymal transition; experimental study; gain of function; knock-down; live cell imaging; loss of function; melanoblast; melanocyte; migration; notochord; novel; nucleosome assembly protein I; precursor cell; protein protein interaction; public health relevance; relating to nervous system; stem-like cell; vertebrate embryos

 DESCRIPTION (provided by applicant): Neural crest cells (NCCs) are a population of highly migratory stem-like cells that have unique characteristics including the ability to undergo an epithelial-to-mesenchymal transition (EMT), migrate extensively throughout the embryo and differentiate into specific derivatives. Defects in neural crest migration can be one of the underlying causes of several human congenital birth defects, termed neurocristopathies including several craniofacial syndromes including DiGeorge and Treacher Collins syndromes. We have identified a new role for the zebrafish (Danio rerio) cell adhesion protein, protocadherin10a (pcdh10a) in which knockdown of pcdh10a causes specific defects in NCC migration. My hypothesis is that Pcdh10a functions in a subset of NCCs to regulate cell migration by the regulation of F-actin distribution. To test this, I will complete the following specific aims:. 1) Determine the cellular localization in the subpopulations of NCCs that require protocadherin10a. I will use immunofluorescence and fluorescent in-situ hybridization to identify exactly which populations of NCCs are expressing pcdh10a and what part of the cell Pcdh10a is localized to in order to determine how it may be regulating cell migration; 2) Investigate pcdh10a requirement in migratory NCCs in regulating F-actin mediated cell motility and cell adhesion. I will determine how F-actin is regulated by pcdh10a by a gain-of-function and loss-of-function approaches. For this experiment I will image fixed samples with the phalloidin, along with live cell imaging using actin binding proteins LifeAct and UtrCH fused to RFP or GFP. This will allow me to determine how F-actin is regulated dynamically during NCC migration in live zebrafish embryos; and 3) Determine if zebrafish Pcdh10a directly interacts with Nap1 and WAVE to regulate cell motility and cell adhesion. Here, I will determine if Pcdh10a binds with any homologues of known proteins that interact with Pcdh10 in mouse, such as proteins of the Nap-1/WAVE complex, which regulates actin. Determining what proteins Pcdh10a is interacting with will allow me to make more specific hypotheses about its function and role in neural crest migration. Together these data will determine a novel role for protocadherins mediating NC cell migration.

 描述（由申请人提供）：神经嵴细胞（NCC）是一群高度迁移的干细胞样细胞，具有独特的特征，包括能够经历上皮间质转化（EMT）、在整个胚胎中广泛迁移并分化成特定的衍生物。神经嵴迁移缺陷可能是几种人类先天性出生缺陷的根本原因之一，称为神经嵴病，包括几种颅面综合征，包括迪乔治综合征和特雷彻柯林斯综合征。我们发现了斑马鱼 (Danio rerio) 细胞粘附蛋白原钙粘蛋白 10a (pcdh10a) 的新作用，其中 pcdh10a 的敲低会导致 NCC 迁移的特定缺陷。我的假设是 Pcdh10a 在 NCC 子集中发挥作用，通过调节 F-肌动蛋白分布来调节细胞迁移。为了测试这一点，我将完成以下具体目标： 1) 确定需要原钙粘蛋白10a 的 NCC 亚群的细胞定位。我将使用免疫荧光和荧光原位杂交来准确识别哪些 NCC 群体表达 pcdh10a 以及 Pcdh10a 位于细胞的哪个部分，以确定它如何调节细胞迁移； 2) 研究迁移性 NCC 中 pcdh10a 在调节 F-肌动蛋白介导的细胞运动和细胞粘附方面的需求。我将通过功能获得和功能丧失的方法确定 pcdh10a 如何调节 F-肌动蛋白。在本实验中，我将使用鬼笔环肽对固定样品进行成像，并使用与 RFP 或 GFP 融合的肌动蛋白结合蛋白 LifeAct 和 UtrCH 进行活细胞成像。这将使我能够确定 F-肌动蛋白在活体斑马鱼胚胎 NCC 迁移过程中如何动态调节； 3) 确定斑马鱼 Pcdh10a 是否直接与 Nap1 和 WAVE 相互作用来调节细胞运动和细胞粘附。在这里，我将确定 Pcdh10a 是否与小鼠中与 Pcdh10 相互作用的已知蛋白质的任何同源物结合，例如调节肌动蛋白的 Nap-1/WAVE 复合物的蛋白质。确定 Pcdh10a 与哪些蛋白质相互作用将使我能够对其在神经嵴迁移中的功能和作用做出更具体的假设。这些数据将共同确定原钙粘蛋白介导 NC 细胞迁移的新作用。