Determining the role of Pcdh10a in zebrafish migratory neural crest cells

 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，包括经历上皮-间充质转化（EMT）、在整个胚胎中广泛迁移并分化为特定衍生物的能力。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，称为神经衰弱，包括几种颅面综合征，包括DiGeorge和Treacher柯林斯综合征。我们已经确定了一个新的作用，斑马鱼（Danio rerio）细胞粘附蛋白，原钙粘蛋白10a（pcdh 10a），其中敲低pcdh 10a导致特定的缺陷，NCC迁移。我的假设是，Pcdh 10a在NCC的一个子集中发挥作用，通过调节F-肌动蛋白分布来调节细胞迁移。为了验证这一点，我将完成以下具体目标：1)确定需要原钙粘蛋白10a的NCC亚群中的细胞定位。我将使用免疫荧光和荧光原位杂交，以确定确切的人群的NCCs表达pcdh 10a和什么部分的细胞Pcdh 10a是本地化，以确定它可能是如何调节细胞迁移; 2）调查pcdh 10a的要求在迁移性NCCs在调节F-肌动蛋白介导的细胞运动和细胞粘附。我将通过功能获得和功能丧失的方法来确定F-肌动蛋白是如何被pcdh 10a调节的。对于这个实验，我将用鬼笔环肽对固定的样品进行成像，沿着使用融合到RFP或GFP的肌动蛋白结合蛋白LifeAct和UtrCH进行活细胞成像。这将使我能够确定在活的斑马鱼胚胎中NCC迁移期间F-肌动蛋白是如何动态调节的; 3）确定斑马鱼Pcdh 10a是否直接与Nap 1和WAVE相互作用以调节细胞运动和细胞粘附。在这里，我将确定Pcdh 10a是否与已知蛋白质的任何同源物结合，这些蛋白质在小鼠中与Pcdh 10相互作用，例如调节肌动蛋白的Nap-1/WAVE复合物的蛋白质。确定Pcdh 10a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。总之，这些数据将确定一个新的作用，原钙粘蛋白介导NC细胞迁移。

 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.