Determining the role of Pcdh10a in zebrafish migratory neural crest cells

 描述(申请人提供)：神经脊细胞(NCC)是一群高度迁移的类干细胞，具有独特的特征，包括经历上皮到间充质转化(EMT)的能力，在胚胎中广泛迁移，并分化为特定的衍生体。神经脊迁移缺陷可能是几种人类先天性出生缺陷的潜在原因之一，这些先天缺陷被称为神经筛查疾病，包括几种颅面综合征，包括DiGeorge和Treacher Collins综合征。我们已经确定了斑马鱼(Danio Rerio)细胞黏附蛋白Protocadherin10a(Pcdh10a)的一个新角色，其中pcdh10a的敲除会导致NCC迁移中的特定缺陷。我的假设是，Pcdh10a在NCC的一个子集中发挥作用，通过调节F-肌动蛋白的分布来调节细胞迁移。为了检验这一点，我将完成以下具体目标：1)确定需要原钙粘附素10a的神经干细胞亚群中的细胞定位。我将使用免疫荧光和荧光原位杂交来准确鉴定哪些NCC群体表达pcdh10a，以及Pcdh10a定位于细胞的哪一部分，以确定它可能如何调控细胞迁移；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黏附中的需求。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pcdh10a调控的。在这项实验中，我将用鬼臼蛋白对固定样本进行成像，同时使用融合到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F-肌动蛋白在活斑马鱼胚胎NCC迁移过程中如何动态调节；以及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黏附。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复合体的蛋白质。确定Pcdh10a与哪些蛋白质相互作用，将使我能够对其在神经脊迁移中的功能和作用做出更具体的假设。这些数据将共同决定原钙粘附素在NC细胞迁移中的新作用。

 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.