The Role of Clustered Protocadherins in Neurite Self-avoidance

 描述（由申请人提供）：神经回路组装的一个关键原理是神经突自我回避，这是一个过程，其中同一神经元的姐妹轴突或树突彼此排斥，但与来自其他神经元的轴突或树突自由相互作用，表明它们可以区分“自我”和“非自我”。在果蝇中，细胞粘附分子Dscam 1已被证明是神经突自我回避的关键介质。Dscam 1前mRNA的选择性剪接产生大量不同的蛋白质亚型，它们以严格的嗜同性方式相互作用，介导导致自我回避的接触依赖性排斥。然而，脊椎动物Dscam基因没有编码显著的多样性。相反，这种多样性和功能似乎是由成簇的原钙粘蛋白（Pcdhs）提供的。我们实验室和其他实验室的研究表明：1）通过选择启动子随机表达成簇的Pcdhs产生单细胞多样性; 2）细胞表面成簇的Pcdhs之间的组合嗜同性相互作用产生单神经元身份; 3）Pcdh- γ蛋白是小鼠星爆无长突细胞和浦肯野细胞中树突状自我回避所必需的。这些观察结果导致了这样的假设，即集群Pcdhs的功能类似于Dscam 1蛋白介导的脊椎动物的自我回避。在此应用中，我们建议进行全面的遗传分析，以确定是否集群Pcdhs在脊椎动物神经系统中的神经突自我回避中发挥核心作用，是否Pcdh多样性是这个过程所需的，以及不同的Pcdh集群是否在特定的细胞环境中相互补偿。为了实现这些目标，我们将产生Pcdh- α、- β和- γ基因簇的单个或全部敲除的单细胞，并研究单个神经元的神经突树枝化中的功能丧失效应。在目标1中，我们将建立和验证两种互补的单细胞敲除方法，双标记嵌合分析（MADM）和嵌合体中的单神经元分析（SNAC）。这两种方法在正常神经网络中稀疏地生成和标记突变细胞，从而允许对单个神经元中的细胞自主基因功能进行稳健的形态学分析和严格的评估。在目标2中，我们将使用Pcdh- γ基因簇的单细胞敲除来验证先前鉴定的树突状自我回避和树枝化表型，通过产生缺乏所有替代同种型的Pcdh- γ等位基因来解决这些过程的多样性要求，并鉴定显示神经突图案化缺陷的脊髓中间神经元谱系，所述神经突图案化缺陷可能与Pcdh- γ簇缺失小鼠中的显著表型相关。运动中枢模式发生器（CPG）完全丧失。在目的3中，我们建议研究所有群集Pcdhs的功能丧失对神经发育的后果，并进行CNS范围的筛选，以寻找轴突或树突状自我回避被破坏的神经元细胞类型。拟议中的研究应该提供重要的新的见解，在脊椎动物神经系统中的神经回路组装的机制，以及到病因的不同神经系统疾病所造成的改变神经回路。

 DESCRIPTION (provided by applicant): A key principle of neural circuit assembly is neurite self-avoidance, a process in which sister axons or dendrites of the same neuron repel each other but interact freely with those from other neurons, indicating they can discriminate "self" from "non-self". In flies the cell adhesion molecule Dscam1 has been shown to be a key mediator for neurite self-avoidance. Alternative splicing of Dscam1 pre-mRNA generates a large number of distinct protein isoforms that interact in a strictly homophilic manner, which mediate contact-dependent repulsion that leads to self-avoidance. However, the vertebrate Dscam genes do no encode significant diversity. Rather, this diversity and function appear to be provided by the clustered protocadherins (Pcdhs). Studies in our lab and others have shown that 1) Stochastic expression of clustered Pcdhs by alternative promoter choice generates single cell diversity; 2) Combinatorial homophilic interactions between clustered Pcdhs at the cell surface generate single neuron identity; 3) Pcdh- γ proteins are required for dendritic self-avoidance in mouse starburst amacrine cells and Purkinje cells. These observations have led to the hypothesis that clustered Pcdhs function similarly as Dscam1 proteins to mediate self-avoidance in vertebrates. In this application, we propose to carry out a comprehensive genetic analysis to determine whether clustered Pcdhs play a central role in neurite self-avoidance in the vertebrate nervous system, whether Pcdh diversity is required for this process, and whether different Pcdh clusters functionally compensate each other in specific cellular contexts. To accomplish these goals, we will generate single cell knockouts of Pcdh- α, - β, and - γ gene clusters individually or altogether, and study the loss of function effects in neurite arborizationof individual neurons. In Aim 1, we will establish and validate two complementary single cell knockout methods, Mosaic Analysis with Double Markers (MADM) and Single Neuron Analysis in Chimeras (SNAC). Both methods sparsely generate and label mutant cells in the otherwise normal neural network, allowing robust morphological analysis and rigorous assessment of cell autonomous gene function in single neurons. In Aim 2, we will use single cell knockouts of the Pcdh- γ gene cluster to validate previously identified dendritic self-avoidance and arborization phenotypes, address the diversity requirement for these processes by generating a Pcdh- γ allele lacking all alternate isoforms, and identify spinal interneuron lineages that display neurit patterning defects that may be associated with a striking phenotype in the Pcdh- γ cluster deletion mice - complete loss of central pattern generator (CPG) for locomotion. In Aim 3, we propose to study the consequences of loss-of-function of all clustered Pcdhs on neural development, and perform a CNS-wide screen for neuronal cell types in which axonal or dendritic self-avoidance is disrupted. The proposed studies should provide significant new insights into mechanisms of neural circuit assembly in the vertebrate nervous system, as well as into etiologies of diverse neurological disorders resulting from altered neural circuitries.