Mechanisms of Cell Adhesion Molecule Function in Retinal Development

摘要 神经回路的形成需要一系列高度多样化和特定的细胞-细胞识别步骤，其中许多步骤是由细胞介导的 黏附分子(凸轮)。事实上，破坏cam或其调节的突变与电路水平有关。 从阅读障碍到精神分裂症的神经发育障碍。我们的模型是小鼠视网膜，它是 中枢神经系统，大约100种类型的神经元组织成专门的电路，编码 视觉世界。我们在这里关注的是伽马原钙粘附素(γ-Pcdhs)，它是从单个基因簇表达的22个CAM 产生数以千计的不同的嗜同者识别复合体。γ-Pcdhs是神经元的重要调节因子 星爆无长突细胞(SACs)的自我回避，以及细胞存活和视网膜中许多其他类型的神经元。这个 γ-Pcdh发挥这些功能的机制以及γ-Pcdh亚型的重要性尚不清楚 多样性。我们使用CRISPR/Cas9方法产生了一系列无偏等位基因的小鼠突变体，其范围在1和 21个完整的γ-Pcdh亚型。从这些研究中，我们了解到一个亚型，γC4，对于神经元的生存是必不可少的，这表明 这个异构体的功能与其他21个不同。我们建议定义自我回避的机制和 神经元存活，并使用我们的等位基因系列来确定正常神经所需的异构体多样性水平 赛道编队。我们的中心假设是：1)高水平的γ-Pcdh亚型多样性使神经元能够 区分“自我”和“非我”，在允许与邻居互动的同时调解自我回避 神经元通过所有异构体共有的机制；和2)相反，神经元生存需要相互作用。 特异性为γC4亚型。在具体目标1中，我们将使用我们减少多样性的突变体的战略子集来 确定囊中自我/非自我区分所需的异构体多样性的程度，囊中的神经元是 视网膜中的运动检测电路。我们将在两个层次上分析该电路：a)触点之间的形态 和B)方向选择性视网膜神经节细胞的电生理功能，下游神经元在 巡回赛。在特定的目标2中，我们将通过体内基因传递来定义自我回避的分子机制 操纵候选路径并绘制基本领域图。在具体目标3中，我们将通过以下方式揭示这些机制 哪种γC4能促进神经元存活。我们将使用视网膜电穿孔来绘制关键的蛋白质结构域， 辅以基于发现的蛋白质组学方法，以寻找γC4的异构体特定蛋白质相互作用。这些 研究将使我们更好地了解γ-Pcdhs如何对细胞-细胞识别和神经回路做出贡献 在视网膜中形成，并提供对被神经发育障碍扰乱的过程的洞察。

ABSTRACT Neural circuit formation requires a series of highly diverse and specific cell-cell recognition steps, many mediated by cell adhesion molecules (CAMs). Indeed, mutations that disrupt CAMs or their regulation are associated with circuit level neurodevelopmental disorders from dyslexia to schizophrenia. Our model is the mouse retina, an extension of the central nervous system where ~100 types of neurons organize into dedicated circuits that encode the features of the visual world. We focus here on the gamma-protocadherins (γ-Pcdhs), 22 CAMs expressed from a single gene cluster that generate many thousands of distinct homophilic recognition complexes. The γ-Pcdhs are critical regulators of neuronal self-avoidance in starburst amacrine cells (SACs), and cell survival and in many other types of neurons in the retina. The mechanisms through which the γ-Pcdhs serve these functions are unknown, as is the importance of γ-Pcdh isoform diversity. We used a CRISPR/Cas9 approach to generate an unbiased allelic series of mouse mutants with between 1 and 21 intact γ-Pcdh isoforms. From these, we learned that one isoform, γC4, is essential for neuronal survival, suggesting that this isoform functions differently from the other 21. We propose to define the mechanisms of self-avoidance and neuronal survival, and to use our allelic series to determine the level of isoform diversity required for normal neural circuit formation. Our central hypotheses are that: 1) a high level of γ-Pcdh isoform diversity enables neurons to distinguish between “self” and “non-self” to mediate self-avoidance while permitting interaction with neighboring neurons through mechanisms common to all isoforms; and 2) neuronal survival, in contrast, requires interactions specific to the γC4 isoform. In Specific Aim 1, we will use a strategic subset of our reduced-diversity mutants to determine the extent of isoform diversity required for self/non-self discrimination in SACs, neurons essential for the motion detection circuit in the retina. We will analyze this circuit at two levels: A) morphology of contacts between SACs, and B) the electrophysiological function of direction-selective retinal ganglion cells, the downstream neurons in the circuit. In Specific Aim 2, we will define the molecular mechanisms of self-avoidance using in vivo gene delivery to manipulate candidate pathways and map essential domains. In Specific Aim 3 we will uncover the mechanisms through which γC4 promotes neuronal survival. We will use retinal electroporation to map critical protein domains, complemented by a discovery-based proteomics approach to find isoform-specific protein interactions for γC4. These studies will allow us to better understand how the γ-Pcdhs contribute to cell-cell recognition and neural circuit formation in the retina and provide insight into processes disrupted by neurodevelopmental disorders.