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异性的细胞-细胞识别步骤，其中许多是由细胞介导的。 粘附分子（CAM）。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， 中枢神经系统，其中约100种神经元组织成专门的电路，编码的功能， 视觉世界我们在这里集中在γ-原钙粘素（γ-Pcdhs），22 CAM表达从一个单一的基因簇， 产生数千种不同的亲同性识别复合物。γ-Pcdhs是神经元的重要调节因子， 星状无长突细胞（SAC）的自我回避，以及视网膜中的细胞存活和许多其他类型的神经元。的 γ-Pcdh发挥这些功能的机制尚不清楚，γ-Pcdh亚型的重要性也是未知的 多样性我们使用CRISPR/Cas9方法来产生小鼠突变体的无偏等位基因系列，其具有1至10个等位基因。 21个完整的γ-Pcdh亚型。从中我们了解到，一种亚型γC4对神经元生存至关重要，这表明 这种亚型的功能与其他21种不同我们建议定义自我回避的机制， 神经元存活，并使用我们的等位基因系列来确定正常神经元存活所需的同种型多样性水平。 电路形成我们的中心假设是：1）高水平的γ-Pcdh亚型多样性使神经元能够 区分“自我”和“非自我”，以调解自我回避，同时允许与邻居的互动 神经元通过所有亚型共同的机制; 2）神经元的存活，相反，需要相互作用 特异于γC4同种型。在具体目标1中，我们将使用我们的减少多样性突变体的战略子集， 确定SAC中自我/非自我辨别所需的同种型多样性的程度， 视网膜中的运动检测电路。我们将在两个层面上分析这个电路：A）之间的接触形态 SACs，和B）方向选择性视网膜神经节细胞的电生理功能，方向选择性视网膜神经节细胞是SACs的下游神经元。 电路。在具体目标2中，我们将使用体内基因传递来定义自我回避的分子机制， 操纵候选通路和绘制必需结构域。在具体目标3中，我们将通过以下方式揭示机制： γC4促进神经元存活。我们将使用视网膜电穿孔来绘制关键蛋白质结构域， 辅以基于发现的蛋白质组学方法，以发现γC4的亚型特异性蛋白质相互作用。这些 研究将使我们更好地了解γ-Pcdhs如何参与细胞间识别和神经回路 视网膜中的形成，并提供洞察神经发育障碍中断的过程。

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.