Combinatorial roles of cadherins in retinal circuit assembly.

 描述(申请人提供)：多种类型神经元之间有序和特定的连接是神经回路功能的基础。我们的工作使用小鼠视网膜作为模型，以确定这种特异性背后的分子和机制。视网膜本身不仅是重要和迷人的，而且也是中枢神经系统中特别容易接近的部分。此外，我们和其他人已经产生了一组转基因株系，可以标记和操纵特定的视网膜细胞类型。我们专注于内丛状层(IPL)，其中数十种类型的中间神经元(无长突细胞和双极细胞)与大约30种类型的视网膜神经节细胞(RGC)形成固定的连接模式，使它们对视觉特征(如特定方向的运动或颜色对比度)具有敏感性。连接这些主要是硬连线的电路似乎需要许多特定细胞类型的分子。在过去的几年里，他已经确定了几个候选基因，并使用功能丧失和功能获得的策略在体内对它们进行了测试。在过去的项目期间，我们发现钙粘素超家族识别分子的成员在IPL的组装中扮演了两个角色。首先，一对经典的钙粘附素(CDH8和CDH9)在 将两种双极细胞类型(BC2、BC5)的轴突乔木定向到IPL内适当的亚层。第二，星爆型无长突细胞(SACs)在侧面的树突构型需要一组簇生的γ-原钙粘附素(Pcdhg‘s)。幸运的是，BC2、BC5和SACS都是方向选择电路的组成部分，该电路还包括开关方向选择RGC，它将四个基本方向上的运动信息发送到大脑的其余部分。这些结果为我们提供了解决一个长期存在的问题的机会：多基因家族的成员如何在组合中共同工作，以形成神经回路的模式。使用新的基因组编辑方法，我们已经产生了缺乏(A)CDH8及其近亲CDH11，(B)CDH9及其近亲CDH6和CDH10，以及(C)Pcdhgs及其近亲α-Pcdhs的双重和三重突变体。重要的是，所有这些基因都是由方向选择回路的细胞表达的。我们现在的目标是分析钙粘附素超家族基因发生单个和组合突变的突变。我们将使用分子、组织学和电生理学的方法来分析这些扰动对方向选择电路的结构和功能的影响。总而言之，这些研究将使我们朝着面对令人不安的现实迈出第一步，即单基因分析不足以理解复杂神经回路的组装。

 DESCRIPTION (provided by applicant): Orderly and specific connections among neurons of multiple types underlie the function of neural circuits. Our work has used mouse retina as a model to identiy molecules and mechanisms that underlie this specificity. The retina is not only important and fascinating in its own right, but is also a particularly accessible portion of the central nervous system. Moreover, we and others have generated a set of transgenic lines that allow specific retinal cell types to be marked and manipulated. We focus on the inner plexiform layer (IPL) in which dozens of types of interneurons (amacrine and bipolar cells) form stereotyped patterns of connectivity with ~30 types of retinal ganglion cells (RGCs), endowing each of them with sensitivity to visual features such as motion in a particular direction or color contrast. Wiring up these largely hard-wired circuits seems likely to require many cell type-specific molecules. Over the past several years he have identified several candidates, and tested them in vivo using loss- and gain-of-function strategies. During the past project period, we found two roles for members of the cadherin superfamily of recognition molecules in assembly of the IPL. First, a pair of classical cadherins (Cdh8 and Cdh9) play instructive roles in directing axonal arbors of two bipolar cell types (BC2, BC5) to appropriate sublaminae within the IPL. Second, a group of clustered gamma protocadherins (Pcdhg's) are required for dendritic patterning of starburst amacrine cells (SACs) in the lateral plane. Fortuitously, BC2, BC5 and SACs are all components of a direction-selective circuit that also includes ON-OFF direction-selective RGCs, which send information about motion in four cardinal directions to the rest of the brain. These results provide us with the opportunity to address a long- standing issue: how do members of multigene families work together in combinations to pattern neural circuits. Using new genome editing methods, we have generated double and triple mutants that lack (a) both Cdh8 and its closest relative Cdh11, (b) Cdh9 and its closest relatives, Cdh6 and Cdh10, and (c) Pcdhgs and their closest relatives, the alpha-Pcdhs. Importantly all of these genes are expressed by cells of the direction-selective circuit. Our aims now are to analyze mutants in which genes of the cadherin superfamily have been mutated singly and in combination. We will use molecular, histological and electrophysiological methods to analyze the consequences of these perturbations on the structure and function of the direction-selective circuit. Together, these studies will allow us to take a first step toward confronting the disturbing reality that analysis of single genes is insufficient to understand the assembly of complex neural circuits.