Screen for determinants of synaptic specificity in outer retina.

 描述（由申请人提供）：视觉信息的神经处理开始于视网膜的第一突触，其由在称为外丛状层（OPL）的薄突触层中具有水平和双极细胞（HC，BC）的视杆和视锥光感受器形成。这些中间神经元，沿着无长突细胞，将信息传递给视网膜神经节细胞，后者再将信息发送到大脑。OPL的连接性至少有三种特异性：视杆细胞和视锥细胞几乎完全分别与视杆细胞和视锥细胞形成突触（细胞特异性）;视杆细胞和视锥细胞分别与毛细胞的轴突和树突形成突触（亚细胞特异性）;视杆细胞和视锥细胞的突触分别局限于OPL的外层和内层（层特异性）。迄今为止，很少有分子被发现介导突触识别的这些方面。本提案的目的是鉴定这些分子。我们的方法是在小鼠体内筛选候选物。在哺乳动物中很少进行过这样的筛选，但OPL突触的大尺寸和可接近性，以及基因转移和基因组修饰的最新技术进步，沿着，现在有可能在可管理的时期内分析数十个基因。为了准备这项筛选，我们已经：（a）鉴定标记OPL中所有突触伙伴的分子标记物;（B）分析它们在突触形成期间的表达;（c）通过体内电穿孔优化基因转移方法;（d）通过体外电穿孔优化基因转移方法。（d）显示这些方法可用于使用shRNA和Cas9/CRISPR有效地减弱视杆细胞和视锥细胞中的基因表达，用于功能丧失研究，并异位表达基因用于功能获得研究;和（e）通过FACS分选纯化发育中的视杆细胞和视锥细胞，并使用RNA-Seq从它们获得转录组信息。我们现在将使用转录组学数据来选择约50个编码由发育中的视杆细胞和视锥细胞差异表达的跨膜或分泌分子的基因。我们将减弱它们在发育中的视网膜中的表达，然后使用多标记共聚焦显微镜来寻找OPL中改变的突触模式。最后，对于这些基因中最有希望的，我们将进行表达分析以及额外的功能丧失和获得研究，以阐明它们在突触形成中的作用。除了在这个临床重要的突触上开始对突触发生和突触选择性进行深入分析外，我们的结果在两个方面也很有用。首先，它们将为研究大脑其他部位不易接触的突触提供试剂和见解。第二，它们可以指导通过光感受器替换来恢复视力的方法的优化。替代方法最近显示出希望，但如果新的光感受器不能形成适当的突触，可能会失败。我们确定的分子可能有助于提高这一策略的效力。

 DESCRIPTION (provided by applicant): Neural processing of visual information begins at the first synapses of the retina, which are made by rod and cone photoreceptors with horizontal and bipolar cells (HCs, BCs) in a thin synaptic layer called the outer plexiform layer (OPL). These interneurons, along with amacrine cells, pass the information to retinal ganglion cells, which send it to the brain. Connectivity in the OPL is specific in at least three ways: rods and cones synapse almost entirely on rod BCs and cone BCs, respectively (cellular specificity); they synapse with axons and dendrites of HCs, respectively (subcellular specificity); and their synapses are confined to outer and inner strata of the OPL, respectively (laminar specificity). To date, few molecules have been found that mediate any of these aspects of synaptic recognition. The objective of this proposal is to identify such molecules. Our approach is to screen candidates in vivo in mice. Few such screens have been performed in any mammal, but the large size and accessibility of OPL synapses, along with recent technical advances in gene transfer and genome modification, now make it possible to analyze dozens of genes in a manageable period. To prepare for this screen, we have: (a) characterized molecular markers that label all synaptic partners in the OPL; (b) analyzed their expression during the period of synapse formation; (c) optimized gene transfer methods by electroporation in vivo; (d) shown that these methods can be used to effectively attenuate gene expression in rods and cones using shRNA and Cas9/CRISPRs for loss of function studies, and to ectopically express genes for gain-of-function studies; and (e) purified developing rods and cones by FACS sorting and used RNA-Seq to obtain transcriptome information from them. We will now use transcriptomic data to select ~50 genes that encode transmembrane or secreted molecules differentially expressed by developing rods and cones. We will attenuate their expression in developing retina, then use multi-label confocal microscopy to seek altered synaptic patterns in the OPL. Finally, for the most promising of these genes, we will conduct expression analysis as well as additional loss- and gain-of-function studies to elucidate their roles in synapse formation. In addition to initiating a deep analysis of synaptogenesis and synaptic selectivity at this clinicall important synapse, our results will be useful in two ways. First, they will provide reagents and insights for studies of less accessible synapses elsewhere in the brain. Second, they may guide optimization of methods to restore vision by photoreceptor replacement. Replacement methods have shown recent promise, but may fail if the new photoreceptors fail to make appropriate synapses. Molecules we identify could be useful in enhancing the efficacy of this strategy.