Screen for determinants of synaptic specificity in outer retina.

 描述(申请人提供)：视觉信息的神经处理始于视网膜的第一个突触，由视杆和视锥感光细胞与水平和双极细胞(HCS，BCS)在称为外丛状层(OPL)的薄突触层中形成。这些中间神经元和无长突细胞一起将信息传递给视网膜神经节细胞，再由视网膜神经节细胞将信息发送到大脑。OPL中的连接至少在三个方面具有特异性：视杆细胞和视锥细胞分别几乎完全在视杆细胞和视锥细胞上突触(细胞特异性)；它们分别与HC的轴突和树突突触(亚细胞特异性)；它们的突触分别局限于OPL的外层和内层(板层特异性)。到目前为止，还没有发现多少分子能在突触识别的这些方面起到中介作用。这项提议的目标是识别这样的分子。我们的方法是在小鼠体内筛选候选基因。在哺乳动物身上进行这种筛查的人很少，但OPL突触的巨大尺寸和可获得性，加上最近在基因转移和基因组修改方面的技术进步，现在使得在可管理的时间内分析数十个基因成为可能。为了准备这次筛选，我们已经：(A)鉴定了标记OPL中所有突触伙伴的分子标记；(B)分析了它们在突触形成期间的表达；(C)通过体内电穿孔优化了基因转移方法；(D)证明了这些方法可有效地利用shRNA和Cas9/CRISPR抑制杆和锥中的基因表达，用于功能丧失研究，并能异位表达用于功能获得研究的基因；和(E)通过FACS分选纯化发育中的杆和锥，并使用RNA-Seq从它们获得转录组学信息。我们现在将使用转录数据来选择~50个编码跨膜或分泌分子的基因，这些分子通过发育中的杆状和锥体而差异表达。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，对于这些最有希望的基因，我们将进行表达分析以及额外的功能丧失和功能获得研究，以阐明它们在突触形成中的作用。除了在这个临床上最重要的突触上启动对突触发生和突触选择性的深入分析外，我们的结果将在两个方面有用。首先，他们将为研究大脑其他部位较难获得的突触提供试剂和见解。其次，它们可能会指导通过光感受器置换恢复视力的方法的优化。替代方法最近显示出了希望，但如果新的光感受器不能产生适当的突触，可能会失败。我们确定的分子可能有助于增强这一策略的有效性。

 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.