Screen for determinants of synaptic specificity in outer retina.

筛选外视网膜突触特异性的决定因素。

基本信息

批准号：
8869733
负责人：
JOSHUA R SANES
金额：
$ 25.35万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2017-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8869733
关键词：
Address Amacrine Cells Antibodies Attenuated Axon Biological Models Blindness Brain Caenorhabditis elegans Candidate Disease Gene Cells Clustered Regularly Interspaced Short Palindromic Repeats Cone Confocal Microscopy Data Dendrites Drosophila melanogaster Electroporation Gene Expression Gene Expression Profile Gene Transfer Genes Genetic Screening Genome Germ Lines Interneurons Intervention Label Mammals Mediating Methods Modification Mus Neurons Pattern Phenotype Photoreceptors Process Reagent Replacement Therapy Retina Retinal Cone Retinal Ganglion Cells Role Sorting - Cell Movement Specificity Synapses Time Transgenic Organisms Vertebrate Photoreceptors Vision Visual system structure Zebrafish base differential expression gain of function horizontal cell in vivo insight loss of function molecular marker mutant outer plexiform layer public health relevance relating to nervous system research study retinal rods selective expression small hairpin RNA success synaptogenesis transcriptome sequencing transcriptomics visual information

项目摘要

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.

描述（由适用提供）：视觉信息的神经处理始于视网膜的第一个突触，该突触是由带有水平和双极细胞（HCS，BCS）的杆和锥形感受器在一个薄薄的合成层中制成的，称为外孔形层（OPL）。这些中间神经元与无聚氨酸细胞一起将信息传递给视网膜神经节细胞，将其发送到大脑。 OPL中的连通性至少在三种方面是特异性的：杆和锥体突触几乎全部完全在杆BC和锥BC上（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的外部和内部（层流特异性）。迄今为止，很少有人发现介导突触识别的任何方面。该提议的目的是确定此类分子。我们的方法是筛选小鼠体内候选者。在任何哺乳动物中，很少有这样的筛选，但是OPL突触的较大尺寸和可访问性，以及基因转移和基因组修饰的最新技术进步，现在可以在可管理的时期内分析数十个基因。为了准备此屏幕，我们有：（a）表征分子标记，将OPL中的所有突触伙伴标记为标记；（b）在突触形成期间分析了它们的表达；（c）通过体内电穿孔优化基因转移方法；（d）表明，这些方法可用于使用SHRNA和CAS9/CRISPR进行功能研究的丧失来有效地衰减棒和锥体中的基因表达，并为功能获得的研究表达基因；（e）通过FACS排序和使用RNA-Seq从中获得转录组信息的纯化开发棒和锥。现在，我们将使用转录组数据选择约50个基因，这些基因编码跨膜或分泌分子通过发育的棒和锥体不同表达的基因。我们将减弱它们在发展视网膜中的表达，然后使用多标签共聚焦显微镜在OPL中寻求改变的突触模式。最后，为了这些基因的最有前途，我们将进行表达分析以及额外的功能损失和功能增益研究，以阐明其在突触形成中的作用。除了对该诊所重要的突触中的突触发生和突触选择性进行深入分析外，我们的结果将在两种方面有用。首先，它们将提供试剂和见解，以研究大脑其他地方其他地方的突触较差的研究。其次，他们可以指导通过替换感受器替换恢复视力的方法的优化。替换方法已显示出最近的承诺，但是如果新的光感受器无法制作适当的突触，可能会失败。我们确定的分子可能有助于提高该策略的效率。