Genetic analyses of complete circuit formation in Caenorhabditis elegans

项目摘要/摘要 尽管神经回路发育对大脑功能和行为至关重要，但我们缺乏基因 组装完整电路所需的信息。为了解决这一知识鸿沟，我们建议开发 标记活体动物完整回路的突触和神经元邻域新技术。使用这些 我们将首次从基因上解剖完整的神经回路，提供基本的遗传学洞察力 电路是如何构建的。我们将利用线虫的生物学-其可用的超微结构 连接体和灵活的遗传学，以解决整个回路中突触连接的问题。我们会 利用线虫咽部广泛的遗传和解剖学知识，线虫咽部是一种小但有功能的 独立电路，作为一种模式。我们将首次创建一个电路中所有神经元的表达图 使用神经元特异的流式细胞仪和转录图谱。然后我们将把这些新的基因表达关联起来 映射到超微结构连接体，以揭示基因表达模式如何与连接性相关。 接下来，我们将构建荧光报告系统来(1)使用GFP标记整个咽部回路 跨突触伙伴的重建(GRAP)和(2)标记相邻神经元的邻域 使用基于CD4的体内细胞间接触生物素标记(IBLINC)方法。使用这些工具，我们 将对细胞表面蛋白进行全基因组RNAi筛选，以确定它们在所有主要步骤中的作用 电路形成-神经元生长、邻域和突触选择。这项提案的结果将是 识别和验证控制连接的遗传因素，这些因素可以在复杂脊椎动物中进一步评估 神经系统和人类。这项提议不仅将改进荧光标记的方法 突触和神经元邻域，但将提供基于地图的精致详细的理解 活体动物的巡回表演。此外，我们的方法和结果将在以下大背景下有用 了解适用于大脑复杂性的电路形成的基本原理。

Project Summary / Abstract Despite the central importance of neural circuit development to brain function and behavior, we lack the genetic information required to assemble a complete circuit. To address this knowledge gap we propose to develop novel synaptic and neuronal-neighborhood techniques to label a complete circuit in live animals. Using these tools we will genetically dissect a complete neural circuit for the first time, providing fundamental genetic insight into how circuits are built. We will take advantage of C. elegans biology - its available ultrastructural connectome and facile genetics, to address questions of synaptic wiring throughout a complete circuit. We will leverage the extensive genetic and anatomical knowledge of the C. elegans pharynx, a small but functionally independent circuit, as a model. We will for the first time create an expression map of all neurons in a circuit using neuron-specific FACS and transcriptomic profiling. We will then correlate these new gene expression maps to the ultrastructural connectome to reveal how patterns of gene expression correlate with connectivity. Next, we will construct fluorescent reporter systems to (1) label the entire pharyngeal circuit using GFP Reconstitution Across Synaptic Partners (GRASP) and (2) label the neighborhoods of adjacent neuronal processes using CD4-based in vivo Biotin Labeling of INtercellular Contacts (iBLINC). Using these tools, we will perform a genome-wide RNAi screen of cell surface proteins to determine their role in all major steps of circuit formation – neuronal outgrowth, neighborhood, and synaptic choice. The results from this proposal will identify and validate genetic factors controlling wiring that can be further evaluated in complex vertebrate nervous systems and humans. This proposal will not only improve methodologies for fluorescent labeling of synapses and neuronal neighborhoods, but will provide an exquisitely detailed map-based understanding of a circuit in live animals. Furthermore, our approach and results will be useful in the broad context of understanding fundamentals of circuit formation applicable across brain complexity.