Genetic analyses of complete circuit formation in Caenorhabditis elegans

秀丽隐杆线虫完整回路形成的遗传分析

• 批准号: 9454707
• 负责人: STEVEN JAY COOK
• 金额: $ 6.13万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-27 至 2020-09-26
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9454707
• 关键词: Address; Affect; Anatomy; Animals; Autistic Disorder; Behavior; Biology; Biotin; Brain; Cadherins; Caenorhabditis elegans; Candidate Disease Gene; Cell Adhesion; Cell Surface Proteins; Cell surface; Cells; Code; Complement; Complex; Confocal Microscopy; Data; Development; Failure; Flow Cytometry; Gene Combinations; Gene Expression; Gene Expression Profile; Gene Family; Genes; Genetic; Genetic Determinism; Human; Immunoglobulins; Individual; Invertebrates; Knowledge; Label; Leucine-Rich Repeat; Link; Logic; Maps; Methodology; Modeling; Neighborhoods; Nervous system structure; Neurites; Neurons; Neurosciences; Pathogenesis; Patients; Pattern; Pattern Formation; Pharyngeal structure; Phenotype; Process; Proteins; Publishing; RNA interference screen; Reporter; Resolution; Role; Schizophrenia; Signal Transduction; Synapses; System; Techniques; Testing; Time; animal imaging; base; connectome; fluorophore; genetic analysis; genetic information; genome-wide; improved; in vivo; innovation; insight; intersectionality; mutant; nervous system disorder; neural circuit; neuron development; neuronal circuitry; neuronal patterning; novel; reconstitution; relating to nervous system; stereotypy; synaptogenesis; theories; tool; transcriptome; transcriptomics

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.

项目总结/摘要 尽管神经回路发育对大脑功能和行为至关重要，但我们缺乏遗传学上的证据。 组装完整电路所需的信息。为了解决这一知识差距，我们建议开发 新的突触和神经元邻域技术来标记活体动物的完整回路。使用这些 我们将第一次从基因上解剖一个完整的神经回路，提供基本的遗传洞察力。 电路是如何构建的我们将利用C。elegans生物学-其可用的超微结构 连接体和简易遗传学，以解决整个电路突触布线的问题。我们将 利用C.线虫咽，一个小的，但功能 独立电路，作为模型。我们将首次创建一个回路中所有神经元的表达图 使用神经元特异性流式细胞术和转录组学分析。然后我们将这些新的基因表达 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，以（1）使用GFP标记整个咽回路 跨突触伙伴重建（GRASP）和（2）标记相邻神经元的邻域 使用基于CD 4的细胞间接触的体内生物素标记（iBLINC）的方法。利用这些工具，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。 回路的形成-神经元的生长、邻近和突触的选择。该提案的结果将 识别和验证控制布线的遗传因素，这些遗传因素可以在复杂脊椎动物中进一步评估 神经系统和人类。这一建议不仅将改善荧光标记的方法， 突触和神经元的邻居，但将提供一个精致详细的地图为基础的理解， 活的动物的电路。此外，我们的方法和结果将有助于在广泛的背景下， 了解适用于大脑复杂性的电路形成的基本原理。