Genetic dissection of cell-cell interaction in neuronal patterning in C. elegans

线虫神经元模式中细胞间相互作用的遗传解剖

• 批准号: RGPIN-2015-04022
• 负责人: Mizumoto, Kota
• 金额: $ 2.55万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=708341
• 关键词: Genetic; dissection; cell; interaction; neuronal

Neurons typically have projections called neurites (axons and dendrites), which are connected with neighboring neurons or other cells to pass signals via special cell-cell junction called synapses. Neurons with branched neurites often arrange their neurites in a tiled pattern by avoiding neurites of their neighboring neurons to maximize coverage while minimizing the overlap with which the territory is innervated. Tiling is observed in many neurons including the somatosensory and visual system of both vertebrates and invertebrates. Despite the notion that each class of neurons utilizes various mechanisms to tile their neurites, little is known about the tiling mechanisms in most systems. The limitation of knowledge on how neurons interact with their neighboring neurons is largely due to the lack of techniques to systematically label individual neurons in living animals. Therefore, we are in need of developing new systems to study inter-neuronal interactions to fully understand the molecular machineries of neural pattern formation.    I propose to tackle this issue using the nematode, Caenorhabditis elegans, as a model system. C. elegans provides numerous experimental advantages. For example, C. elegans has a simple nervous system that consists of only 302 neurons, a stereotyped cell lineage. Most important for this study, individual neurons can be visualized with cell-specific markers in living animals, making C. elegans a powerful genetic model system to study neuronal circuit assembly and architecture.    By combining two cell-specific promoters and two fluorescent proteins, I generated a genetic marker that stably labels two neighboring motor neurons, DD5 and DD6. Both of these cells belong to the same class of D-type GABAergic motor neurons and tile their axons and dendrites with their neighboring DDs. Using this genetic marker strain, I propose to identify genes responsible for axonal/dendritic tiling, and conduct functional analyses of the isolated genes to understand the cellular and molecular mechanisms of tiling. Proposed research involves basic and advanced genetics, molecular biology, genomic analyses using next generation sequencing (NGS) technology and imaging with state-of-the-art microscopes. This research program will provide an excellent platform for students to experience basic and advanced technologies in diverse disciplines.    I believe this is a timely proposal considering the current enthusiasm and interest in studying mammalian brain structure and function. As molecular mechanisms regulating neuronal development and function are well conserved from C. elegans to mammals, the proposed program will identify fundamental mechanisms of how local inter-neuronal communication contribute to neural circuit assembly. The insights from a simple nervous system will help us understand the principles of neurodevelopment in the mammalian brain.

神经元通常具有称为神经突（轴突和树突）的突起，它们与邻近的神经元或其他细胞连接，通过称为突触的特殊细胞-细胞连接传递信号。具有分支神经突的神经元通常通过避开其神经突将其神经突排列成平铺模式。在许多神经元中观察到区域受神经支配的重叠，包括脊椎动物的体感和视觉系统。尽管人们认为每一类神经元都利用不同的机制来平铺它们的神经突，但对大多数系统中的平铺机制知之甚少，关于神经元如何与其相邻神经元相互作用的知识的限制很大程度上是由于缺乏技术。因此，我们需要开发新的系统来研究神经元间的相互作用，以充分了解神经模式形成的分子机制。 我建议使用线虫（秀丽隐杆线虫）作为模型系统来解决这个问题，秀丽隐杆线虫具有许多实验优势，例如，秀丽隐杆线虫具有仅由 302 个神经元组成的简单神经系统，这是一种典型的细胞谱系。对于这项研究很重要的是，可以通过活体动物中的细胞特异性标记来可视化单个神经元，使秀丽隐杆线虫成为研究神经回路组装和结构的强大遗传模型系统。 通过结合两个细胞特异性启动子和两个荧光蛋白，我生成了一个稳定标记两个相邻运动神经元 DD5 和 DD6 的遗传标记，这两个细胞属于同一类 D 型 GABA 能运动神经元，并平铺它们的轴突和轴突。使用这种遗传标记菌株，我建议鉴定负责轴突/树突平铺的基因，并对分离的基因进行功能分析，以了解其细胞和分子机制。拟议的研究涉及基础和高级遗传学、分子生物学、使用下一代测序（NGS）技术的基因组分析以及使用最先进的显微镜成像该研究项目将为学生提供一个体验基础和高级的绝佳平台。不同学科的技术。 考虑到当前对研究哺乳动物大脑结构和功能的热情和兴趣，我相信这是一个及时的提议，因为调节神经发育和功能的分子机制从线虫到哺乳动物都得到了很好的保存，所提出的计划将确定局部相互作用的基本机制。 -神经元通讯有助于神经回路组装。来自简单神经系统的见解将帮助我们了解哺乳动物大脑中神经发育的原理。