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

神经元通常有被称为神经突（轴突和树突）的突起，它们与邻近的神经元或其他细胞相连，通过被称为突触的特殊细胞-细胞连接点传递信号。具有分支神经突的神经元通常将其神经突排列成平铺状，以避开相邻神经元的神经突，从而最大化覆盖范围，同时最小化其受支配区域的重叠。在许多神经元中都观察到平铺现象，包括脊椎动物和无脊椎动物的体感觉和视觉系统。尽管人们认为每一类神经元利用不同的机制来平铺它们的神经突，但在大多数系统中，人们对平铺机制知之甚少。关于神经元如何与邻近神经元相互作用的知识有限，主要是由于缺乏系统地标记活体动物中单个神经元的技术。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。我建议用秀丽隐杆线虫（Caenorhabditis elegans）作为模型来解决这个问题。秀丽隐杆线虫提供了许多实验优势。例如，秀丽隐杆线虫有一个简单的神经系统，仅由302个神经元组成，这是一种刻板的细胞谱系。对于这项研究最重要的是，单个神经元可以通过活体动物的细胞特异性标记可视化，使秀丽隐杆线虫成为研究神经元电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，我生成了一个遗传标记，可以稳定地标记两个相邻的运动神经元DD5和DD6。这两种细胞属于同一类d型gaba能运动神经元，它们的轴突和树突与邻近的dd相连接。利用这一遗传标记菌株，我建议鉴定轴突/树突平铺的基因，并对分离的基因进行功能分析，以了解平铺的细胞和分子机制。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不同学科基础和先进技术的良好平台。考虑到目前研究哺乳动物大脑结构和功能的热情和兴趣，我认为这是一个及时的建议。由于从秀丽隐杆线虫到哺乳动物调节神经元发育和功能的分子机制都得到了很好的保守，因此该计划将确定局部神经元间通信如何促进神经回路组装的基本机制。从一个简单的神经系统中获得的见解将帮助我们理解哺乳动物大脑中神经发育的原理

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.**