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

神经元通常具有称为神经突（轴突和树突）的突起，其与邻近神经元或其他细胞连接，以通过称为突触的特殊细胞-细胞连接来传递信号。具有分支神经突的神经元通常通过避免其相邻神经元的神经突以平铺模式排列其神经突，以最大化覆盖范围，同时最小化受神经支配的区域的重叠。平铺在许多神经元中观察到，包括脊椎动物和无脊椎动物的体感和视觉系统。尽管每类神经元都利用各种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局限性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。 我建议使用线虫，秀丽隐杆线虫，作为一个模型系统来解决这个问题。C.秀丽线虫提供了许多实验优势。例如，C.秀丽线虫的神经系统很简单，只有302个神经元，这是一种定型的细胞谱系。对于这项研究来说，最重要的是，在活体动物中，单个神经元可以用细胞特异性标记物进行可视化，使C。elegans是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，我产生了一个遗传标记，稳定地标记两个相邻的运动神经元，DD 5和DD 6。这两种细胞都属于同一类D型GABA能运动神经元，它们的轴突和树突与相邻的DD平铺。使用这种遗传标记菌株，我建议确定负责轴突/树突平铺的基因，并进行功能分析的分离基因，以了解平铺的细胞和分子机制。拟议的研究涉及基础和先进的遗传学，分子生物学，使用下一代测序（NGS）技术的基因组分析和最先进的显微镜成像。该研究计划将为学生提供一个很好的平台，体验不同学科的基础和先进技术。 我相信，考虑到目前对研究哺乳动物大脑结构和功能的热情和兴趣，这是一个及时的建议。由于调节神经元发育和功能的分子机制是从C. elegans到哺乳动物，拟议的计划将确定如何本地神经元间的通信有助于神经电路组装的基本机制。从一个简单的神经系统的见解将帮助我们了解哺乳动物大脑神经发育的原则。

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.