Discovery and analysis of the C. elegans neuronal gene expression network (CENGEN)

项目摘要 目前缺乏对神经系统内差异基因表达的了解。理想一 我想知道，在所有神经元类型中，基因组是如何转录和加工成 功能性RNA。这一信息是非常重要的，因为差异基因表达决定了 单个神经元的形式和功能，决定了单个神经元如何对电路生理学做出贡献， 行为，并影响个体神经元如何受到损伤和疾病的影响。此外，详细和 对神经系统内不同基因表达的完整了解将有助于阐明 以及产生神经元多样性的细胞机制，包括基因表达的调节， 剪接和miRNA功能。然而，在这一领域的进展是有限的：对于大多数神经系统，确切的 不同类型的神经元的数量是未知的，因此神经元特异性基因表达的全局图 是无法实现的。在这里，我们建议在一个项目中解决这个问题，以发现和分析C。elegans 神经元基因表达图谱和网络（CeNGEN）。梭线虫的神经系统含有302 包括118类不同神经元类型的总神经元。我们建议利用这一独特的属性， 在每一种神经元类型中高精度地分析基因表达。CeNGEN将在四个月内 具体目标。目的1）建立118株转基因菌株，每株转基因菌株表达的荧光标记物都是唯一的， 标记单一类型神经元。目的2）使用创新的细胞解离和FACS方法分离每种类型的细胞， 神经元，并使用RNA-seq方法评估全局编码转录本和miRNA 表达以及剪接多样性。目的3）利用单细胞测序技术对基因进行精确定位 多参数空间上的表达式。目的4）构建以细胞为中心和基因为中心的表达图谱， 并寻求与C.线虫的神经系统包括 接线图，细胞谱系，神经递质身份和功能。CeNGEN代表了一个典型的 神经遗传学的进展，并提供了一个独特的机会，阐明全球控制神经元特异性 基因表达，并将基因表达与神经元布线和功能联系起来。预期重大成果 包括：鉴定产生神经元特异性和多样性的保守调节机制; 详细了解选择性剪接和miRNA在神经系统中的功能; 差异基因表达对神经元谱系、解剖学、功能和连接性的影响。CeNGEN还将为 为进一步研究C. elegans神经科学，并将提供一个框架，解决全球 在更复杂的神经系统中的差异基因表达，目前还不适合这种情况。 全面的方法。

PROJECT SUMMARY There is a current lack of understanding of differential gene expression within the nervous system. Ideally one would like to know, across all neuron types, exactly how the genome is transcribed and processed into functional RNAs. This information is fundamentally important because differential gene expression defines the form and function of individual neurons, determines how individual neurons contribute to circuit physiology and behavior, and influences how individual neurons are affected by injury and disease. Further, detailed and complete knowledge of differential gene expression within the nervous system would help elucidate the logic and cellular mechanisms that generate neuronal diversity, including regulation of gene expression, alternative splicing, and miRNA function. Yet progress in this area has been limited: For most nervous systems, the exact number of distinct types of neurons is unknown and therefore a global map of neuron-specific gene expression is not achievable. Here we propose to address this problem in a project to discover and analyze the C. elegans Neuronal Gene Expression Map & Network (CeNGEN). The C. elegans nervous system contains precisely 302 total neurons comprising 118 classes of distinct neuronal types. We propose to exploit this unique attribute to analyze gene expression with high accuracy in every individual neuronal type. CeNGEN proceeds in four specific aims. Aim 1) Establish 118 transgenic strains, each one expressing fluorescent markers that uniquely label a single type of neuron. Aim 2) Use innovative cell dissociation and FACS methods to isolate each type of neuron from age-matched adults, and use RNA-seq approaches to assess global coding transcript and miRNA expression, as well as splicing diversity. Aim 3) Utilize single cell sequencing technology to precisely map gene expression over multiple parameter spaces. Aim 4) Build cell-centered and gene-centered expression maps, and seek connections with other uniquely known features of the C. elegans nervous system including the wiring diagram, the cell lineage, neurotransmitter identity, and function. CeNGEN represents a paradigmatic advance in neurogenetics, and provides a unique opportunity to elucidate the global control of neuron-specific gene expression and to relate gene expression to neuronal wiring and function. Expected significant outcomes include: Identification of conserved regulatory mechanisms that generate neuronal specificity and diversity; Detailed understanding of alternative splicing and miRNA function across the nervous system; Relationship of differential gene expression to neuronal lineage, anatomy, function and connectivity. CeNGEN will also serve as a resource for future studies in C. elegans neuroscience, and will provide a framework for addressing global differential gene expression in more complex nervous systems that are currently not amenable to this comprehensive approach.