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

项目总结 目前对神经系统内差异基因的表达缺乏了解。理想情况下是一个 我想知道，在所有类型的神经元中，基因组到底是如何转录和处理成 功能核糖核酸。这一信息非常重要，因为差异基因表达定义了 单个神经元的形态和功能，决定了单个神经元如何对电路生理学和 并影响单个神经元如何受到损伤和疾病的影响。进一步、详细和详细地 对神经系统内差异基因表达的完整了解将有助于阐明其中的逻辑 以及产生神经元多样性的细胞机制，包括基因表达的调节，可供选择 剪接和miRNA功能。然而，这方面的进展是有限的：对于大多数神经系统来说，准确的 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球图谱 是不可能实现的。在这里，我们建议在一个发现和分析线虫的项目中解决这个问题 神经元基因表达图谱与网络(CENGEN)。线虫的神经系统正好含有302 神经元总数包括118类不同的神经元类型。我们建议利用这一独特的属性来 对每种神经元类型的基因表达进行高精度分析。CeNGen在四个月内取得进展 明确的目标。目的1)建立118株转基因菌株，每株表达独特的荧光标记 标记一种单一类型的神经元。目的2)使用创新的细胞分离和流式细胞术分离每种类型的 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精确定位基因 多个参数空间上的表达式。目的4)构建以细胞为中心和以基因为中心的表达图谱， 并寻求与线虫神经系统的其他独特已知特征的联系，包括 接线图、细胞谱系、神经递质特性和功能。成根代表了一个典范 神经遗传学的进展，并为阐明神经元特异性的全球控制提供了独特的机会 基因表达，并将基因表达与神经元连接和功能联系起来。预期的重大结果 包括：识别产生神经元特异性和多样性的保守调控机制； 详细了解神经系统中的选择性剪接和miRNA功能； 差异基因表达与神经元谱系、解剖、功能和连接性的关系。CeNGen还将为 作为线虫神经科学未来研究的资源，并将提供一个框架，以解决全球 在更复杂的神经系统中的差异基因表达，目前还不符合这一点 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.