Profiling the circadian transcriptome of Drosophila nervous system using RNA sequ

使用 RNA 序列分析果蝇神经系统的昼夜节律转录组

基本信息

批准号：
8060138
负责人：
Michael Evan Hughes
金额：
$ 5.47万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2012-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8060138
关键词：
Address Adult Alternative Splicing Automobile Driving Behavior Biological Models Biological Process Blood Pressure Brain Cells Circadian Rhythms Complex Data Set Development Disease susceptibility Dissection Dissociation Drosophila genus Event Feedback Fluorescence-Activated Cell Sorting Gene Expression Gene Expression Profile Genes Goals Head Health Heart Diseases Hour Human Intrinsic drive Label Length Life Liver Maintenance Malignant Neoplasms Mammals Measures Mediating Membrane Mental disorders Metabolism Molecular Molecular Profiling Motor Activity Myocardial Infarction Nervous system structure Neuraxis Neuroglia Neurons Neuropeptides Output Pathogenesis Pathway interactions Physiological Physiology Pigments Population Predisposition Procedures Process RNA RNA Sequence Analysis RNA Sequences Regulation Signal Transduction Skeletal Muscle Sleep Wake Cycle Statistical Methods System Technology Tissues Transcript Transcriptional Regulation Transgenic Organisms Translating Work analytical tool base brain tissue cell type circadian pacemaker compound eye drug metabolism feeding fly human disease meetings neural circuit neuroregulation novel novel strategies receptor research study response tool

项目摘要

DESCRIPTION (provided by applicant): Circadian rhythms are daily oscillations of behavior and physiology that regulate many important aspects of human health, including blood pressure, drug metabolism, and sleep / wake cycles, as well as influencing susceptibility to cancer, heart disease, and some mental illnesses. The circadian clock regulates these processes by driving rhythmic expression of genes throughout the body; however, the mechanisms which translate circadian rhythms into normal or pathological states are largely unknown. Genes which mediate circadian physiologies, especially in the nervous system are often cell type specific, which necessitates experimental approaches that focus on functionally and anatomically distinct subpopulations of cells. Because of the significant similarity between the human and fly circadian system, as well as the ease of experimental manipulations in the fly, this project proposes developing RNA sequencing technology in the fly to identify rhythmic gene expression in purified circadian clock neurons. The long-term objective is to develop and exploit an experimental system in which the transcriptional regulation underlying circadian rhythms can be directly assessed in purified cell populations from complex tissues, with the ultimate goal of deepening our understanding of how circadian rhythms contribute to disease susceptibility. To this end, transgenic technologies will be employed to label and purify distinct subpopulations of cells in the fly central nervous system. Gene expression will be directly measured using RNA sequencing, and established statistical methods will be employed to identify circadianly-regulated genes. Three specific aims are proposed to achieve these goals: (1) Profile the circadian transcriptome in the fly brain using RNA sequencing. (2) Identify transcriptional differences between purified neurons and glia (3) Identify transcripts regulated by Pigment Dispersing Factor (PDF) signaling. The first aim will develop the experimental and analytical tools necessary for carrying out RNA sequencing- based experiments in the fly brain, while revealing genes regulated by the circadian clock in brain tissue. The second aim will pioneer the purification and expression profiling of different cell types and provide the first systematic analysis of gene expression in fly neurons versus glia. The third aim will identify genes regulated by PDF, a neuropeptide essential for normal circadian rhythms, specifically in a subset of circadian clock neurons. These aims are conceptually independent, while working together to meet increasingly difficult technical challenges. Successful completion of these aims will provide a highly detailed analysis of the transcriptional regulation underlying circadian rhythms, neuron versus glia differentiation, as well as PDF signaling in isolated circadian neurons. Moreover, these studies will pioneer an approach to isolating and sequencing the transcriptome of distinct subpopulations of neurons. PUBLIC HEALTH RELEVANCE: Circadian rhythms regulate many aspects of human health, including susceptibility to cancer, heart attacks, and mental illness. These rhythms are established by the rhythmic expression of genes in many different tissues in the body; however, the identify of these cycling genes as well as the mechanisms by which they influence human health are often unknown. To bridge this gap, this project will develop technologies to identify and characterize rhythmically-expressed genes in purified subpopulations of neurons.

描述（由申请人提供）：昼夜节律是行为和生理学的每日振荡，可以调节人类健康的许多重要方面，包括血压，药物代谢和睡眠 /唤醒周期，以及影响癌症，心脏病和一些精神疾病的易感性。昼夜节律时钟通过在整个体内推动基因的节奏表达来调节这些过程。但是，将昼夜节律转化为正常状态或病理状态的机制在很大程度上未知。介导昼夜节律生理的基因，尤其是在神经系统中通常是细胞类型的特异性，这需要实验方法，这些方法专注于功能和解剖上不同细胞的亚群。由于人类和飞行昼夜节律系统之间的显着相似性，以及飞行中的实验操作的易于性，该项目提出了飞行中的RNA测序技术，以识别纯化的昼夜节律钟表神经元中的节奏基因表达。长期目标是开发和利用实验系统，在这种实验系统中，可以直接在纯化的细胞种群中直接评估从复杂组织的纯化细胞种群中评估的转录调节，其最终目标是加深我们对昼夜节律如何有助于疾病易感性的理解。为此，将采用转基因技术来标记和净化中枢神经系统中细胞的不同亚群。将使用RNA测序直接测量基因表达，并将采用已建立的统计方法来鉴定圆周调节的基因。提出了三个特定的目的来实现这些目标：（1）使用RNA测序介绍了蝇脑中的昼夜节律转录组。（2）确定纯化神经元和神经胶质之间的转录差异（3）识别由色素分散因子（PDF）信号传导调节的转录本。第一个目的将开发用于在蝇脑中进行基于RNA测序实验的实验和分析工具，同时揭示由大脑组织中昼夜节律调节的基因。第二个目的将开拓不同细胞类型的纯化和表达分析，并对蝇神经元与神经胶质的基因表达进行首次系统分析。第三个目标将鉴定由PDF调节的基因，PDF是正常昼夜节律必不可少的神经肽，特别是在昼夜节律神经元的一部分中。这些目标在概念上是独立的，同时共同应对越来越困难的技术挑战。这些目标的成功完成将对昼夜节律，神经元与神经胶质分化以及孤立的昼夜节律神经元中的PDF信号传导提供高度详细的分析。此外，这些研究将开创一种分离和测序神经元亚群的转录组的方法。公共卫生相关性：昼夜节律调节人类健康的许多方面，包括对癌症，心脏病发作和精神疾病的敏感性。这些节奏是通过体内许多不同组织中基因的节奏表达来确定的。但是，这些自行车基因的识别以及影响人类健康的机制通常是未知的。为了弥合这一差距，该项目将开发技术，以识别和表征神经元纯化亚群中的节奏表达基因。