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.