7. PROJECT SUMMARY AND ABSTRACT Astrocytes comprise a large and heterogeneous population of cells in the mammalian central nervous system (CNS) whose diverse roles in brain function have gained increasing recognition. Astrocytes facilitate synapse formation and maturation, maintain ion homeostasis, perform critical clearance of neurotransmitters in the synapse, and mediate hemodynamic control between active neurons and local cerebral blood flow. Nevertheless, large gaps remain in our fundamental understanding of astrocyte biology that preclude a deeper understanding of how these cells participate in and contribute to neural circuit function and dysfunction. Whereas the molecular and functional diversity of distinct neuronal populations has long been recognized, such frameworks remain poorly defined for astrocytes. Our previous study identified discrete subpopulations of protoplasmic astrocytes in the adult forebrain expressing the transcription factor, Gli1, indicating active Sonic hedgehog signaling. This suggests that Gli1 astrocytes possess unique gene expression programs that distinguish them from Gli1 negative astrocytes. In this study, we will use a combinatorial approach that incorporates the covalent tagging of nascent RNA in vivo, with next generation sequencing and bioinformatics analysis to perform transcriptional profiling of Gli1 astrocytes. Specifically, this project focuses on identifying differential gene expression programs 1) between Gli1 positive and negative astrocyte populations, and 2) between Gli1 astrocyte populations localized in different regions throughout the forebrain. This discovery-based approach will enable us to identify gene expression programs that are specific to molecularly and regionally defined subpopulations of astrocytes, providing unprecedented insight into the nature of astrocyte diversity in vivo. The results from this study will generate a transcriptional database that will be an important resource for designing future experiments aimed at elucidating the role of Shh signaling in astrocytes, and understanding the precise role of Gli1 astrocytes in CNS function. Understanding the molecular and functional heterogeneity of astrocytes is a critical step towards a deeper understanding of brain function and dysfunction.