PROJECT SUMMARY RNA regulatory networks in neuronal cell type diversity and function The mammalian brain is probably the most complex organ in the body and its proper function requires coordination of many diverse types of excitatory and inhibitory neurons that form distinct functional circuitries. Characterization of neuronal cell types is fundamental to understand not only how the brain works, but also how specific cell types are selectively affected in multiple neuronal disorders and how this process can be reversed. A large number of neuronal cell types were recently defined based on their gene expression profiles in bulk and single cells, and these cell types are organized into a hierarchical cell taxonomy. Alternative splicing is a mechanism to generate multiple transcript and protein variants with distinct functions, thus providing a major driving force of the molecular diversity in mammals. The overarching goal of my research group is to understand the contribution of alternative splicing and the underlying RNA-regulatory networks in the brain and brain-related disorders. Previous work from multiple groups, including ours, unambiguously demonstrated that the brain has unique splicing-regulatory programs compared to non-neuronal tissues. Our studies also revealed the establishment of a pan-neuronal splicing program regulated by multiple RNA-binding proteins (RBPs) during neural development, and demonstrated the important role of the Rbfox protein family in regulating axonal maturation and neuronal excitability. However, how alternative splicing contributes to the distinct molecular profiles of diverse neuronal cell types in the cortex is currently poorly understood. We hypothesize that the transcriptome diversity generated by highly regulated alternative exons is a major component that specifies neuronal cell type identity and function. In this application, we describe our preliminary analysis of neuronal cell type-specific alternative splicing regulation in mouse cortex, which provides strong support for the following specific aims we would like to pursue: 1) Perform systematic analysis of neuronal cell type-specific alternative splicing to identify novel neuronal subclasses and regulators using deep sc-RNA-seq data. 2) Validate our computational predictions and characterize mechanisms of neuronal cell type-specific splicing regulation and function using two complementary model systems: the distinction of two major subclasses of GABAergic interneurons originating from caudal (CGE) and medial (MGE) ganglionic eminences, and a GABAergic neuron- specific microexon in Ank3/Ankyrin G. To achieve our goal, we will use a multidisciplinary approach that integrates cutting-edge statistical an machine learning methods and multiple in vitro and in vivo experimental models. This study will generate a global and mechanistic view of precise alternative splicing regulation across diverse neuronal cell types and illuminate its impact on neuronal structur...