PROJECT SUMMARY The adult human brain is comprised of numerous cell types exhibiting specific transcriptomic and epigenomic signatures associated with their spatial location, connectivity, and function. Although systematic efforts are underway to characterize cell types in the adult human brain, the transitional cell types and cell states in developing human brains are not fully defined. Our project will use epigenomic and three-dimensional (3D) chromosomal architectural information to classify developmental cell types and identify key regulatory dynamics that may underlie cellular lineage commitment and maturation. Single-cell DNA methylation and chromatin accessibility profiles have been successfully used to de novo identify distinct cell types in developing and adult brains. The single-nucleus joint profiling of DNA methylation and chromatin conformation by sn-m3C-seq provides unique information to identify enhancer-gene looping and provides a resource to link genetic variants associated with neuropsychiatric disorders to genes. Cell types identified with epigenomic information will be integrated with spatial transcriptomic signatures using our innovative single-cell joint profiling method of transcriptome and DNA methylation snmCT-seq. We will determine the spatial and temporal dynamics of developing brain cell types using multiplexed RNA in situ and Digital Spatial Profiling (DSP) assays. We will apply state-of-the-art approaches to integrate multiple data modalities to identify progenitor and transitional cell types, lineage-specific regulatory elements, and enhancer-gene 3D interactions. We will construct cell-type- specific predictive models of cellular trajectory and gene regulation during brain development by integrating transcription factor-gene interaction information with time- and pseudotime-series genomics data. The cell type and regulatory elements catalog of human brain development generated by our project will aid the identification of developing brain cell types involved in neuropsychiatric disorders and regulatory regions susceptible to perturbation by disease-associated variants.