PROJECT ABSTRACT/SUMMARY Brain blood vessel cells play key roles in both physiological and pathological states. Endothelial and mural cells compose the main structural and functional elements of the vasculature. Situated between the endothelial cell lumen and the surrounding astrocytes, microglia, and neurons, mural cells serve as a critical signaling hub of the neurovascular unit. Mural cells are required for the development of the blood brain barrier and regulate blood flow in response to neuronal activity. However, most studies of brain blood vessels use adult mice and therefore little is known about the developing vasculature or human brain blood vessels at any age. These knowledge gaps impact patients. During development, both genetic and sporadic disorders of the vasculature can have a severe impact on long-term neurological function, and we currently have no treatments. To molecularly define the stages and subtypes of mural cells in the developing brain, we have developed an integrated approach to enrich for vascular mural cells, profile them, and functionally characterize their effects. In the prenatal human brain, Fluorescence Activated Cell Sorting (FACS) followed by single cell RNA sequencing (scRNAseq) identified four different types of mural cells during the second trimester: mitotic, classic pericyte, fibroblast, and smooth muscle cells. Trajectory analysis of our transcriptomic data showed smooth muscle cells as potential progenitors that give rise to classic pericytes and fibroblasts. The four mural cell subtypes had recently been reported in the adult human brain, and the presence of their full repertoire at such a young age suggests a highly dynamic and time-consuming maturation process for these cells. In the embryonic mouse brain, previous studies had not been able to define subtypes of mural cells. Our application of FACS followed by scRNAseq at embryonic day 13.5 uncovered the same subtypes as the prenatal human brain. In sum, the above experiments lead me to hypothesize that smooth muscle cells are evolutionarily conserved mural stem cells in the developing brain which give rise to classic pericytes, the differentiated effector cells of the blood brain barrier. My short-term goals for the proposed DP2 are the following: 1. Construct a spatiotemporal atlas of brain mural cells during development using scRNAseq and RNA and protein detection in tissue. 2. Perform lineage tracing with mouse and human cells to interrogate stage-specific roles in blood brain barrier development. As a neuroscientist, vascular biologist, and neonatologist, I am uniquely suited to pioneer our understanding of brain vascular development and the application of this knowledge towards future therapy.