PROJECT SUMMARY The blood-brain barrier (BBB) plays a crucial role in brain health, but its role in brain development and the cellular interactions which govern its formation remain unknown. Recent studies have revealed specific time points during which microglia-like progenitors exit the prenatal liver and migrate into the brain, suggesting a complex development and function of the BBB throughout gestation. Furthermore, once in the brain, these progenitors differentiate into a population of cells similar to microglia, which are traditionally thought to arise from progenitors that exit the yolk sac and migrate directly into the brain. Understanding the developmental changes in BBB permeability is essential to unraveling the mechanisms by which it regulates cellular trafficking into the brain. Dr. MacKenzie's lab published these findings and is currently investigating the migration of these prenatal liver-derived (PL-derived) immune cells into the brain using an innovative in utero prenatal liver injection model. Additionally, Dr. Crouch's lab has developed a novel strategy to isolate endothelial and mural cells, key vascular cell types of the developing brain vasculature, for in vitro study and single-cell RNA sequencing analysis. With these techniques, this proposal aims to elucidate the cellular and molecular development of the BBB and its impact on cellular trafficking into the brain. Our overall hypothesis is that immature venous-like vasculature mediates the entry of PL-derived immune cells into the brain due to its heightened BBB permeability during development. To address this hypothesis, we will first characterize the spatiotemporal development of the BBB, identifying cell types and anatomical regions associated with increased vascular permeability. Second, we will trace the entry of PL-derived immune cells into the brain and track changes in their cell identity over time. The outcomes of this research will advance our understanding of brain vascular development and potentially inform the development of therapeutics targeting the unique selective permeability of the prenatal brain vasculature.