Postnatal lung growth during alveolarization markedly increases gas exchange surface area. Rapid growth of the pulmonary vasculature during early alveolarization drives distal lung growth. As alveolarization slows, the vasculature transitions from a phase of angiogenic growth to quiescence, however the molecular mechanisms regulating this transition remain poorly defined. This gap in knowledge confounds efforts to develop targeted therapies to treat diseases of dysregulated angiogenesis and impaired alveolarization, including bronchopulmonary dysplasia, the most common complication of preterm birth. We recently employed single cell transcriptomics to define endothelial cell (EC) diversity during postnatal lung development and to identify novel mechanisms regulating pulmonary angiogenesis and quiescence. Our preliminary data identified a tremendous increase in EC diversity after birth, marked by the appearance of numerous transcriptionally distinct clusters. A highly proliferative EC cluster is abundant before birth, virtually disappears just after birth, but peaks again at early alveolarization, a time of exponential pulmonary angiogenesis. The microvascular EC (MEC) broadly separated into Car4 expressing (Car4+) and Car4- MEC. In contrast with gradual changes in gene expression in the Car4+ MEC over time, gene expression changed dramatically in the Car4- MEC, with separation of this population into two transcriptionally distinct clusters of “early” (P1-P7) and “late” (P21) Car4- MEC. High expression of the paternally imprinted gene-3 (Peg3), a gene expressed by self-renewing progenitor cells, distinguished the “early” from the “late” Car4- MEC. Peg3 also enhances NFkB signaling, a pathway we previously identified as essential for pulmonary angiogenesis during early alveolarization. Of note, the expression of receptor-ligand pairs suggested that cross-talk stemming from the Car4+ MEC may promote pro-proliferative and pro-angiogenic signaling in the Car4- MEC. Taken together, our data suggest the overall hypothesis that the early Car4- MEC represent a specialized, highly proliferative and angiogenic EC population required for the rapid growth of the pulmonary vasculature during early alveolarization, which will be tested through three specific aims. Aim 1 will utilize transgenic and cell-specific knock out mice, advanced imaging, and loss of function studies in primary EC to probe the role of Peg3 in promoting proliferation, angiogenesis and NFkB activation in Car4- MEC. Aim 2 will use FACS sorted Car4+ and Car4- MEC and a novel mouse model permitting targeting of Car4+ MEC to test if interaction between these two distinct MEC promotes postnatal angiogenesis. Finally, Aim 3 will employ computational ligand-receptor analysis, ATAC-Seq, and EC-specific knock out mice to determine if chronic hyperoxia impairs angiogenesis by impairing Car4- MEC proliferation, Peg3-mediated self-renewal and Car4+ and Car4- MEC cross-talk. The successful completion of ...