PROJECT SUMMARY Neonates born during the saccular stage of lung development (23-32 wks gestation) are at highest risk for bronchopulmonary dysplasia (BPD), a leading preterm birth complication. The mechanisms underlying this vulnerability are poorly defined, a knowledge gap we consider foundational to the lack of curative BPD therapies. To understand the irreversibility of arrested alveologenesis in BPD, we require a refined, mechanistic understanding of the normal saccular to alveolar transition. Our preliminary data from 4- dimensional live imaging and single-cell transcriptomics support a new model of alveologenesis in which myofibroblast ring structures support the extrusion of AT2s (alveolar type 2 cells) followed by their differentiation into AT1s (alveolar type 1 cells). According to our model, mature AT1s produce ECM proteins and other factors that recruit specialized endothelial cells to become the alveolar capillary bed. Sequential, spatiotemporally restricted signaling pathways, including Wnt and BMP, coordinate cell movement, proliferation, and architecture. We have developed a neonatal injury model with a phenotype of impaired alveologenesis that is relevant to human BPD by exposing neonatal mice to hyperoxia and inflammation during the saccular stage. Our preliminary data from this model associate overexpression of Wnt5A/Wnt11 with impaired alveologenesis. Post-injury deficits include decreased BMP production and activity in alveolar epithelial cells and impaired AT2 to AT1 cell differentiation and decreased expression of extracellular matrix (ECM) components by AT1 cells. Based on preliminary and published data, we hypothesize that alveologenesis involves formation of a ring of myofibroblasts that express Wnt5a and Wnt 11 to drive AT2 proliferation and promote extrusion through the ring. Subsequent epithelial BMP production down-regulates Wnt, promoting AT2 to AT1 differentiation and generation of an extracellular scaffold for capillary assembly. Injury dysregulates Wnt and Bmp signaling, perturbing the precise spatiotemporal patterning during this critical timeframe and resulting in arrested alveologenesis and long-term functional deficits. We will test this hypothesis in the following specific aims: 1) Define the role of myofibroblast Wnt expression in regulating AT2 proliferation and alveolar development; 2) Characterize mechanisms by which BMP signaling regulates AT2-to-AT1 cell differentiation; 3) Determine the mechanisms whereby nascent AT1 cells generate a scaffold for the developing alveolus. Successful completion of this proposal is anticipated to transform our understanding of alveologenesis, identifying new molecular targets to promote post-injury alveolar restoration and the optimal time windows for deployment of such newly directed therapies.