PROJECT SUMMARY The alveolar epithelium plays a critical role in maintaining normal lung function and is comprised of type 1 and type 2 alveolar epithelial cells (AEC1s and AEC2s, respectively). AEC1s provide broad coverage of the alveolar surface and serve as the interface for gas exchange. AEC2s produce pulmonary surfactant to regulate alveolar surface tension and, importantly, also function as progenitor cells that can differentiate into AEC1s after lung injury. Defects in AEC1 regeneration underlie severe pulmonary diseases such as idiopathic pulmonary fibrosis (IPF), a progressive scarring disease of the lungs that results in gradual deterioration of lung function leading to respiratory failure and death within 3-5 years. The lack of curative therapies stems from disease pathogenesis remaining poorly understood. Our laboratory recently demonstrated that the specific defect in epithelial regeneration that drives the pathogenesis of fibrosis is impaired AEC2 to AEC1 differentiation. However, there remains a critical gap in knowledge of the cellular instructions that promote AEC1 differentiation after alveolar damage, although it is known that downregulation of the transcription factors ETV5 and CTNNB1 promote differentiation. To identify signaling pathways that may regulate AEC2 to AEC1 differentiation, we performed scRNA-Seq on lineage-labeled AEC2s during regeneration after alveolar injury and observed that the Notch2 receptor and canonical Notch target gene, Hes1, were highly upregulated in AEC2s differentiating into AEC1s. scRNA-Seq datasets from normal human lungs similarly revealed high expression of NOTCH2 and HES4 (a paralog of HES1 not found in mice) in AEC1s. These preliminary findings led us to speculate that activation of the canonical Notch signaling pathway is required for AEC2 to AEC1 differentiation. This proposal will test our central hypothesis that NOTCH2 activation promotes AEC2 to AEC1 differentiation via HES-mediated repression of ETV5 and CTNNB1 expression via the following two aims: (1) Test the hypothesis that Notch2 signaling is required for AEC2 to AEC1 differentiation after alveolar injury and (2) Test the hypothesis that HES4 promotes human AEC2 to AEC1 differentiation by repressing ETV5 and CTNNB1 expression. We will utilize conditional knockout mouse models in addition to primary human and human iPSC-derived organoid systems to explore the dynamics of Notch/HES signaling in AECs. The results of these studies will advance our current understanding of how Notch/HES regulates differentiation in AECs and, in doing so, establish a foundation for investigating how the Notch pathway can be leveraged to promote restoration of normal alveolar structure and function after lung injury. By defining key pathways involved in alveolar regeneration, this proposal may inform the development of targeted therapies for IPF and other pulmonary diseases characterized by ineffectual restoration of AEC1s.