Abstract Unlike many mammalian vital organs, the lung exhibits a robust regenerative response to severe injuries such as influenza infection, which primarily targets epithelial cells airways and alveoli. Quiescent lung-resident epithelial progenitors enter the cell cycle, proliferate, and differentiate following lung injury, participating in two distinct regenerative pathways: functionally beneficial regeneration and maladaptive tissue remodeling. Intralobular airway-resident distal p63+ progenitors are one such progenitor cell type that migrates into the alveoli, adheres to the denuded alveolar basement membrane, and rapidly proliferates to generate ectopic bronchiolar- like tissue, forming honeycomb-like cysts that fail to resolve after injury and that do not participate in gas exchange. Though ultimately a maladaptive injury response, this ectopic bronchiolization does appear to benefit individuals with severe alveolar injuries by providing an “emergency” epithelial barrier. Distal p63+ progenitors are the only cells in the distal lung that express the master epithelial regulator Trp63, specifically the ΔN isoform (ΔNp63). ΔNp63 is highly active in the proliferative basal stem cells of other epithelial tissues such as the skin, mammary, prostate, and trachea, in which it confers basal cells with their stem-like identity and transcriptionally regulates the cellular processes of migration, adhesion, and proliferation. In my own preliminary data, I have found that influenza-injured mice with broad ΔNp63 deletion in the airway epithelium display a completely abrogated maladaptive alveolar remodeling response. Besides this data, there have been no studies directly investigating the role of ΔNp63 in maladaptive remodeling and the mechanisms by which it promotes this regenerative pathway. Aim 1 of this proposal will utilize conditional deletion of ΔNp63 in and lineage-tracing of distal p63+ progenitors to investigate if loss of ΔNp63 causes a cell identity change in distal p63+ progenitors following pulmonary injury. Intracellular flow cytometry, immunohistochemistry, and qPCR will be used to assess the fate decisions of ΔNp63-/- distal p63+ progenitors upon deletion both prior to and following influenza injury; pulmonary function tests will additionally be used to evaluate the physiological consequences of ΔNp63 knockout. Aim 2 will employ CRISPRa-mediated overexpression of p63 in tandem with in vitro and ex vivo migration assays to determine if cell motility is affected by ΔNp63 overexpression in distal p63+ progenitors as it is in other ΔNp63-expressing cell and tissue types. Finally, upon confirmation/identification of known and/or previously unidentified ΔNp63 migration targets in distal p63+ progenitors, CRISPR-mediated knockout of these targets followed by in vitro and ex vivo migration assays will be utilized to evaluate the importance of the ΔNp63- driven migration program in injury-activated distal p63+ progenitor motility. These experiment...