Alveolar epithelial progenitor cells drive lung regeneration via dynamic changes in chromatin topology modulated by lineage-specific Nkx2-1 activity

Lung epithelial regeneration after acute injury requires coordination of extensive cellular and molecular processes controlling proliferation and differentiation of specialized alveolar cells to pattern the morphologically complex alveolar gas exchange surface. During regeneration, specialized Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells, though the precise molecular and epigenetic determinants of these processes remain unclear. Here, we report a refined primary murine alveolar organoid assay which recapitulates critical aspects of in vivo regeneration, providing a tractable model to dissect these regenerative processes. Clonal expansion of single AEPs generate complex alveolar organoids with extensive structural maturation and organization. These organoids contain properly patterned AT1 and AT2 cells surrounding numerous alveolar-like cavities with minimal structural contribution from mesenchymal cells, implying extensive cell autonomous regenerative function encoded in adult AEPs. Leveraging a time series of paired scRNAseq and scATACseq, we identified the AEP state at single cell resolution and described two distinct AEP to AT1 intermediate states: a widely reported Krt8+ transitional state defined by cell stress markers and a second state defined by differential activation of signaling pathways mediating AT1 cell differentiation. Transcriptional regulatory network (TRN) analysis demonstrated that these AT1 transition states were driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8+ state characterized by disorganized, uncontrolled growth. Finally, AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of AEP state, clonal expansion, and disorganization of alveolar structure, implying a continuous requirement for Nkx2-1 in maintenance and function of adult lung progenitors. Together, these data provide new insight into cellular hierarchies in lung regeneration and implicate dynamic epigenetic maintenance via lineage transcription factors as central to control of facultative progenitor activity in AEPs. ### Competing Interest Statement The authors have declared no competing interest.