Understanding the cellular and molecular dynamics of lung progenitors is necessary to achieve the ultimate therapeutic goal of tissue regeneration in the context of debilitating lung diseases. Alveolar type 2 cells (AT2s) of the distal lung epithelium are considered the predominant facultative progenitors of adult murine lung alveoli, the tissues responsible for gas exchange in mammals. While normally quiescent at homeostasis, AT2s can enter cell cycle and transdifferentiate into alveolar type 1 cells (AT1s) during lung repair. The kinetics of AT2 transdifferentiation into AT1s following mild insults, where alveolar architectural integrity is preserved and lung function is restored, is not completely understood. Additionally, the transcriptomic programs across all cell lineages that might regulate this process largely remain unknown. To identify putative molecular pathways activated in the epithelium and its overall niche, cell-type specific responses were characterized in a mouse model during adult lung compensatory growth following the mild injury of unilateral pneumonectomy (PNX). Histologic analyses showed proliferation of AT2s as a primary response, followed by differentiation into AT1s that peaked around day 10-12 post-PNX. Time-series single-cell RNA sequencing profiles revealed that AT2s express transitional markers such as Krt8 and Areg, consistent with previous studies. Trajectory analysis using a Continuous State Hidden Markov Model (CSHMM) predicted Hippo pathway effector Tead1 and Notch signaling target Hes1 as active transcription factors associated with the transdifferentiation of AT2s to AT1s. To verify involvement of Notch post PNX, AT2s were assessed for Notch activation using a genetic reporter, which showed an increase in the percentage of Notch-responsive AT2s post-PNX. To test potential function of Notch in AT2 transdifferentiation into AT1s, Notch signaling was inhibited in vitro in a mouse cell co-culture model which showed loss of AT2 transdifferentiation into AT1s. Single-cell RNA sequencing profiles post-PNX revealed an activated lung mesenchymal subset unique to injury that was characterized by high expression of extracellular matrix-related (ECM) genes along with low expression of Acta2 and Pdgfra. Interestingly, this mesenchymal subset included Cthrc1+ fibroblasts that expressed the highest levels of ECM genes and transcripts associated with BMP and TGFβ signaling pathways, including Fstl1, Grem1, and Tgfβ3, factors previously linked to epithelial differentiation. Immunostaining combined with in situ hybridization assays identified Cthrc1+ fibroblasts adjacent to Krt8+ transitional epithelial cells. Genetic lineage tracing of Cthrc1+ cells post-PNX revealed the persistence of their descendants after completion of compensatory lung regrowth. In summary, analysis of AT2 global transcriptomes and differentiation kinetics post-PNX revealed activated/transitional states, nascent AT1 cells, and the emergence of Cthrc1+ mesenchymal subpopulations. These results suggest that AT2s are one of the early responders to PNX stimuli and identified mesenchymal cells states that may assist in overall compensatory lung regrowth post-PNX. / 2025-09-17T00:00:00Z