2025 Theses Doctoral

Transitional cell states in lung development and injury-repair

Ke, Xiangyi

Cells in transitional states are found in multiple biological systems and sit at the crossroadof important events, such as development and regeneration. These cells are enigmatic due to their mixed identities, transient gene expression signature, and harboring key regulators of cell-fate switch. Despite their importance, it is challenging to understand the regulation of these transitional states in developmental systems, where dynamic tissue interactions lead to morphogenetic changes that rapidly generate new compartments and boundaries.

One paradigm for the study of these states is the developing murine lung. During lung morphogenesis, distal epithelial progenitors in growing buds undergo dramatic changes in fate and cytoarchitecture as they transition into airway progenitors. Lineage studies in mice show that distal epithelial cells identified by expression of Id2 or Sox9 expand to form the future alveolar compartment but also give rise to the Sox2 epithelial tubules that form the bronchial tree. Formation of these distinct and well-balanced compartments labeled by Sox9 or Sox2 is a key event during formation of the bronchial tree overall preceding the appearance of defined differentiated epithelial cell phenotypes.

Cells undergoing transition are at the most plastic state as they are being subjected to signals that allow them to progressively initiate a distinct program while repressing their initial program. A variety of studies have investigated the overall gene expression signatures associated with the distal and proximal identity of these compartments as they expand. But it remains unclear what characterizes the epithelial cells that lie in the transition between Sox9-Sox2 fates, their regulation, and impact in the developing lung. Although studies in mice have identified signals such as the Hippo-Yap as regulators of this transition, the identity of these cells and their phenotypic features and behavior remain elusive.

We have developed a system to identify and characterize cells undergoing transition from the Sox9 to the Sox2 compartments in the developing murine lung epithelium. Using an unbiased single cell genomic approach and bioinformatic analysis, we identified markers to isolate the cells during the period when branching morphogenesis is most active and most representative of the transitional cell state. The analyses revealed a previously uncharacterized population of NKX2- 1/ICAM1 double-positive epithelial progenitors with an expression signature distinct from all others in the lung. Surprisingly, their signature showed extensive overlap with that of transitional cells reported in the adult murine alveolar epithelium undergoing repair after severe injury. Analysis of human fetal lungs showed that this signature was largely conserved, labeling transitional cells at the sites of segregation of Sox9 and Sox2 fates in the developing distal epithelium.

Functional analysis in lung explant cultures and organoids showed that these cells are remarkably plastic and respond to morphogenetic cues by changing their domain and abundance, reflecting dynamic changes in proximal-distal cell fate. Progression of NKX2-1/ICAM1 throughout these transitional states was regulated by balanced activation of signals such as Fgf, Tgfβ, Hippo-Yap, p53, and AP1. Moreover, analysis of additional injury models in the adult lung identified cells undergoing a similar transitional cell state during repopulation of the airway epithelium after severe injury. These observations broaden the understanding of these transitional states and suggest they may be relevant in the development of regenerative strategies for managing pulmonary conditions.