Epithelial-mesenchymal cell competition coordinates fate transitions across tissue compartments during lung development and fibrosis

Klinkhammer, Kylie; Warren, Rachel; Knopp, Joseph; Nguyen, Toan; De Langhe, Stijn P.

doi:10.1038/s41467-025-66690-z

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Published: 21 November 2025

Epithelial-mesenchymal cell competition coordinates fate transitions across tissue compartments during lung development and fibrosis

Nature Communications volume 16, Article number: 10956 (2025) Cite this article

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Abstract

The coordination between epithelial progenitors and their mesenchymal niche is critical for organogenesis and repair, yet the mechanisms governing their competitive interactions remain unclear. Here, we reveal a paradigm of tissue-scale fitness sensing in the lung, where mesenchymal Yap levels antagonize epithelial Yap levels to dictate epithelial stem cell fate. We show that reduced fitness in alveolar fibroblasts (AF1s) via Yap/Taz or Myc deletion leads to their apoptotic elimination and a collapse of the alveolar stem cell niche. This niche collapse triggers a pathological competitive response from the epithelium, which undergoes aberrant bronchiolization that phenocopies human pulmonary fibrosis. Mechanistically, we uncovered a molecular switch that controls mesenchymal fate. During development and fibrosis resolution, Snail1/2 sequesters Yap/Taz to drive an adipogenic program, generating niche-supportive AF1s. Conversely, Yap/Taz-TEAD-Myc binding instructs a myogenic, pro-fibrotic program. Our findings demonstrate that inter-tissue cell competition, governed by a Snail/Yap rheostat, orchestrates lung architecture and provides a framework for targeting the mesenchymal niche to treat fibrotic disease.

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Introduction

The human lung’s intricate structure optimizes gas exchange through its extensive branching network of airways, culminating in 700 million alveoli¹. Spanning an impressive 70 square meters, this network forms during the branching morphogenesis phase of development, where mesenchymal cells encircle the epithelial monolayer². This crosstalk between epithelial and mesenchymal cells drives branching and differentiation processes. Interactions between these cell populations, along with their matrix, regulate the branching program and transition to the alveolar differentiation phase^3,4,5. This epithelial-mesenchymal crosstalk remains essential during adult homeostasis and repair following injury^2,6,7.

Early tissue recombination studies revealed that the mesenchyme plays a crucial role in instructing the epithelium, imparting its regional identity⁸. This instructive function becomes particularly evident in the injured adult lung. Following injury, developmental programs are reactivated to repair tissue damage and restore the organ to homeostasis. This paradigm applies to the lung, involving various signaling pathways such as Fgf, Hippo, and Wnt, which are either upregulated or downregulated in response to lung injury^9,10,11. These signals form the foundation for communication and crosstalk between different cell types, both within the epithelium and between epithelial and mesenchymal cells.

In the adult lung, cartilage, airway smooth muscle, alveolar fibroblast 1 (AF1), and pericytes establish various mesenchymal stem cell niches. These niches are essential for maintaining and/or activating adult epithelial stem cells during homeostasis or initiating repair after injury^{6,9,10,12,13,14}. Any disruptions in these interactions during lung development, homeostasis, or repair after injury can lead to the development of pulmonary diseases such as bronchopulmonary dysplasia (BPD), emphysema, and pulmonary fibrosis.

Fibrotic diseases, a major contributor to global morbidity and mortality, remain poorly understood. These diseases can affect various organs, ultimately leading to organ failure¹⁵. Fibrosis is believed to account for up to 45% of global deaths, with chronic lower respiratory fibrosis being a leading cause of death in the United States^15,16. Idiopathic pulmonary fibrosis (IPF), a prevalent form of interstitial lung disease (ILD), results in alveolar remodeling and progressive loss of pulmonary function, respiratory failure, and death within five years of diagnosis^17,18. IPF pathogenesis involves fibrotic remodeling, inflammation, and the loss of lung architecture¹⁹. Despite the underlying causes being elusive, genetic and experimental evidence supports the notion that chronic alveolar epithelial injury and inadequate repair of the respiratory epithelium are intrinsic to IPF disease pathogenesis. Histologically, respiratory epithelial cells in the lung parenchyma express atypical proximal airway epithelial and indeterminate cell type markers^20,21, including goblet and basal cell (BC) markers, which are typically restricted to conducting airways. Fibrotic lesions and honeycomb structures replace alveoli, which are normally lined by alveolar type 1 (AT1) and AT2 cells. The bronchiolization process that leads to honeycomb structures is driven by intra-epithelial cell competition, where distal airway stem cells with high Yap/Myc levels become supercompetitors and replace the alveolar epithelium with bronchial epithelium²². Some abnormal airway/alveolar epithelial cells attempting to aid in regeneration persist in Krt8+ transitional states, which express genes associated with pro-fibrotic phenotypes^23,24,25,26.

In this work, we reveal a paradigm of tissue-scale fitness sensing in the lung, where mesenchymal Yap levels antagonize epithelial Yap levels to dictate epithelial stem cell fate. Mechanistically, we uncovered a molecular switch that controls mesenchymal fate. During development and fibrosis resolution, Snail1/2 sequesters Yap/Taz to drive an adipogenic program, generating niche-supportive AF1s. Conversely, Yap/Taz-TEAD-Myc binding instructs a myogenic, pro-fibrotic program. Our findings demonstrate that inter-tissue cell competition orchestrates lung architecture and provides a framework for targeting the mesenchymal niche to treat fibrotic disease.

Results

Yap/Taz bind to Snail1/2 to drive alveolar fibroblast 1 differentiation during lung development

The pulmonary mesenchyme includes multiple distinct cell lineages with various functions in lung development and in the pathogenesis and progression of debilitating respiratory conditions like idiopathic pulmonary fibrosis (IPF)^27,28. Lung mesenchymal lineages are increasingly recognized as highly heterogeneous, including alveolar fibroblast 1 (AF1), adventitial fibroblasts or alveolar fibroblasts 2 (AF2s), peribronchial fibroblasts, airway and vascular smooth muscle cells, pericytes, mesothelial cells. The AF1 lineage which forms the niche for AT2 stem cells in the adult lung arises around E16.5 during lung development. During lung fibrosis, AF1s take on multiple molecular states with distinct roles in facilitating responses to fibrotic lung injury giving rise to injury-induced fibrotic, inflammatory and stress activated fibroblasts populations which then either undergo apoptosis or revert back to AF1s during fibrosis resolution^29,30,31. Yap/Taz signaling^32,33 mediates the acquisition of some of these molecular states. However, little is still known about how these different lineages arise during lung development and are regulated during lung fibrosis and resolution³⁴.

The role of Hippo signaling during mesenchymal lineage commitment during lung development remains largely unknown. To investigate this, we genetically inactivated Yap/Taz (Yap1^f/f;Wwtr1^f/f) or the Hippo kinases Mst1/2 (Stk4^f/f;Stk3^f/f) from the undifferentiated lung mesenchyme using Tbx4^rtTa;Tet^Cre starting from E9.5. Subsequently, we performed combined single nuclear RNAseq and ATACseq on E15.5 and E18.5 lungs. For each sample, we sequenced between 5200 and 8200 cells.

To our surprise, we found that inactivating Yap1/Wwtr1 in the lung mesenchyme from E9.5 onwards impairs AF1 differentiation (marked by Scube2), which was confirmed by immunostaining for Plin2, a well-known marker for AF1s (Fig. 1A–F, Supplementary Fig. S1, Fig. 2A). Conversely, inactivating Stk3/4 in the lung mesenchyme from E9.5 onwards accelerates AF1 differentiation (Fig. 1A–F, Supplementary Fig. S1). Neither inactivation of Yap1/Wwtr1 or Stk3/4 using Tbx4^rtTa;Tet^Cre starting from E9.5 affected endothelial differentiation (Supplementary Fig. S4B), though E18.5 Tbx4^rtTa;Tet^Cre;Yap1^f/f;Wwtr1^f/f lungs featured increased subpleural vascularization (Supplementary Fig. S4A). These findings suggest that mesenchymal Yap/Taz signaling plays a pivotal role in driving AF1 lineage commitment. This finding is intriguing because upon bleomycin injury, increased mesenchymal Yap/Taz-Tead signaling is believed to promote AF1 to fibrotic fibroblast differentiation^32,33.

**Fig. 1: Single nuclei combined RNA and ATAC analysis of E15.5 and 18.5 mouse lungs.**

**Fig. 2: Snail1/2 and Yap/Taz are required for AF1 differentiation.**

We were intrigued to explore whether Yap/Taz signaling in AF1 differentiation is partially mediated by a different transcriptional cofactor besides Teads. Snail1 and Snail2, which are highly expressed in AF1s and undifferentiated distal mesenchyme (Supplementary Fig. S2A–E), have been demonstrated to bind Yap and Taz in bone marrow-derived mesenchymal stem cells (MSCs) to regulate their self-renewal and differentiation^35,36. In these MSCs, Snail1 and Snail2 form binary complexes with Yap/Taz that not only control Yap/Taz protein levels but also regulate the expression of specific Yap/Taz-target genes^35,36. To investigate a potential role for Snail1/2 in regulating mesenchymal lineage commitment during lung development, we added E15.5 and E18.5 Tbx4^rtTa;Tet^Cre;Snai1/2^f/f lung samples, induced from E9.5, to our combined single nuclear RNAseq and ATACseq analysis.

We discovered that mesenchymal Snail1/2 binds to Yap/Taz to orchestrate an adipogenic differentiation program towards AF1. This is evident from reduced chromatin accessibility of CEBP binding motifs in lung AF1s from Tbx4^rtTa;Tet^Cre;Snai1/2^f/f and Tbx4^rtTa;Tet^Cre;Yap1^f/f;Wwtr1^f/f mice (Fig. 1G, Supplementary Fig. S2F). Notably, the absence of Snail1/2 increases chromatin accessibility of Tead binding motifs, suggesting that Snail1/2 inhibits Yap/Taz-mediated Tead signaling (Fig. 1G, F, Supplementary Fig. S2F). A closer examination of Snail1/2 knockout mesenchyme compared to control mesenchyme reveals that the loss of Snail1/2 promotes mesenchymal differentiation along a myogenic differentiation program towards a myofibroblast lineage. This lineage exhibits increased chromatin accessibility of Stat1, Stat3, and Stat4^37,38,39,40 as well as myogenic regulator factors including MyoD, Myf5, and MyoG^41,42,43 binding motifs (Fig. 1G, Supplementary Fig. S2F). This suggests that the Snail/Yap rheostat governs a fundamental fate decision between adipogenic and contractile mesenchymal lineages. The specific outcome—differentiation towards myofibroblasts versus airway smooth muscle—is likely dependent on additional developmental cues and the specific mode of Yap/TEAD activation.

Spectra⁴⁴ (supervised pathway deconvolution of interpretable gene programs) analysis also demonstrates decreased activation of adipogenic or fat cell differentiation pathways and increased activation of myofibroblast differentiation pathways in AF1s upon Snai1/2 inactivation (Supplementary Fig. S3I, Supplementary Data 1). Inactivation of Snai1/2 using Tbx4^rtTa;Tet^Cre starting from E9.5 did not appear to drastically affect endothelial cell differentiation (Supplementary Fig. S4).

Mesenchymal Yap antagonizes epithelial Yap to coordinate fate transitions across tissue compartments

We discovered that accelerated AF1 differentiation in the mesenchymal Stk3/4 knockout lungs is accompanied by accelerated alveolar epithelial differentiation (Fig. 1E, Supplementary Fig S3A–H). Conversely, impaired AF1 differentiation in the mesenchymal Yap/Taz knockout lungs hinders the transition from a branching program to an alveolar differentiation program^3,4,5 (Fig. 2B, Supplementary Fig. S3A–H), which occurs around E16.5 in parallel with AF1 differentiation. Notably, increased epithelial Yap signaling during lung development also impairs this transition⁴⁵, implying that mesenchymal Yap levels may antagonize epithelial Yap levels. Indeed, we observe increased Yap levels in the epithelium of Tbx4^rtTa;Tet^Cre;Yap1^f/f;Wwtr1^f/f lungs but decreased epithelial Yap levels in lungs in which we overexpress a dominant, active version of the Hippo transcriptional effector Yap1^S112A in the lung mesenchyme (Fig. 2C). This overexpression also activates a robust myogenic differentiation program, leading to increased smooth muscle cell differentiation (Fig. 2D). Together, our findings indicate that lung mesenchymal Yap levels antagonize epithelial Yap levels during lung development to coordinate airway versus alveolar epithelial differentiation.

The transition from a branching program to an alveolar differentiation program^3,4,5 during lung development bears a striking resemblance to the binary decision made by distal airway stem cells in response to bleomycin injury. This decision involves either generating more airway epithelium, leading to bronchiolization, or alveolar epithelium. Notably, bronchiolization, a hallmark of pulmonary fibrosis, is mediated by an increase in airway epithelial Yap/Myc signaling²². Since mesenchymal Yap levels antagonize epithelial Yap levels during lung development, thereby coordinating airway versus alveolar epithelial differentiation, we hypothesized that mesenchymal Yap levels could also antagonize bronchiolization in pulmonary fibrosis. To investigate this, we induced bleomycin injury in adult lungs in which we inactivated Yap/Taz in AF1 cells using three distinct Cre lines: Plin2^CreERT2, Tbx4^rtTa;Tet^Cre, and Scube2^CreERT2, while simultaneously lineage tagging them with Rosa26-mTmG. Surprisingly, all three mouse models exhibited a striking increase in bronchiolization 6 weeks after bleomycin injury. This was characterized by an increase in aberrant basaloid (Krt8) and/or basal cells (Krt5) and increased pulmonary fibrosis based on hydroxyproline content compared to control lungs (Fig. 3A–K, Supplementary Fig. S5A–K). We found that inactivating Yap/Taz profoundly affects the fitness and survival of AF1s in the injured environment. Lineage tracing revealed a dramatic reduction in GFP-positive AF1s in Plin2^CreERT2;Yap^f/f;Wwtr^f/f;mTmG lungs post-injury (Fig. 3E), and TUNEL staining confirmed these cells were undergoing apoptosis (Fig. 3L, M). As AF1s form the essential niche for AT2 stem cells, this loss of the mesenchymal support structure was accompanied by AT2 cell loss. We propose that this collapse of the alveolar niche triggers a pathological regenerative response from distal airway stem cells, which, in the absence of proper mesenchymal cues, fill the damaged parenchyma through bronchiolization rather than functional alveolar repair (Fig. 3L, M). Furthermore, since AF1s form the niche for AT2 cells, we observed that this loss of AF1s also leads to AT2 cell death. In contrast, the opposite phenotype was observed in Plin2^CreERT2;Stk3/4^f/f;mTmG lungs, where we inactivated the Hippo kinases Mst1/2 in AF1 cells. These lungs exhibited decreased bronchiolization 6 weeks after bleomycin injury and no AF1 loss (Fig. 3D, H, J, Supplementary Fig. S5A–K). We therefore propose that bronchiolization in pulmonary fibrosis also involves an epithelial-mesenchymal cell competition based on Yap levels between distal airway stem cells and AF1/myofibroblasts. Note that in the absence of injury Plin2^CreERT2;Yap1^f/f;Wwtr1^f/f;mTmG and Plin2^CreERT2;Stk3/4^f/f;mTmG lungs appeared normal 9 weeks after tamoxifen induction (Supplementary Fig. S6).

**Fig. 3: Loss of Yap/Taz in AF1s promotes bronchiolization after bleomycin injury.**

Next, we performed single-cell RNAseq on non-injured control and bleomycin-injured control lungs, as well as bleomycin injured Plin2^CreERT2;Yap1^f/f;Wwtr1^f/f lungs. Notably, only Plin2^CreERT2;Yap1^f/f;Wwtr1^f/f lungs exhibited a prominent aberrant basaloid cell population which based on trajectory inference analysis appears to be derived from club cells (Supplementary Fig. S5A–K). This population displayed increased expression of Krt5, Krt17, Yap1, and Gdf15 (Fig. 4A,B, Supplementary Fig. S5A–K) and increased inferred Myc transcriptional activity (Fig. 4C). Spectra analysis also suggests increased Myc target gene expression in the aberrant basaloid cells that appear in Plin2^CreERT2;Yap1^f/f;Wwtr1^f/f lungs after bleomycin injury (Supplementary Fig. S7). Deeper Spectra analysis indicates that this aberrant basaloid population is highly proliferative and metabolically active, likely undergoing rapid division and growth. The combination of active pathways such as Glycolysis, Pyruvate Metabolism, Citric Acid (TCA) Cycle, Oxidative Phosphorylation, Ribosome Biogenesis, Amino Acid Metabolism, Fatty Acid and Ketone Metabolism, Nucleotide Synthesis, Myc target gene activation, mTORC1/PI3K-AKT Signaling,G1/S Transition, DNA Synthesis and Repair, G2/M Transition and the quality control p53 pathway etc., suggests a state often seen in cancer cells, activated stem cells, or rapidly dividing progenitor cells (Supplementary Fig. S7, Supplementary Data 2). A significant number of the active pathways are dedicated to energy production and the creation of molecular building blocks. Taken together, the transcriptional and pathway analyses reveal that the aberrant basaloid population has undergone a profound metabolic reprogramming. They have adopted a hyper-proliferative, cancer-like metabolic state, providing a clear molecular engine for their ‘supercompetitor’ phenotype and their ability to drive pathological tissue remodeling.

**Fig. 4: Loss of Yap/Taz in AF1s promotes the generation of aberrant basaloid cells after bleomycin injury.**

Surprisingly, at 6 weeks post bleomycin injury, we found no difference between the AF1 populations from control versus Plin2^CreERT2;Yap1^f/f;Wwtr1^f/f injured lungs. This suggests that increased pulmonary fibrosis is a result of the rise in aberrant basaloid cells. The lack of significant transcriptional differences in AF1 populations from control versus Plin2^CreERT2;Yap1^f/f;Wwtr1^f/f injured lungs at 6 weeks post injury could be because most mutant AF1s have already been outcompeted at this time point.

To compare the aberrant basaloid populations observed in Plin2^CreERT2;Yap1^f/f;Wwtr1^f/f lungs after bleomycin injury with those observed in human IPF we integrated our mouse data sets with a human data set²⁶ containing aberrant basaloid cells and found that both cell populations cluster together indicative of their similarity (Fig. 5). Together these data illustrate that this aberrant basaloid population bears striking resemblance to that observed in IPF^{22,23,24,25,26,46}.

**Fig. 5: Loss of Yap/Taz in AF1s promotes the generation of aberrant basaloid cells after bleomycin injury similar to aberrant basaloid cells in IPF.**

Alveolar fibroblast 1 fitness levels are determined by their Myc levels

Yap and Myc work together to control genes essential for cell proliferation. When Myc is activated, it binds extensively to its genomic targets, which are largely pre-associated with TEAD⁴⁷. At these sites, Yap recruitment appears to depend on Myc and is necessary for complete transcriptional activation. This partnership between Yap and Myc is considered vital for initiating the cell cycle and supporting organ growth⁴⁷. Since we have recently demonstrated that Myc and Yap cooperate in the lung epithelium to drive cell competition²² we wondered whether inactivation of Myc in AF1s prior to bleomycin injury would also render them vulnerable to becoming outcompeted by bronchial epithelium (Fig. 6A). Indeed, we observe increased bronchiolization based on Krt8 and Krt5 expression (Fig. 6B, D, E) and increased pulmonary fibrosis based on hydroxyproline content (Fig. 6C) in Plin2^CreERT2;Myc^f/f;mTmG mice 6 weeks after bleomycin injury.

**Fig. 6: Loss of Myc in AF1s promotes bronchiolization after bleomycin injury.**

Myofibroblast to alveolar fibroblast 1 dedifferentiation requires a Yap/Taz/Snail1/2 transcriptional program

Finally, we wondered if Snail1 and Snail2 are also crucial for the dedifferentiation of myofibroblasts into AF1s during the resolution of bleomycin-induced pulmonary fibrosis. To investigate this, we inactivated Snai1 and Snai2 in myofibroblasts using Acta2^CreERT2;Snai1/2^f/f,mTmG mice, starting 2 weeks after bleomycin injury and harvesting the lungs 4 weeks later (Fig. 7A). Single-cell RNA sequencing analysis of these lungs and lineage tracing of myofibroblasts revealed that inactivating Snai1/2 from myofibroblasts after bleomycin injury hinders their dedifferentiation into AF1s and/or impairs their clearance through apoptosis. This is demonstrated by an increase in lineage labeled AF1s/Myofibroblasts (Fig. 7E, F) and a cluster of myofibroblast-like cells (AF1-SS) with elevated expression of fibrosis-related genes (myofibroblast) population, including Mt1, Mt2, and Thbs1⁴⁸ in our single cell analysis (Fig. 7B, C). Note that the Snai1^f/f mice we used only have exon3, which is predicted to encode the two C-terminal zinc finger domains as well as the polyadenylation sequence, of the Snai1 gene flanked by LoxP sites⁴⁹ and therefore a truncated Snai mRNA including exon1 and 2 is still being produced that is detected by 2 of the 3 probes of the 10X flex platform for single cell analysis. Since Snail1 is known to negatively regulate its own expression⁵⁰, we therefore see an increase in the expression of this truncated Snai1 mRNA in our AF1ss population.

**Fig. 7: Snail1/2 are required for myofibroblast dedifferentiation into AF1s during the resolution of pulmonary fibrosis.**

Spectra analysis of the AF1-SS population revealed a cell state locked in a pro-fibrotic, pro-inflammatory loop (Fig. S8, Supplementary Data 3). These cells showed sustained activation of myofibroblast differentiation and collagen synthesis pathways, alongside robust signatures of cellular stress (Unfolded Protein Response), inflammation (TNF-α and IL6-JAK-STAT3 signaling), and hypoxia. This molecular signature is indicative of a stalled pathological state that actively prevents the resolution of fibrosis. Consequently, we observed increased pulmonary fibrosis 6 weeks after bleomycin injury, as measured by hydroxyproline content (Fig. 7D).

To further characterize the AF1-SS population we integrated our datasets with those recently published by Tsukui et al.^30,48,51 and found that AF1-SS population clusters together with inflammatory fibroblasts in these datasets (Supplementary Figs. S9, S10).

Discussion

The mechanical properties of the lung, such as tension and stiffness, are tightly regulated to maintain organ form and function. These bulk features originate from the mechanical properties of individual cells, which are determined by actomyosin-dependent contractility and intercellular adhesion. Dynamic regulation of these processes allows cells to exert forces on their neighbors, and during regeneration, this machinery serves to sense the growth rates of neighboring cells, ensuring uniform tissue growth. This study uncovers a Snail/Yap rheostat that functions as a core molecular switch controlling the fitness of mesenchymal cells. We demonstrate that this rheostat governs a process of inter-tissue cell competition, providing a new mechanistic framework for how the lung coordinates its architecture during development and repair.

Hippo/Yap signaling serves as a molecular sensor for cell density and a key regulator of tissue force balance. While Yap activity varies between individual cells, the collective pattern of its localization reflects the tissue’s overall mechanical state, making Yap signaling a potent determinant of cellular fitness. Our data suggest that inactivating Yap/Taz in alveolar fibroblast 1 cells (AF1s) reduces their fitness and disrupts the lung’s overall mechanical properties. This perturbation allows distal airway stem cells to increase their own Yap levels and initiate a pathological bronchiolization of the lung parenchyma. We recently observed a similar response when inactivating Yap/Taz in AT2 stem cells, and these findings collectively reveal that different lung cell populations engage in a competitive process to remodel the lung, aligning with the classical cell competition model^52,53.

A central finding of this work is that the pathological bronchiolization seen in our injury models is not driven by a primary increase in epithelial aggression, but rather by the primary collapse of the mesenchymal niche. Our data support a clear causal sequence: genetic perturbations that reduce the fitness of AF1s, such as the deletion of Yap/Taz or Myc, lead to their elimination via apoptosis. The loss of these essential niche-supporting cells leads to the secondary death of the alveolar type 2 (AT2) stem cells they maintain. This destruction of the niche creates an epithelial void and generates a powerful, but misdirected, regenerative signal. In the absence of the proper instructive cues from the now-absent AF1s, distal airway stem cells mount a default pathological response, filling the damaged parenchyma with aberrant airway-like structures instead of functional alveoli. This “niche collapse” model provides an alternative possibility of our understanding of fibrotic remodeling, shifting the focus from the “winner” epithelium to the “failing” mesenchyme, such as observed with aging⁵⁴, as the possible initiating pathological event.

We further identify tissue-scale mechanical cooperation as a crucial factor in orchestrating organ formation and regeneration. This cooperation involves mesenchymal and epithelial cell populations sensing the Yap-defined fitness levels in their neighbors. We find that during both lung development and repair after bleomycin injury, mesenchymal Yap levels and fitness antagonize epithelial Yap levels and stemness. Our data therefore suggest that tissues strive to restore tissue-wide homeostatic Yap levels as quickly as possible following injury. However, attempting to maintain tissue-level function at all costs may lead to aberrant remodeling when there is a great disturbance in the force, such as when millions of cells die off because of a catastrophic injury.

Our analysis of the aberrant basaloid cells that emerge in this context provides a stunning molecular explanation for their “supercompetitor” status. Our pathway analysis of this population reveals a profound metabolic reprogramming, characterized by the coordinated upregulation of glycolysis, the TCA cycle, oxidative phosphorylation, nucleotide synthesis, and Myc target gene activation. This is the classic signature of a hyper-proliferative state, analogous to the Warburg effect in cancer cells or the bioenergetics of rapidly dividing stem cells. This metabolic rewiring provides the functional engine for the “winner” phenotype, allowing these cells to generate the biomass and energy required to rapidly proliferate and overwhelm the compromised alveolar region, connecting the mechanisms of pathological lung remodeling to the broader fields of cancer metabolism and stem cell bioenergetics.

Furthermore, our experiments inactivating Snai1/2 during the resolution phase of fibrosis provide a molecular basis for the persistence of a non-resolving, pathological state. The resulting AF1-SS cell population is trapped in a vicious cycle, defined by the simultaneous activation of pro-fibrotic (collagen synthesis), pro-inflammatory (TNF-α and IL6-JAK-STAT3 signaling), and cellular stress (Unfolded Protein Response) pathways. This molecular signature is indicative of a stalled state that actively prevents the resolution of fibrosis. This finding has direct therapeutic implications, as it suggests that strategies aimed at pharmacologically promoting the Snail1/2-high/Yap-low state in myofibroblasts could represent a different class of pro-resolution therapies for treating pulmonary fibrosis.

In conclusion, our work demonstrates that the lung maintains its form and function through a system of tissue-scale cooperation, where epithelial and mesenchymal populations sense each other’s fitness levels via the Hippo/Yap pathway. These findings provide new insights into how organ size is regulated and how regeneration is coordinated across different tissue compartments. Moving forward, future studies aiming to better characterize this contractile heterogeneity and explore the tissue-level mechanisms that coordinate force balance will be important to find a cure for pulmonary fibrosis.

Methods

Experimental model and subject details

Both male and female mice were used in all experiments. Mice were bred in a pathogen-free environment on a standard laboratory setting of a 12 h light, 12 h dark exposure to mimic the day-night cycle. Room temperature was kept between 21 °C and 24 °C. Stk3/4^f/f (RRID:IMSR_JAX:017635), Yap1^f/f;Wwtr1^f/f⁵⁵, Tet-Yap1^S112A-H2BGFP (RRID:IMSR_JAX:031279), Plin2^CreERT2 (kind gift from Dr. Ahlbrecht⁵⁶), Acta2^CreERT2 (kind gift from Dr. Chambon⁵⁷), Scube2^CreERT2 (kind gift from Dr. Sheppard³⁰), Rosa26-mTmG (RRID:IMSR_JAX:007676), Rosa26-Confetti (RRID:IMSR_ JAX:017492), Snai1^f/f (kind gift from Dr. Weiss⁴⁹), Snai2^f/f (RRID:IMSR_JAX:037485), Tet^Cre (RRID:IMSR_JAX:006234), Tbx4^rtTA (kind gift from Dr. Shi⁵⁸).

Summary information of genetic mouse models and experiments can be found in Supplementary Table 1. Bleomycin mice were intratracheally instilled with 50 µL bleomycin (0.8–2 U/kg body weight optimized for each strain, batch of bleomycin, and gender) as adults, 8–12 weeks old. For tamoxifen induction, Acta2^CreERT2, Plin2^CreERT2, Scube2^CreERT2 were placed on tamoxifen chow (rodent diet with 400 mg/kg tamoxifen citrate; Harlan Teklad TD.130860). Plin2^CreERT2 were placed on tamoxifen chow for 10 days at 2–3 months of age and were intratracheally instilled with bleomycin 3 weeks after being returned to normal rodent chow. Scube2^CreERT2 mice were placed on tamoxifen chow at 2 months old for 3 weeks with an additional intraperitoneal tamoxifen injection (0.20 mg/g body weight, Millipore Sigma) on the last day of tamoxifen citrate feed, and after 3 weeks on normal laboratory chow, they were injured with bleomycin. Acta2^CreERT2 received bleomycin at 2–3 months of age and were put on tamoxifen chow 3 weeks later for 3 weeks until euthanasia. Tbx4^rtTA;Tet^Cre mice were put on doxycycline containing chow (rodent diet with 625 mg/kg doxycycline; Harlan Teklad TD.09761) at weaning and remained on doxycycline chow until euthanasia and were intratracheally instilled with bleomycin at 2–3 months of age on doxycycline. All injured mice were euthanized 6 weeks post bleomycin injury.

Dams of embryonic mice were put on doxycycline at embryonic day 9.5 (E9.5), and embryos were dissected at E13.5, E15.5, and E18.5. All experiments were approved by the Mayo Clinic Institutional Animal Care and Use Committee.

Immunohistochemistry and fluorescence

All staining was done on paraffin sections of formalin-fixed lungs. Immunofluorescent staining was performed with the following primary antibodies: chicken anti-GFP (1:500; GFP-1020; RRID:AB_10000240; Aves Labs Inc.), rabbit anti-Keratin 5 (1:200; clone EP1601Y; MA5-14473; RRID:AB_10979451; Thermo Fisher Scientific), rabbit anti-SFTPC (1:200; WRAB-9337; RRID:AB_2335890; Seven hills bioreagents), goat anti-RAGE (1:500; AF1145; RRID:AB_354628; R&D Systems), rat anti-Keratin 8 (1:100; TROMA-I; RRID:AB_531826; Developmental Studies Hybridoma Bank), mouse anti-alpha actin (smooth muscle actin (SMA), Acta2; 1:500; Clone 1A4; sc-32251; RRID:AB_262054; Santa Cruz Biotechnology Inc.), rabbit anti-Yap (1:100; 4912; RRID:AB_2218911; Cell Signaling), rabbit anti-PLIN2 (1:200, ab52356, RRID:AB_2223599, Abcam), mouse anti-Cadherin (E Monoclonal) (1:200, Clone 36, 610181, RRID:AB_397580, BD Biosciences), rabbit anti-Snail2 (1:100, Clone C19G7, 9585, RRID:AB_2239535). TUNEL assays were performed on paraffin sections of formalin-fixed lungs using the In-Situ Cell Detection Kit, TMR Red (12156792910, Roche).

After deparaffinization, slides were rehydrated through a series of decreasing ethanol concentrations, antigen unmasked by either microwaving in citrate-based antigen unmasking solution (Vector Labs, H-3300) or by incubating sections with proteinase K (7.5 μg/ml) (Invitrogen, 25530-049) for 7 min at 37 °C. Tissue sections were then washed in TBS with 0.1% Tween-20 and blocked with 3% Bovine Serum Albumin (BSA), 0.4% Triton X-100 in TBS for 30 min at room temperature followed by overnight incubation of primary antibodies diluted in 3% BSA, 0.1% Triton X-100 in TBS. The next day, slides were washed in TBS with 0.1% Tween-20 and incubated with secondary antibodies diluted in 3% BSA, 0.1% Triton X-100 in TBS for 3 h at room temperature. All fluorescent staining was performed with appropriate secondary antibodies from Jackson Immunoresearch. Slides were mounted using Vectashield (Vector Labs, H-1000).

Microscopy and imaging

Tissue was imaged using a micrometer slide calibrated Zeiss LSM800 Laser scanning confocal microscope using ZEN imaging software or Leica Stellaris 5 confocal microscope with LASX imaging software. Lungs were imaged using tiled stitched 20× images covering the entire cross-section of the left or lower right lung lobe from ≥6 different lungs. Representative images were chosen. Images were processed and analyzed using Zen blue (Zeiss), LASX (Leica), ImageJ (NIH) and Adobe Photoshop 2024 (Adobe) software. The total area of Krt5, Krt8 or GFP was determined using ImageJ (NIH) and analyzed based on the total lung area. Image quantification and analysis was performed in a double blinded fashion. Each quantification was ≥3 different mouse lungs.

Quantitative real-time PCR

Total mRNA was extracted from lung accessory lobes stored in RNALater (Invitrogen, AM7021) and using Total RNA Kit I (Omega Biotek, R6834-02) according to the manufacturer’s instructions. RNA concentration was determined by spectrophotometry. cDNA was generated using Maxima™ First Strand cDNA Synthesis (Fisher Scientific, FERK1642) according to the manufacturer’s instructions. Gene expression was analyzed by quantitative RT-PCR using Taqman Gene Expression Assays (Applied Biosystems, 4369016) directed against the mouse targets β-glucuronidase (Mm00446953_m1), Krt5 (Mm01305291_g1), Trp63 (Mm00495788_m1). Quantitative real-time PCR was performed using a StepOne Plus system (Applied Biosystems). Data were presented as 2^−ΔΔCt with β-glucuronidase as the internal sample control normalized to control group. Each experiment was repeated with samples obtained from ≥3 different lung preparations.

Nanostring

RNA was isolated from lung accessory lobes as described above. 100 ng of RNA was hybridized with a custom RNA probe panel designed by NanoString (NanoString Technologies; DL_1206_C9662) for 16 h according to manufacturer’s instructions. The RNA-probe hybridization was loaded on a NanoString cartridge and processed in a NanoString nCounter. Data was analyzed with Rosalind.bio (Rosalind, Inc) and Log2 Fold Changes were calculated and graphed. Each experiment was repeated with samples obtained from ≥3 different lung preparations.

Hydroxyproline assay

The right lobes were flash frozen in dry ice at the time of harvest and stored at −80 °C. For acid hydrolysis, the lobes were baked in a 70 °C oven without lids for 2 days until completely dry. The weights of dry lobes were measured and 500 µl of 6 N HCl were added to each sample. The lungs were then hydrolyzed in an 85 °C oven for 2 days with occasional vortexing. The hydrolysates were cooled at room temperature and centrifuged at maximum speed for 10 min. The supernatants then were transferred to fresh 1.5 mL tubes and centrifuged at maximum speed for 10 min. Each sample or standard was diluted with citrate-acetate buffer (5% citric acid, 1.2% glacial acetic acid, 7.24% sodium acetate, and 3.4% sodium hydroxide) in a 96-well plate. Chloramine-T solution (1.4% chloramine-T, 10% N-propanol, and 80% citrate-acetate buffer) was added, and the mixture was incubated for 20 min at room temperature. Then, Ehrlich’s solution (1.27 M p-dimethylaminobenzaldehyde, 70% N-propanol, 20% perchloric acid) was added to each sample and the samples were incubated at 65 °C for 20 min. Absorbance was measured at 550 nm. Standard curves were generated for each experiment using reagent hydroxyproline (Sigma H-1637) as a standard. The amount (µg) of hydroxyproline were calculated by comparison to the standard curve.

10× genomics single cell multiome ATAC+ gene expression sequencing

Embryonic day E15.5 or E18.5 lungs were collected and flash-frozen in a dry ice/ethanol slurry. Frozen tissues were homogenized with a dounce homogenizer and nuclei were isolated according to 10× Genomics Nuclei Isolation from Embryonic Mouse Brain for Single Cell Multiome ATAC+ Gene Expression Sequencing. Nuclei viability was assessed, and total nuclei were counted. ATAC and gene expression libraries were then constructed from the nuclei according to the 10× Genomics Chromium Next GEM Single Cell Multiome ATAC+ Gene Expression User Guide. Sequencing was performed on a NextSeq500. Alignment was performed using the ‘Cellranger count’ function provided in 10× Genomics single-cell gene expression software. For secondary analysis we used the R-package ArchR⁵⁹ to apply standard quality control metrics and unsupervised clustering for the generation of initial UMAP projections and integrate multiomics data.

10× genomics single cell fixed RNA profiling (flex) analysis

Cells from 2 50 μm formalin fixed paraffin embedded (FFPE) tissue sections were deparaffinized and manually dissociated with a pellet pestle using 1.0 mg/mL Liberase TH in RPMI according to manufacturer’s directions (10× Genomics).Cells were counted using Countess II FL Automated Cell Counter (Thermo Fisher Scientific) and up to 10,000 cells per sample were hybridized with mouse WTA Probes (10× Genomics; PN-2000703-2000718) for 17 h according to manufacturer’s instructions. Post-hybridization cells were counted and pooled with equal number of cells per sample for a target recovery of 128,000 cells. GEM generation and final library preparations and sequencing were completed by the Mayo Genomics Research Core according to manufacturer’s instructions on an Illumina NextSeq. Fastq files were analyzed with Cell Ranger Pipeline using 10× Genomics Cloud Analysis. For secondary analysis we used Scanpy⁶⁰ to apply standard quality control metrics and unsupervised clustering for the generation of initial UMAP projections and integrate data using Harmonypy⁶¹ or Scanorama⁶², trajectory inference was determined using Monocle 3⁶³, whereas (supervised pathway deconvolution of interpretable gene programs) analysis was performed using Spectra⁴⁴. Data from the current study (CTRL NI, CTRL d42 Bleo, Acta2^CreERT2;Snai1/2^f/f d42 Bleo) was integrated with multiple published lung fibrosis datasets (GSE218666, GSE173878, GSE235212, GSE218665, GSE210341, GSE132771, GSE276546, GSE229523)^30,48,51.

In situ for Snai1 and Snai2

Whole-mount in situ hybridization was performed as previously described⁶⁴ with antisense probes against full length Snai1 or Snai2.

Statistics and reproducibility

All results are expressed as mean values ± SEM. The ‘n’ represents biological replicates and can be found in the figure legends. The significance of differences between 2 sample means was determined by two-tailed unpaired t-test (assuming unequal or equal variances as determined by the F-test of equality of variances). All datasets followed a normal distribution and P values less than 0.05 were considered statistically significant. The number of samples to be used was based on the number of experimental paradigms multiplied by the number in each group that is necessary to yield statistically significant results (based on power analysis, to reject the null hypothesis with 80% power (type I error = 0.05).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

10x Genomics Multiome and Flex RNA profiling generated in this study have been deposited in Zenodo Repository (10.5281/zenodo.17468266). Previously published datasets use in this study include: GSE218666, GSE173878, GSE235212, GSE218665, GSE210341, GSE132771, GSE276546, GSE229523. Further information and requests for resources, data, and reagents should be directed to and will be fulfilled by the Lead Contact, Stijn De Langhe: delanghe.stijn@mayo.edu.

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Acknowledgements

This study was supported by NIH R01 HL146461 (S.D.L.), HL132156 (S.D.L.), NIH R35 HL161169 (S.D.L.), NIH T32 HL105355 (R.W.) and Mayo Clinic Brewer Family Career Development Award (R.W.).

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These authors contributed equally: Kylie Klinkhammer, Rachel Warren, Joseph Knopp, Stijn P. De Langhe.

Authors and Affiliations

Department of Medicine, Division of Pulmonary and Critical Medicine, Mayo Clinic, Rochester, MN, USA
Kylie Klinkhammer, Rachel Warren, Joseph Knopp, Toan Nguyen & Stijn P. De Langhe
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA
Stijn P. De Langhe

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Kylie Klinkhammer
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Rachel Warren
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Joseph Knopp
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Toan Nguyen
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K.K., R.W., and J.K. designed and performed experiments, interpreted the data and prepared the manuscript. T.N. performed experiments. S.D.L. conceived, designed and supervised the study, analyzed and interpreted the data and prepared the manuscript.

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Correspondence to Rachel Warren or Stijn P. De Langhe.

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Klinkhammer, K., Warren, R., Knopp, J. et al. Epithelial-mesenchymal cell competition coordinates fate transitions across tissue compartments during lung development and fibrosis. Nat Commun 16, 10956 (2025). https://doi.org/10.1038/s41467-025-66690-z

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Received: 14 April 2025
Accepted: 12 November 2025
Published: 21 November 2025
Version of record: 08 December 2025
DOI: https://doi.org/10.1038/s41467-025-66690-z