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[Preprint]. 2025 May 2:rs.3.rs-6189965.
doi: 10.21203/rs.3.rs-6189965/v1.

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

Kylie Klinkhammer  1 , Rachel Warren  1 , Joseph Knopp  1 , Toan Nguyen  1 , Stijn P De Langhe  1   2
Affiliations

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

Kylie Klinkhammer et al. Res Sq. .

Abstract

Morphogenesis and cell state transitions must be coordinated in time and space to produce a functional tissue. In this study, we reveal that lung mesenchymal Yap levels and fitness antagonize epithelial Yap levels and stemness during lung development and repair following bleomycin injury. Elevated mesenchymal Yap signaling and fitness antagonize epithelial Yap levels and stemness, accelerating alveolar epithelial differentiation while impairing branching during lung development or bronchiolization after bleomycin injury. Conversely, mesenchymal Snail/Slug sequesters Yap/Taz to direct an adipogenic differentiation program towards alveolar fibroblast 1 (AF1) during both lung development and the resolution of pulmonary fibrosis. On the other hand, Yap/Myc-Tead binding instructs a myogenic differentiation program. Through our experiments and modeling, we identify tissue-scale mechanical cooperation as a pivotal factor in orchestrating organ formation and regeneration.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Single nuclei combined RNA and ATAC analysis of E15.5 and 18.5 mouse lungs.
(A) Graph-based clustering and cell-type or sample annotation of integrated datasets from E15 and E18 ctrl and mutant lungs based on enriched gene expression and chromatin accessibility profiles by using ArchR. AF1ss (AF1 population in Snai1/2 inactivated mesenchyme), AF1yt (AF1 population in Yap/Taz inactivated mesenchyme). (B,C) Subsetting and clustering of (B) mesenchyme and (C) epithelium. (D,E) Graph-based clustering and cell-type annotation on individual samples of (D) mesenchyme and (E) epithelium. (F) Tead motif accessibility and representative gene expression for main mesenchymal and epithelial clusters. (G) Heatmap of differential transcription factor motif accessibility showing which regulatory factors are predicted to most active in each cell type. AF1ss (AF1 population in Snai1/2 inactivated mesenchyme) contains enrichment for myogenic differentiation and Tead transcription factors (green), AF1 is enriched for adipogenic factors (yellow), AF1yt (AF1 population in Yap/Taz inactivated mesenchyme) lacks Tead binding activity.
Figure 2
Figure 2. Snail1/2 and Yap/Taz are required for AF1 differentiation.
(A) Immunostaining for Adrp and Acta2 on E15.5 control (n=4), Tbx4-rtTa;Tet-Cre;Yap1f/f;Wwtr1f/f (n=3), Tbx4-rtTa;Tet-Cre;Snai1/2f/f (n=3), and Tbx4-rtTa;Tet-Cre;Stk3/4f/f (n=2) lungs. Quantification of Adrp expression from images represented. (B) Co-immunostaining for Rage and Sftpc on E18.5 control, Tbx4-rtTA;Tet-Cre;Yap1f/f;Wwtr1f/f, Tbx4-rtTA;Tet-Cre;Snai1/2f/f and Tbx4-rtTA;Tet-Cre;Stk3/4f/f lungs doxycycline induced from E9.5. Nanostring nCounter RNA profiling on control (n=19) and Tbx4-rtTA;Tet-Cre;Yap1f/f;Wwtr1f/f (n=21) lungs at E18.5. (C) Co-immunostaining for Yap and E-cadherin on E15.5 control (n=6), Tbx4-rtTA;Tet-Cre;Yap1f/f;Wwtr1f/f (n=3) and Tbx4-rtTA;Tet-YapS112A (n=5) lungs doxycycline induced from E9.5. Scale bar 50μm. Quantification of epithelial Yap intensity on represented images. (D) Co-immunostaining for Yap and Acta2 on E15.5 control (n=5) and Tbx4-rtTA;Tet-YapS112A (n=5) lungs doxycycline induced from E9.5. Scale bar 100μm. Quantification of average pixel intensity of Acta2 staining on whole lung represented images.
Figure 3
Figure 3. Loss of Yap/Taz in AF1s promotes bronchiolization after bleomycin injury.
(A-D) Immunostaining for GFP and Krt5 on (A) control (n=7), (B) Adrp-CreERT2; Yap1f/f;Wwtr1f/f;mTmG (n=12) lungs 6 weeks after bleomycin injury. (C) Nanostring nCounter RNA analysis for Krt5 on control (n=42), Adrp-CreERT2; Yap1f/f;Wwtr1f/f;mTmG (n=13) and Adrp-CreERT2;Stk3/4f/f (n=11). (D) Image quantification of GFP in images represented in (A,B). (E-H) Immunostaining for Krt8 in (E) control, (F) Adrp-CreERT2;Yap1f/f;Wwtr1f/f, (G) Adrp-CreERT2;Stk3/4f/f, and (H) Scube2-CreERT2;Yap1f/f;Wwtr1f/f lungs 6 weeks after bleomycin injury. Scale bar 500μm. (I) Image quantification of Krt8 in images represented in (E-H). (J) Hydroxyproline analysis on bleomycin injured Scube2-CreERT2;mTmG controls (n=43) and Scube2-CreERT2;Yap1f/f;Wwtr1f/;mTmG (n=30). (K-M) TUNEL staining and quantification on bleomycin injured control AdrpCreERT2;mTmG (n=3) and AdrpCreERT2;Yap1f/f;Wwtr1f/f;mTmG (n=3) lungs.
Figure 4
Figure 4. Loss of Yap/Taz in AF1s promotes the generation of aberrant basaloid cells after bleomycin injury.
(A) clusters from scRNA-seq on control not injured (NI), bleomycin injured control, and bleomycin injured Adrp-CreERT2;Yap1f/f;Wwtr1f/f. Aberrant basaloid cell cluster is circled. (B) Gene expression of marker genes. (C) Myc transcription factor activity.
Figure 5
Figure 5. Loss of Yap/Taz in AF1s promotes the generation of aberrant basaloid cells after bleomycin injury similar to aberrant basaloid cells in IPF.
(A) clusters from scRNA-seq on control not injured (NI), bleomycin injured control, bleomycin injured Adrp-CreERT2;Yap1f/f;Wwtr1f/f and human IPF. (B) Gene expression of marker genes.
Figure 6
Figure 6. Loss of Myc in AF1s promotes bronchiolization after bleomycin injury.
(A) Immunostaining for Krt8 and Krt5 on control (n=6) and Adrp-CreERT2; Mycf/f;mTmG (n=11) lungs 6 weeks after bleomycin injury. Scale bar 200μm. (B) Hydroxyproline analysis on bleomycin injured AdrpCreERT2;mTmG (n=23) and Adrp-CreERT2;Mycf/f;mTmG (n=34) lungs. (C) Image quantification of Krt8 and Krt5 in images represented in (A). (D) qPCR analysis on RNA from bleomycin injured Cre- controls (n=19) and Adrp-CreERT2;Mycf/f;mTmG (n=22) lungs for Krt5 and Tp63 genes.
Figure 7
Figure 7. Snail1/2 are required for myofibroblast dedifferentiation into AF1s during the resolution of pulmonary fibrosis.
(A) Mesenchymal cell clusters from scRNAseq on non injured control and bleomycin injured control and Acta2-CreERT2;Snai1/2f/f lungs tamoxifen induced 2 weeks after bleomycin injury. (B) Cell specific gene expression. (C) Fibrosis promoting pathway activities are upregulated in AF1-SS (AF1 population in Snai1/2 inactivated mesenchyme). (D) Hydroxyproline analysis of soluble collagen on control (n=30) and Acta2-CreERT2;Snai1/2f/f(n=7). (E) Co-immunostaining for GFP and Acta2 on bleomycin injured control (n=3) and Acta2-CreERT2;Snai1/2f/f (n=7) lungs tamoxifen induced 2 weeks after bleomycin injury. Scale bar 100 μm. (F) Quantification of GFP in images represented in (E).
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