Non-canonical Wnt signaling promotes epithelial fluidization in the repairing airway

Abstract

Concerted migration of basal stem cells (BCs) in the airway, also known as epithelial fluidization, has been implicated in epithelial repair after injury. How BC migration is regulated, and how it influences the success of epithelial repair, remains unclear. Here we have identified non-canonical Wnt signaling through Ptk7, Fzd7, and YAP as a critical regulator of BC migration in the mouse trachea. Using live imaging and genetic studies in the mouse, we find that Ptk7 is required for the concerted movement of BCs after injury, and that this requirement extends to BC proliferation and subsequent restoration of epithelial homeostasis after injury. We demonstrate that Ptk7 exerts this function in conjunction with Wnt5a and Fzd7, and through YAP activation in BCs. Our data provide mechanistic insight into the regulation of epithelial repair in the airway.

Fig. 1. BCs migrate after airway injury.

a Diagram of tracheal section and orientation for immunostaining and live imaging. b Montage of BC migration in undamaged versus polidocanol-injured versus actomyosin-inhibited, polidocanol-injured trachea. Quantification of BC migration as determined by percentage of migratory tdTomato+ BCs, and the change of the combined area of all BCs between 3 h after acquisition versus start of acquisition. Created in BioRender. Cai, X. (2025) https://BioRender.com/7evq4zuc Montage of wound closure. Quantification of wound closure as determined by the change of wound diameter between 4 h post ablation versus immediately following ablation. Red line indicates diameter of the wound. mean ± SD; n = sample size as follows, b 4 (Mock Undamaged and Cytochalasin B 1dp polidocanol) and 6 (Mock 1dp polidocanol) mice, c 5 (Mock) and 3 (Cytochalasin B) mice. **P < 0.01 (b–Δarea of BCs: Mock Undamaged vs Mock 1dp polidocanol = 0.0054, Mock 1dp polidocanol vs Cytochalasin B 1dp polidocanol = 0.0083), ***P < 0.001 (b–%migrating BCs, c < 0.0001), based on one-way ANOVA with Tukey’s multiple comparison test (b) and two-tailed, unpaired Student’s t-test (c). Scale bar = 10 µm. Timestamp indicated as hours:minutes, with 00:00 recorded immediately following ablation. See also Supplementary Figs. 1 and 2.

Fig. 2. Loss of function of non-canonical Wnt co-receptors impairs BC migration.

a Montage and quantification of BC migratory behavior after 1d polidocanol injury in control versus Ptk7 loss of function trachea. Genetic controls of undamaged and 1d post polidocanol-injured trachea were taken from Fig. 1b. b Montage and quantification of BC migratory behavior in laser-ablated in control versus Ptk7 loss of function trachea. Genetic controls were taken from Fig. 1c. Red line indicates diameter of the wound. mean ± SD; n = sample size as follows, a 4 (Control Undamaged and Ptk7 KO 1dp polidocanol) and 6 (Control 1dp polidocanol), b 5 (Mock) and 3 (Ptk7 KO and dFzd7-21) mice. *P < 0.05 (a–Δarea of BCs: Control 1dp polidocanol vs Ptk7 KO 1dp polidocanol = 0.0159), **P < 0.01 (a–Δarea of BCs: Control Undamaged vs Control 1dp polidocanol = 0.0062), ***P < 0.001 (b Mock vs dFzd7-21 = 0.0002, a–%migrating BCs, b Mock vs Ptk7 KO < 0.0001), based on one-way ANOVA with Tukey’s multiple comparison test. Scale bar = 10 µm. Timestamp indicated as hours:minutes, with 00:00 recorded immediately following ablation.

Fig. 3. Fzd7 and Dvl1 localize to the BC cortex after injury.

a Staining and quantification of Fzd7 localization in tdTomato+ BCs reveals cortical decoration in BCs 1d after polidocanol injury, but spare localization in BCs from undamaged tissue. b Staining and quantification of Dvl1 localization in tdTomato+ BCs reveals cortical decoration in BCs 1d after polidocanol injury, but sparse localization in BCs from undamaged tissue. Cortical Dvl1 remains absent after Ptk7 loss of function in BCs from damaged tissue. mean ± SD; n = sample size as follows, a 30 cells from 3 mice, b 30 cells from 3 mice (Control Undamaged and Ptk7 KO 1dp polidocanol, 33 cells from 4 mice (Control 1dp polidocanol). ***P < 0.001 (a, b < 0.0001), based on two-tailed, unpaired Student’s t-test (a) and one-way ANOVA with Tukey’s multiple comparison test (b). Scale bar = 10 µm. See also Supplementary Fig. 3.

Fig. 4. Wnt5a promotes BC migration.

a Montage and quantification of BC migratory behavior in laser-ablated trachea after treatment with DMSO, porcupine inhibitor, β-catenin inhibitor, recombinant Wnt3a, or recombinant Wnt5a. DMSO controls were taken from Fig. 1c. Red line indicates diameter of the wound. b Staining and quantification of Wnt5a localization in tdTomato+ BCs reveals cortical decoration in BCs 2 h after polidocanol injury, but low relative cortical localization in BCs from undamaged tissue. Cortical decoration of Wnt5a in 2 h post injured BCs is no longer present after Ptk7 depletion. c Staining and quantification of Wnt5a puncta after RNAscope for Wnt5a and PDGFRa reveal an increase of Wnt5a transcript levels in PDGFRa+ cells 2 h after polidocanol injury. Depletion of Ptk7 in BCs did not affect increase of Wnt5a puncta after injury. White dotted line represents the basement membrane. mean ± SD; n = sample size as follows, a 5 (Mock) and 3 (LGK974, iCrt14, Wnt5a, Wnt3a) mice, b 30 cells from 3 mice (Control Undamaged), 33 cells from 3 mice (Control 2hrp polidocanol), 32 cells from 4 mice (Ptk7 KO 2hrp polidocanol), c 62 (Control Undamaged), 63 (Control 2hrp polidocanol), 56 (Ptk7 KO 2hrp polidocanol) cells from 3 mice. N.S. not significant (a Mock vs iCrt14 = 0.9904, Mock vs Wnt3a = 0.9573, c Control 2hrp polidocanol vs Ptk7 KO 2hrp polidocanol = 0.1956), **P < 0.01 (a Mock vs Wnt5a = 0.011), ***P < 0.001 (a Mock vs LGK974, b, c Control Undamaged vs Control 2hrp polidocanol <0.0001), based on one-way ANOVA with Tukey’s multiple comparison test. Scale bar = 10 µm. Timestamp indicated as hours:minutes, with 00:00 recorded immediately following ablation. See also Supplementary Figs. 3 and 4.

Fig. 5. Ptk7-dependent YAP activation promotes BC migration.

a Staining and quantification of YAP in tdTomato+ BCs reveals an increase of YAP activity after 1d post polidocanol injury. Ptk7 loss of function decreases YAP activation after injury. b Montage and quantification of BC migratory behavior in laser-ablated trachea after treatment with DMSO or YAP inhibitor. DMSO controls were taken from Fig. 1c. Red line indicates diameter of the wound. mean ± SD; n = sample size as follows, a 3 mice (quantified cells pooled from two sections per mouse), b 5 (Mock) and 4 (Verteporfin) mice. **P < 0.01 (a Control Undamaged vs Control 1dp polidocanol = 0.0026, Control 1dp polidocanol vs Ptk7 KO 1dp polidocanol = 0.0049), ***P < 0.001 (b < 0.0001), based on one-way ANOVA with Tukey’s multiple comparison test (a) and two-tailed, unpaired Student’s t-test (b). Scale bar = 10 µm. Timestamp indicated as hours:minutes, with 00:00 recorded immediately following ablation.

Fig. 6. Ptk7 loss of function impairs airway regeneration.

a Staining of tracheal epithelium from genetic controls or Krt5-specific Ptk7 knock out. Quantification of tracheal epithelium thickness as determined by tdTomato or overexposure of Pdpn or Foxj1 staining. b Quantification of the percentage of ciliated cells/total cells in the tracheal epithelium, as determined by Foxj1 staining. c Staining of club cells, as determined by CC10, and quantification of the percentage of club cells/total cells in the tracheal epithelium. d Staining of mitotic cells, as determined by phospho-histone H3 (PH3), and quantification of mitotic index (percentage of PH3+ BCs/total BCs). e A model of non-canonical Wnt-mediated regulation of BC migration after injury. Wnt5a is secreted by fibroblasts, which activates non-canonical Wnt signaling through Ptk7, Fzd7, and Dvl. Non-canonical Wnt signaling, in turn, activates YAP signaling and promotes BC migration and proliferation during tissue repair. Created in BioRender. Cai, X. (2025) https://BioRender.com/jgqkk9i. mean ± SD; n = 3 mice. N.S. not significant (a Control Undamaged vs Ptk7 KO Undamaged = 0.9082, Control 1dp polidocanol vs Ptk7 KO 1dp polidocanol = 0.9757, b Control Undamaged vs Ptk7 KO Undamaged = 0.9547, Control 1dp polidocanol vs Ptk7 KO 1dp polidocanol >0.9999, c Control Undamaged vs Ptk7 KO Undamaged = 0.8836, Control 1dp polidocanol vs Ptk7 KO 1dp polidocanol >0.9999, d Control Undamaged vs Ptk7 KO Undamaged = 0.8732), ***P < 0.001 (a Control Undamaged vs Control 1dp polidocanol = 0.0001, a Control 1dp polidocanol vs Control 5dp polidocanol, Control 5dp polidocanol vs Ptk7 KO 5dp polidocanol <0.0001, b, c Control Undamaged vs Control 1dp polidocanol, Control 1dp polidocanol vs Control 5dp polidocanol, Control 5dp polidocanol vs Ptk7 KO 5dp polidocanol <0.0001, d Control Undamaged vs Control 2dp polidocanol = 0.0003, d Control 2dp polidocanol vs Ptk7 KO 2dp polidocanol = 0.0006), based on one-way ANOVA with Tukey’s multiple comparison test. Scale bar = 10 µm. See also Supplementary Figs. 5 and 6.