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

Hu, Daniel Jun-Kit; Cai, Xiaoyu Tracy; Simons, Jesse; Yun, Jina; Elstrott, Justin; Jasper, Heinrich

doi:10.1038/s41467-025-59320-1

Download PDF

Article
Open access
Published: 03 May 2025

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

Nature Communications volume 16, Article number: 4124 (2025) Cite this article

3493 Accesses
2 Citations
2 Altmetric
Metrics details

Subjects

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.

Life-long functional regeneration of in vivo airway epithelium by the engraftment of airway basal stem cells

Article 05 November 2024

Non-canonical Wnt signaling promotes directed migration of intestinal stem cells to sites of injury

Article Open access 09 December 2021

Repair of airway epithelia requires metabolic rewiring towards fatty acid oxidation

Article Open access 13 February 2023

Introduction

The airway epithelium serves vital gas-exchange functions to ensure oxygen delivery to the rest of the organism. As barrier epithelia, the trachea and lungs are exposed to frequent environmental challenges and must maintain regenerative capacity for proper tissue function. The trachea is lined with a pseudostratified epithelium primarily consisting of Krt5+ basal cells (BCs), CC10+ club cells, and Foxj1+ ciliated cells¹. A number of other cell types, such as Ascl1+ pulmonary neuroendocrine cells and FoxI1+ pulmonary ionocytes, are found at significantly smaller frequencies^2,3. Basal cells (BCs) serve as stem cells in the airway, and their activation and differentiation has to be coordinated to replace lost cells^1,4. BCs in the trachea are largely quiescent during homeostasis, but enter the cell cycle in response to airway injury^5,6. While BCs are normally located in the trachea and primary bronchi during homeostasis, BCs populate the distal airway after injury⁷, and it is believed that they migrate to injured regions of the epithelium to promote repair^8,9,10. This migration occurs in a coordinated process termed ‘epithelial fluidization’ that needs to be carefully regulated to avoid tissue disorganization. The underlying mechanisms regulating BC migration are only beginning to be explored. Migration of BCs along the distal airway in response to H1N1 viral infections has recently been found to be triggered by interleukin 6 (IL-6), and to be promoted by YAP signaling^11,12. It is unclear whether BC migration shares similar regulation after other forms of airway injury or within the proximal airway.

Wnt signaling is critical for the regulation of mammalian BC activity, and for maintaining airway homeostasis^13,14,15,16. In canonical Wnt signaling, secreted Wnt ligands (primarily Wnt3a) interact with the cell surface receptors Frizzled5 (Fzd5) and LRP5/6 to activate β-catenin through Disheveled (Dvl)^17,18. In contrast, during non-canonical Wnt signaling, Dvl is activated by other Wnt ligands (such as Wnt5a) interacting with co-receptors Frizzled7 (Fzd7), Ptk7, and Ror2^19,20,21. Dvl activation triggers a cascade of Rho GTPase activation, actin rearrangement, and lamellipodia formation^22,23. Rho activation also mediates relocation of YAP to the nucleus, which then forms a complex with TEAD transcription factors to transcriptionally regulate cytoskeleton dynamics^24,25. In the mammalian intestine, Wnt5a, Ptk7 and Fzd7 have been found to regulate cell fate through YAP activation^26,27.

Here, we report that Ptk7-induced non-canonical Wnt signaling promotes mammalian BC migration after injury. We have previously identified a role of non-canonical Wnt signaling through the Drosophila homolog of Ptk7 in regulating stem cell migration in the adult Drosophila intestine²⁸. Using live imaging in the mouse trachea, we find that the same signaling mechanism regulates BC migration in the mouse trachea after injury by polidocanol treatment or laser ablation. Our data indicate that BC migration is induced by Wnt5a-mediated activation of Fzd7 and Ptk7, promoting YAP activity. We demonstrate that these signaling events are required for directed BC migration towards sites of injury, and contribute to wound healing. Impairing BC migration hinders the regenerative process, decreasing BC division, the generation of differentiated cells, and overall epithelial repair. Our results further support the notion that BC migration is critical for the repair of the conducting airway, and provide mechanistic insight into this regenerative response after injury.

Results

Basal cells undergo actin-dependent migration towards site of injury

Previous studies have investigated the collective migration of airway epithelial cells in precision-cut lung slices or in vitro air-liquid interphase cultures^11,29,30,31. To visualize BC behavior directly in the proximal airway, we imaged live tracheal sections ex vivo. Trachea were sectioned along the coronal plane, and the most ventral (i.e., lateral) section was imaged from an en face view (Fig. 1a). BCs were visualized with tdTomato expression after tamoxifen-inducible excision of a transcriptional STOP cassette using Krt5::Cre^ERT2 (Krt5::Cre^ERT2; lox>STOP>lox tdTomato; Supplementary Fig. 1a)^1,32. 97% of tdTomato+ cells co-expressed the BC marker, podoplanin (Pdpn), after the tamoxifen treatment regimen (Supplementary Fig. 1b). Similarly, 94% of Pdpn+ cells were also tdTomato+ (Supplementary Fig. 1b). A regenerative response was induced in the tracheal epithelium by treating mice with polidocanol, a detergent demonstrated to damage differentiated club and ciliated cells^33,34 (Supplementary Fig. 1b). In the course of 5–7 days after injury, BCs undergo proliferation to replace these differentiated cells and restore epithelial homeostasis. BCs were largely immotile under uninjured conditions, but, 1 day (d) after polidocanol injury, BCs underwent dynamic and concerted migration, resulting in increased fluidization as observed by an increase in the combined area of all BCs. (Fig. 1b, Supplementary Fig. 2a, and Supplementary Movies 1–5).

**Fig. 1: BCs migrate after airway injury.**

Polidocanol and other widely-used injury models, such as H1N1 infection and bleomycin, induce tissue-wide damage^{11,33,35,36,37}. Because we wanted to investigate the BC response to local injury, and specifically examine directionality of migration, we employed a more spatially and temporally controlled form of damage. We utilized high-powered lasers to ablate a localized ~35 µm wound in the tracheal section. After injury by laser ablation, we observed local migration of BCs towards the site of ablation, which resulted in gradual closure of the wound (Fig. 1c, Supplementary Fig. 2b, and Supplementary Movies 6–8). Similar to migration observed after polidocanol damage, BCs extended protrusions followed by translocation of the cell body towards the site of injury.

The presence of protrusion formation during BC migration suggests a role for actomyosin activity. To confirm this, we inhibited actin polymerization with Cytochalasin B³⁸. We found that BCs in both polidocanol-injured and laser-ablated tissue failed to migrate (Fig. 1b, c and Supplementary Movies 9 and 10). Inhibiting actin impaired BC migration after polidocanol injury, and dramatically reduced closure rates of the wound after laser ablation.

Altogether, these data support a model in which BCs undergo concerted, actin-dependent migration towards sites of injury to facilitate wound healing after detergent or laser-induced damage, consistent with previously described collective BC migration after viral injury¹¹. The absence of BC migration under homeostatic conditions suggests that this behavior is activated as part of the regenerative response. The signaling mechanisms regulating BC migration after injury remained unclear, however. We have previously shown that injury-induced stem cell migration in the Drosophila intestine²⁸ is regulated by non-canonical Wnt signaling, and hypothesized that the signaling mechanisms involved in triggering and promoting stem cell migration in barrier epithelia may be evolutionarily conserved. Given that non-canonical Wnt signaling is also important in maintaining airway homeostasis¹³, we tested whether this pathway also regulates BC migration.

Non-canonical Wnt signaling regulates BC migration

Both Fzd7 and Ptk7 serve as co-receptors of Wnt, and act in non-canonical Wnt signaling^{20,21,26,39,40}. Therefore, to more specifically target non-canonical Wnt signaling, we used a Krt5::Cre^ERT2 tamoxifen-inducible driver to knock out Ptk7 in BCs in combination with the Rosa26:LSL-tdTomato transgene (Supplementary Fig. 1a)^1,26,32. Knocking out Ptk7 was sufficient to impair BC migration in both polidocanol and ablation injury models (Fig. 2 and Supplementary Movies 11 and 12). As another strategy to specifically disrupt non-canonical Wnt signaling, we impaired Fzd7 function with an inhibitory peptide, dFzd7-21, that had previously been shown to inhibit Fzd7 but not Fzd5 or Fzd8 signaling^26,41. Treatment of tracheal sections with dFzd7-21 similarly impaired directed BC migration after ablation (Fig. 2b and Supplementary Movie 13).

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

To further examine a role of non-canonical Wnt signaling in promoting BC migration, we assessed protein expression of the pathway’s components. Ptk7 was enriched in BCs as determined by immunostaining of medial-located sections (Fig. 1a and Supplementary Fig. 3a). Importantly, this signal was dramatically reduced in BCs after knocking out Ptk7, demonstrating effective knock out efficiency. Under uninjured conditions, both Dvl1 and Fzd7 were undetectable in BCs, potentially due to low expression or a dispersed localization (Fig. 3). However, 1d after polidocanol injury, both Dvl1 and Fzd7 strongly localized to the cell cortex. The presence of cortical Dvl1 is attributed to activation of non-canonical Wnt signaling^42,43. Consistent with this, disrupting non-canonical Wnt activity by knocking out Ptk7 was sufficient to abolish Dvl1 localization at the BC cortex (Fig. 3b). We also examined whether the changes in Dvl1 localization can be attributed to its transcriptional regulation. We performed RT-qPCR on sorted tdTomato + BCs, and found that Dvl1 mRNA levels were increased modestly but insignificantly 1d after polidocanol injury (Supplementary Fig. 3b). Interestingly, Dvl1 transcript levels in injured BCs were reduced after depleting Ptk7, suggesting that non-canonical Wnt signaling may regulate Dvl transcriptionally in addition to post-translationally (Supplementary Fig. 3b).

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

We then examined the role of Wnt ligands during BC migration by inhibiting Wnt secretion with the porcupine inhibitor LGK974⁴⁴. Treatment with LGK974 reduced the migratory capacity of BCs (Fig. 4a and Supplementary Movie 14). Specifically inhibiting ß-catenin with iCRT14 did not greatly affect BC migration (Fig. 4a, Supplementary Fig. 3c, and Supplementary Movie 15), suggesting that canonical Wnt signaling does not play a large role in BC migration. To further explore this, we tested the effect of recombinant Wnt molecules from both canonical and non-canonical Wnt pathways: Wnt3a, which is associated with canonical Wnt signaling, and Wnt5a, which is associated with non-canonical Wnt signaling^26,27,45. Addition of Wnt3a to ablated tissue did not affect wound closure (Fig. 4a, Supplementary Fig. 3c, and Supplementary Movie 16). In sharp contrast, the addition of Wnt5a increased the rate of wound closure (Fig. 4a and Supplementary Movie 17).

**Fig. 4: Wnt5a promotes BC migration.**

We then compared protein expression of Wnt3a versus Wnt5a. Because differentiated cells of the epithelium are lost shortly after polidocanol injury³³, Wnt localization was assayed 2 h (hr) after injury. Wnt3a was expressed sparsely in uninjured BCs, and did not significantly differ in localization or intensity after injury (Supplementary Fig. 3d). Wnt5a was similarly expressed sparsely in uninjured BCs, but, unlike Wnt3a, was found localized at the BC cortex 2 h after polidocanol (Fig. 4a). Knocking out Ptk7 was sufficient to decrease Wnt5a localization to the cortex of injured BCs (Fig. 4b), which is consistent with Ptk7’s function as a Wnt co-receptor^21,26. Wnt5a localization was also analyzed in Ptk7 knockout mice with lower recombination efficiency, enabling comparison of Ptk7-depleted BCs with control BCs in the same trachea (Supplementary Fig. 4a). Wnt5a cortical decoration was observed 2 h after polidocanol injury in control, tdTomato- BCs, but was absent in neighboring Ptk7-depleted, tdTomato+ BCs (Supplementary Fig. 4a).

To better understand the source of secreted Wnt5a within the regenerating trachea, we measured Wnt5a transcript levels using RNAscope. Puncta representing mRNA was observed within the epithelium and mesenchyme of undamaged trachea. However, the number of puncta in Krt5+ BCs did not significantly change 2 h after polidocanol (Supplementary Fig. 4b). Instead, we found that Wnt5a transcript levels were increased in PDGFRa+ fibroblasts 2 h after injury, and this increase was not affected by knocking out Ptk7 in BCs (Fig. 4c). PDGFRa+ fibroblasts are abundant within the tracheal mesenchyme⁴⁶, and in previous studies, PDGFRa+ fibroblasts in the tracheal mesenchyme were reported to secrete multiple Wnt ligands to regulate BC proliferation¹⁴. PDGFRa+ fibroblasts in alveoli were further reported to secrete Wnt5a to regulate ATII stem cell proliferation¹⁵. Altogether, these data suggest that BC migration is induced by Wnt5a secreted from mesenchymal fibroblasts that activate non-canonical Wnt signaling in epithelial BCs.

Non-canonical Wnt-dependent YAP activity is required for BC migration

Previous studies have demonstrated that non-canonical Wnt signaling plays critical roles in activating the YAP pathway^26,27. Furthermore, it has been reported that YAP activity is required for BC migration within the distal airway after H1N1 viral infection¹¹. We tested whether non-canonical Wnt signaling promotes BC migration through the activation of YAP. YAP expression is diffuse in BCs from uninjured trachea (Fig. 5a). After 1d polidocanol-induced injury, YAP localizes to the nucleus of BCs, which is characteristic of YAP activation^24,25. Importantly, YAP localization to the nucleus is lost when Ptk7 is depleted in injured BCs (Fig. 5a). We then determined whether YAP activation is required for BC migration. Inhibition of YAP/TEAD transcriptional activity by Verteporfin was sufficient to greatly impair wound closure after laser ablation (Fig. 5b and Supplementary Movie 18). These data suggest that YAP is activated in a non-canonical Wnt-dependent manner after injury, and that this process is critical for regulating BC migration.

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

Impairing BC migration causes regenerative decline

To determine whether migration is critical for tissue repair, we tested whether impairing BC migration affects regeneration of the tracheal epithelium. Epithelial thickness is drastically reduced after damage by polidocanol, but regenerates after five days of recovery (Fig. 6a and Supplementary Fig. 5a). Knocking out Ptk7 did not affect epithelial thickness of uninjured trachea, but decreased recovery of epithelial thickness 5d after injury (Fig. 6a). We then tested whether impaired regeneration was due to the effect of Ptk7 loss of function on tissue composition. In genetic controls, both differentiated Foxj1+ ciliated and CC10+ club cells were completely absent 1d after polidocanol injury, but returned by 5d post injury (Fig. 6a–c and Supplementary Fig. 5b, c). In contrast, knocking out Ptk7 resulted in reduced differentiated cell numbers 5d after injury (Fig. 6a–c and Supplementary Fig. 5c). Non-canonical Wnt does not seem to play a major role in the survival of differentiated cells, as knocking out Ptk7 did not affect relative differentiated cell numbers under homeostatic conditions (Fig. 6a–c and Supplementary Fig. 5b, c). The consequence of knocking out Ptk7 on epithelial thickness and tissue composition was similar along the whole proximal/distal axis of the trachea (Supplementary Fig. 6). Decreased recovery of epithelial thickness and Foxj1+ ciliated cell number in 5d post injured trachea was observed in proximal, middle, and distal regions after BC-specific depletion of Ptk7 (Supplementary Fig. 6a). The decreased epithelial thickness after Ptk7 depletion was also observed when analyzing the entire length of H&E stained trachea (Supplementary Fig. 6b).

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

We then tested whether the observed decrease in differentiation and regeneration after disrupting non-canonical Wnt signaling was, in part, due to effects on BC proliferation. Mitotic activity, as determined by phospho-histone H3 (PH3), is very low under homeostatic conditions, but increases greatly 2d after polidocanol injury (Fig. 6d). Knocking out Ptk7 in BCs from uninjured trachea did not significantly affect the number of mitotic cells. In stark contrast, knocking out Ptk7 in BCs from injured trachea dramatically reduced mitotic figures (Fig. 6d), indicating that non-canonical Wnt influences cell cycle progression in BCs.

Discussion

Our study demonstrates that BCs migrate towards sites of injury in response to non-canonical Wnt signaling, which is activated via a Wnt5a/Fzd7/Ptk7/Dvl1 signaling axis, and concludes with YAP activation (Fig. 6e). Disrupting non-canonical Wnt signaling impairs regeneration, suggesting a critical role of this pathway during airway repair. Our focus on BC migration within the trachea offers additional insight compared to previous studies in in vitro cultures or distal airway explant models^8,29,30,31. BCs are present in the proximal airway under homeostatic condition, but are only generated in the distal airway after injury^1,7,8,9,10. Our tracheal explant model enables the study of regulatory mechanisms and migratory patterns more immediately following injury, as we can examine the transition from static to motile BCs in homeostatic versus injury conditions.

Wnt signaling plays a critical regulatory function in maintaining airway homeostasis^13,14,15,16. In the proximal airway, canonical Wnt signaling has been identified to promote airway regeneration by regulating BC proliferation and ciliated cell differentiation^14,47,48. Wnt5a is required during alveolar regeneration to regulate self-renewal versus differentiation of alveolar type (AT) II cells¹⁵. We observe that knocking out Ptk7 decreases dividing BC numbers in the trachea, resulting in fewer club and ciliated cells. This suggests that non-canonical Wnt activity regulates BC cell cycle progression, but it remains unclear whether the pathway also regulates the differentiation process or if the loss of ciliated cells after Ptk7 depletion is a result of impaired mitotic progression. In contrast to BC proliferation, which requires both canonical and non-canonical Wnt signaling, our data suggest that the regulation of BC migration relies on non-canonical rather than canonical Wnt signaling, as manipulating Wnt3a levels and β-catenin function does not grossly affect wound closure. Importantly, we cannot rule out the possibility that canonical Wnt signaling plays a role in longer-range cell migration.

Previous studies have reported heterogeneity of BC regenerative function along the dorsal-ventral axis of the trachea^49,50. Our live imaging analyses were performed on the most ventral region of the trachea, as the cartilage provides additional stability during live imaging. In future studies, it will be interesting to assess differences in motility between ventral and dorsal regions, which will require additional method development. Along the proximal-distal axis, Ptk7 depletion consistently decreased recovery of ciliated cell numbers and epithelial size in all regions, suggesting that non-canonical Wnt signaling serves as a general regulatory mechanism of BCs. While we cannot rule out the possibility that non-canonical Wnt activation and the kinetics of BC migration may be different in the dorsal region of the trachea, the capabilities of BCs to migrate to more distal parts of the airway¹¹ suggests that BC migration is part of the general regenerative response.

Our data identify Wnt5a as the key Wnt in regulating BC migration, and suggest that fibroblasts in the mesenchyme function as a source of Wnt5a. In the alveoli, Wnt5a is secreted from neighboring fibroblasts after injury to regulate alveolar stem cell renewal¹⁵. In the trachea, spatiotemporal control of Wnt secretion has been demonstrated to regulate regeneration after polidocanol injury¹⁴. Wnt secretion from mesenchymal fibroblasts is required to activate canonical Wnt signaling for BC proliferation in the early stages of regeneration, and Wnt secretion from BCs activates canonical Wnt at later stages to promote differentiation. Our data builds on these findings, as the original study did not investigate non-canonical Wnt signaling or identify the role of specific Wnt ligands. We find that Ptk7 KO resulted in BC migratory and proliferative defects early during the regenerative response, which coincides with our observed Wnt5a upregulation in fibroblasts. The spatiotemporal dynamics of Wnt5a secretion, whether non-canonical Wnt signaling remains activated at later stages of regeneration, and whether BCs negatively regulates Wnt5a secretion after completion of migration, however, remains unclear. The interplay between canonical and non-canonical Wnt signaling during regeneration, and the extent in which Ptk7 contributes to canonical Wnt regulation are important areas of study for future research. Both canonical and non-canonical Wnt signaling has been observed to be dysregulated in chronic airway pathologies such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease^{51,52,53,54,55,56}. Abundant BC migration has also been characterized during IPF, and it would be interesting to examine whether these migratory defects are contributed by over-activation of non-canonical Wnt signaling^11,12.

Our study in the trachea aligns with previous reports identifying a role of YAP signaling in regulating BC migration along the distal airway¹¹. YAP was observed to be activated by IL-6 secretion after H1N1 infection and bleomycin injury¹². While the addition of IL-6 or Wnt5a increased BC motility, it is unclear whether BC migration requires both Wnt5a and IL-6 secretion. The nature of the challenge to the airway could lead to a more dominant role of Wnt5a versus IL-6. For example, viral infection and resulting inflammation may trigger greater IL-6 secretion versus Wnt5a secretion following polidocanol injury or ablation. Furthermore, regulatory mechanisms may be different depending on the time frame following perturbation, as BC migration in this study was assayed within a day following injury, while H1N1-mediated migration was assayed 11 days following infection. Severity of damage, particularly to the mesenchyme, could also contribute to activation of different signaling pathways as minimal damage in the mesenchyme occurs after polidocanol^33,34 and ablation compared to the fibrotic damage caused by bleomycin³⁷. We suspect ablation largely spares the mesenchyme as BCs are still capable of migrating into the wound, suggesting that the epithelial basement membrane and the surrounding mesenchymal extracellular matrix are intact. Given the clear importance of both non-canonical Wnt and IL-6 signaling in migration, it would be interesting to study the sequential timing of these pathways. One possible model could be that YAP signaling is first activated by non-canonical Wnt signaling to promote more immediate BC migration within the proximal airway, before IL-6 signaling is activated for long-term and long-range BC migration along the distal airway. Coordination of cell migration and division is important to prevent premature division before cells complete migration towards sites of injury. We have previously identified that migration and division of stem cells in the Drosophila intestine are closely linked, and that affecting one process affects the other²⁸. We observe that knocking out Ptk7 impairs mitosis of BCs, but it is not clear whether this is a consequence of impairing migration or whether non-canonical Wnt plays additional roles in regulating cell cycle-dependent kinases. Identifying strategies to more specifically inhibit BC migration will be important in understanding the timing and regulation of migration and proliferation during tissue repair.

Advances in our understanding of dynamic cellular behavior within the airway have been limited by the ability to monitor tissue-wide responses to injury in real time. As live imaging strategies became more sophisticated in recent years, the ability to characterize stem cell migration in the airway has improved. As such, the importance of stem cell migration in remodeling the airway, and the link between migratory dysfunction and airway pathology, have been increasingly clear. Understanding the regulatory mechanism(s) of BC migration and how these mechanisms become dysregulated during disease, will thus inform the identification of interventions for airway pathologies.

Methods

Mice transgenics and chemical administration

All mice were used in accordance with protocols approved by Genentech’s Institutional Animal Care and Use Committee, and adhere to the NRC Guidelines for the Care and Use of Laboratory Animals. Male and female mice between 3 and 5 months of age were used for this study. Only male mice were used to quantify epithelium thickness (detailed below). Krt5::Cre^ERT2 was previously generated¹, and bred into Rosa26-tdTomato mice (Jackson Laboratory, JAX:007914) to enable expression in Krt5+ BCs and their progeny. Ptk7^fl/fl (Gene ID: 71461) mice were purchased from genOway²⁶. Krt5::CreERT2; Rosa26-tdTomato mice were crossed with Ptk7^fl/fl mice to conditionally knock out Ptk7 in Krt5+ BCs.

Tamoxifen (Sigma, T5648) dissolved in corn oil was administered intraperitoneal every other day for five days at 0.16 mg/g body weight, for a total of three injections. Mice were euthanized by CO₂ 7–11 days after the final tamoxifen injection (Supplementary Fig. 1a). 12.5 µL of 2% polidocanol (Sigma, 88315) in phosphate-buffered saline (PBS) was administered by intracheal instillation into isoflurane-anesthetized mice 6 days after the final tamoxifen injection. For uninjured controls, PBS was administered instead.

Live imaging of tracheal sections

Trachea were dissected in 50% DMEM and 50% F12 media (DMEM:F12) with 5% fetal bovine serum (FBS). Trachea were embedding in 3% low melting agarose in DMEM:F12, 5% FBS, 1.5mM L-glutamine, 1× insulin-transferrin-selenium-G (Gibco, 41400045), 0.1 µg/ml cholera toxin, 0.025 µg/ml murine EGF, and 0.03 mg/ml bovine pituitary extract (mouse tracheal epithelial cell media, MTEC)⁵⁷ for sectioning. 300 µm thick longitudinal tracheal sections were processed on a Leica VT1200 vibratome at 0.18 mm/s, with 1.8 mm amplitude, and in DMEM:F12. The agarose is not removed from the tracheal section before imaging, and contributes to physically stabilizing the sample.

The most ventral tracheal section was transferred to a 35 mm glass bottom dish (MatTek, P35G-1.5-14-C), restrained with a metal harp (Warner Instruments, 641418), and submerged in MTEC. The cartilage on the ventral section, and the increased volume compared to a medial section increases the stability of the section during imaging. Tracheal sections were imaged at 10 min intervals for at least 3 h on a Yokogawa CSU-W1/Zeiss 3i Marianas spinning disk confocal microscopy system with a 40× PlanFleur objective, or, for laser ablation experiments, imaged at 30 min intervals for at least 4 h on a Bruker Ultima In Vivo Multiphoton/Newport MaiTai DeepSee Spectra Physics 2-photon microscopy system with a 20x Olympus 1.0 water immersion objective. Tracheal sections remained incubated at 37 °C with 5% CO₂ throughout the imaging process.

Laser ablation of tracheal sections

Ablated sections and corresponding undamaged controls were imaged with a 960 nm wavelength, 2.8 µs dwell time, and ~25 mW power, using a Bruker Ultima In Vivo Multiphoton/Newport MaiTai DeepSee Spectra Physics 2-photon microscopy system with a 20× Olympus 1.0 water immersion objective. A wound of a ~ 35 µm diameter was created in the epithelium with a 880 nm wavelength, 20µs dwell time, ~278 mW power, and 60× zoom on the same system. Sections were imaged immediately following ablation on the 2-photon system at intervals of 30 min for ~4hrs.

Small molecule inhibitor and recombinant protein treatment of tracheal sections

Tracheal sections were treated with small molecule inhibitors or recombinant protein in MTEC at 37 °C with 5% CO₂ for 1 h prior to imaging, and throughout the live imaging process. Mock-treated sections were incubated in 0.1% DMSO in MTEC at 37 °C with 5% CO₂ for 1 h prior to imaging, and throughout the live imaging process.

Small molecule inhibitors and concentrations used in this study: Cytochalasin B (10 µg/ml, Sigma Aldrich, C6762), dFzd7-21 (100 µg/ml, Genentech, Inc.)^25,40, LGK974 (20 µM, Sigma Aldrich Chemicals, 531091), iCrt14 (30 µM, Millipore, SML0203), Verteporfin (10 µM, Selleck Chemical, S178610), Wnt5a (500 ng/ml, Novus, 645-WN-010), and Wnt3a (500 ng/ml, Novus, 1324-WN-010).

Immunostaining and histology of tracheal sections

Trachea were dissected in DMEM:F12 with 5% fetal bovine serum (FBS), and fixed overnight in 4% paraformaldehyde in PBS at 4 °C. Trachea were embedded in 3% low melting agarose in PBS, and sectioned longitudinally on a Leica VT1200 vibratome at 50 µm thickness, 0.18 mm/s, 1.8 mm amplitude, and in PBS. More medially-located sections were chosen to provide a cross sectional view. Tracheal sections were blocked with PBS, 0.1% Triton X-100 supplemented with 5% donkey serum. Sections were incubated in primary antibody overnight at 4 °C antibody, washed in PBS + 0.01% Tween-20, and incubated in secondary antibodies and Hoechst stain (1:1000) for 3 h at room temperature. The agarose remained around the tissue sample throughout the immunostaining and slide mounting process.

For RNAscope experiments and immunostaining experiments for analyses along the proximal/distal axis (Fig. 4c, Supplementary Figs. 4b and 6), mouse trachea were collected and fixed with 10% formalin at room temperature overnight, and washed with 70% ethanol twice the following day. Samples were embedded in paraffin and sectioned at 7μm thickness. Whole lung sections stained with hematoxylin and eosin (H&E) were scanned using a Hamamatsu Nanozoomer S360 at 20× magnification using NZAcquire 3.10.10. For immmunostaining of paraffin sections, sections were baked on a slide warmer at 60 °C for 30 min, dewaxed, and rehydrated. After heat-induced antigen retrieval in citrate buffer (Millipore, S1699), immunostaining continued as described above.

Primary antibodies used in this study: Mouse monoclonal against Wnt5a (1:300, Invitrogen, MA5-15502, RRID:AB_10985211) and CC10 (1:300, Santa Cruz, sc-365992, RRID:AB_10915481); rabbit polyclonal against Disheveled1 (Dvl1; 1:300, Abcam, ab233003), Wnt3a (1:300, Abcam, ab219412, RRID:AB_2924383), YAP (1:300, Cell Signaling, 4912, RRID:AB_2218911), Foxj1 (1:300, Abcam, ab235445), Krt5 (1:300, Abcam, ab52635, RRID:AB_869890), and phospho-Histone H3 (1:1000, EMD Millipore, 06-570, RRID:AB_310177); rat monoclonal against Frizzled7 (Fzd7; 1:300, R&D Systems, MAB1981, RRID:AB_2247464); goat polyclonal against Ptk7 (1:300, R&D Systems, AF4499, RRID:AB_2174357) and tdTomato (1:500, SICGEN, ab8181, RRID:AB_2722750); and Syrian hamster monoclonal against podoplanin (Pdpn; 1:300, DSHB, 8.1.1, RRID:AB_531893). Secondary antibodies against mouse, rabbit, and goat were Alexa Fluor dyes from Invitrogen (1:300). Secondary antibodies against Syrian hamster were Alexa Fluor dyes from Abcam (1:300). Secondary antibodies against rat were cyanine dyes from Jackson ImmunoResearch Laboratories.

Microscopy and image analysis

Images of fixed tissue were taken with a Yokogawa CSU-W1/Zeiss 3i Marianas spinning disk confocal microscopy system using a 40× or 63× PlanFleur objective. Images were analyzed and processed using ImageJ (NIH, Bethesda, MD) and Adobe Photoshop. Figures were composed in Adobe Illustrator.

RNA in situ hybridization co-detection

Freshly cut paraffin sections (7 μm thick) were baked on a slide warmer at 60 °C for 30 min, dewaxed, and rehydrated by serial washes with xylene, 100% ethanol, 90% ethanol, and 70% ethanol for 5 min each. Antigen retrieval was performed with an RNA-protein co-detection ancillary kit (Advanced Cell Diagnostics, 323180). Sections were incubated with primary antibodies against tdTomato (1:500, SICGEN, ab8181, RRID:AB_2722750) and, when applicable, Krt5 (1:300, Abcam, ab52635, RRID:AB_869890) at 4 °C overnight. Signals from Wnt5a and, when applicable, PDGFRa probes (Advanced Cell Diagnostics) were developed and amplified by the RNAscope multiplex assay kit (Advanced Cell Diagnostics, 323100) the following day.

FACs sorting and RT-qPCR

Mouse tracheas were collected and shredded with scissors. Samples were dissociated with Papain dissociation system (Worthington Biochemical Corporation, LK003150) at 37 °C for 30 min. After vigorous pipetting, cells were filtered through 40um sterile filters followed by red blood cell lysis at room temperature for 5 min. After washing, cells were stained with anti-Epcam PE/Cy7 (1:100, Biolegend, 103114), anti-CD45 FITC (1:100, BD Biosciences, BD553080), anti-SSEA-1 APC (1:100, Biolegend, 125618), and anti-CD24 Alexa594 (1:100, Biolegend, 101834, RRID:AB_2565427) on ice for 30 min. Cells were resuspended in 2% bovine serum albumin (BSA) containing Sytox blue (1:1000, Thermo Fisher Scientific, S34857), and filtered through 40μm filters. tdTomato+ basal cells (CD45-/Epcam + /tdTomato + /SSEA1-/CD24-) were sorted by a FACS Aria (BD Biosciences) with a 100μm nozzle using the 8-4-0 sorting mode. RNA was extracted using Qiagen RNeasy Micro kits. RNA quantity and quality was measured using a Nanodrop (Thermo Scientific). cDNA was made using the iScript cDNA Synthesis Kit (BioRad, 1708890). For Taqman gene expression assays, reactions were run on a QuantStudio 6 Flex Real-Time PCR System (ThermoFisher). The following Taqman probes were used: Dvl1 (Mm00438592_m1, mouse, FAM-MGB) and GADPH (Mm99999915_g1, mouse, FM-MGB). Threshold cycle values (Ct) of Dvl1 were normalized to a housekeeping gene, GAPDH (ΔCT).

Quantification and statistical analysis

All quantifications were performed manually using ImageJ software (NIH, Bethesda, MD).

Analysis of BC migration

BCs were identified by tdTomato fluorophore expression, as 97% and 100% of tdTomato+ cells were BCs (Supplementary Fig. 1b), as determined by Pdpn co-staining, in undamaged and 1d post polidocanol-treated trachea respectively. BCs were scored as migratory based on a change of cell shape and translocation of the cell body > 5 µm during the time lapse. Regions where individual BCs were not easily identifiable were not quantified, with an average of 80 BCs scored in a 150 µm × 150 µm region per movie. BC migration after polidocanol injury was also assayed by quantifying the relative change of the combined area of all BCs in a 300 µm × 300 µm region between the beginning of the time lapse acquisition and 3 h later. Masks were generated around tdTomato+ cells with the Huang thresholding method in ImageJ. The areas of all ROIs were then totaled to obtain the combined area of all BCs. Relative change of combined area (Δarea of BCs) was calculated as such: relative change in BC area = BC area at 3hr time point/BC area at beginning of acquisition. A relative change in BC area >1 would represent epithelial fluidization as BCs migrate into more space throughout the duration of the time lapse.

BC motility in ablation experiments was assayed by closure of the wound. The diameter of the wound was measured immediately following ablation, and then 4hrs after ablation. Wound closure was measured by the difference in wound diameter between these two time points (Δdiameter of wound).

Analysis of immunostaining

Epithelium thickness was measured by the distance from the basement membrane, as determined by basal tdTomato fluorophore expression (since BCs line the basement membrane), to the most apical region of the epithelium, as determined by either basal tdTomato fluorophore expression (in 1d or 5d post polidocanol) or overexposure of Pdpn or Foxj1 antibody (Supplementary Fig. 4a) to visualize background of the epithelium. tdTomato fluorophore expression was not used to visualize the apical end of the epithelium in undamaged tissue, as the vast majority of tdTomato+ cells are BCs at this stage (Supplementary Fig. 1b). Epithelium thickness was only measured in male mice.

To quantify relative cell numbers, cell fate of cells was identified by the following markers: BCs by Pdpn staining or tdTomato fluorophore expression, ciliated cells by Foxj1 staining, and club cells by CC10 staining. Mitotic cells were identified by phospho-histone H3 staining (PH3). Total cell number was determined by DAPI.

To quantify relative cortical protein levels, ImageJ was used to measure intensity levels across a line passing through the cortex of the cell. A 5 µm length line was drawn centered and perpendicular to the cortex, starting from the cytoplasm and ending outside the cell. The cortex was estimated either by Pdpn staining or tdTomato fluorophore expression. Cortical intensity was determined as the highest intensity within 0.2 µm of the line center (2.3–2.7 µm). Cytoplasmic intensity was determined by the average intensity from 0.6 µm to 1.4 µm of the line starting point within the cytoplasm. Relative cortical protein levels was quantified by normalizing cortical intensity with cytoplasmic intensity (relative cortical levels = cortical intensity/cytoplasmic intensity).

Statistical analysis and reproducibility

Each sample, ‘n’, is from at least three independent experiments, and is defined in the Figure Legends depending on experiment. The exception is when Wnt5a cortical localization was compared between tdTomato- and tdTomato+ BCs within the same trachea (Supplemental Fig. 4a), as obtaining mice with low but still present levels of recombination was stochastic. The number of independent experiments performed for these data was two. Additional statistical details for each experiment are also noted in the Figure Legends. Statistical analyses were performed with Prism (GraphPad Software, La Jolla, CA, USA). A Student’s t-test was used to determine statistical significance between two independent groups. A one-way ANOVA with Tukey test was used to determine statistical significance with multiple comparisons between three or more independent groups. Significance was accepted at the level of p < 0.05. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those generally employed in the field. All experiments (from both live and fixed samples) were quantified blindly.

Reporting summary

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

Data availability

This study did not generate any large data sets. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Heinrich Jasper ([email protected]). Unique biological materials generated in this study are readily available upon request, but will require an MTA. Any data generated from experiments are available on request. Source data are provided with this paper.

References

Rock, J. R. et al. Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc. Natl. Acad. Sci. USA 106, 12771–12775 (2009).
Article ADS CAS PubMed PubMed Central Google Scholar
Montoro, D. T. et al. A revised airway epithelial hierarchy includes CFTR-expressing ionocytes. Nature 560, 319–324 (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Mou, H. et al. Airway basal stem cells generate distinct subpopulations of PNECs. Cell Rep. 35, https://doi.org/10.1016/j.celrep.2021.109011 (2021).
Rock, J. R. et al. Notch-dependent differentiation of adult airway basal stem cells. Cell Stem Cell 8, 639–648 (2011).
Article CAS PubMed PubMed Central Google Scholar
Rock, J. R., Randell, S. H. & Hogan, B. L. Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling. Dis. Model. Mech. 3, 545–556 (2010).
Article CAS PubMed PubMed Central Google Scholar
Wu, M., Zhang, X., Lin, Y. & Zeng, Y. Roles of airway basal stem cells in lung homeostasis and regenerative medicine. Respir. Res. 23, 122 (2022).
Article CAS PubMed PubMed Central Google Scholar
Kumar, P. A. et al. Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection. Cell 147, 525–538 (2011).
Article CAS PubMed PubMed Central Google Scholar
Vaughan, A. E. et al. Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury. Nature 517, 621–625 (2015).
Article ADS CAS PubMed Google Scholar
Ray, S. et al. Rare SOX2+ airway progenitor cells generate KRT5+ cells that repopulate damaged alveolar parenchyma following influenza virus infection. Stem Cell Rep. 7, 817–825 (2016).
Article CAS Google Scholar
Kathiriya, J. J., Brumwell, A. N., Jackson, J. R., Tang, X. & Chapman, H. A. Distinct airway epithelial stem cells hide among club cells but mobilize to promote alveolar regeneration. Cell Stem Cell 26, 346–358.e344 (2020).
Article CAS PubMed PubMed Central Google Scholar
Stancil, I. T. & Michalski, J. E. Interleukin-6-dependent epithelial fluidization initiates fibrotic lung remodeling. Sci. Transl. Med. 14, eabo5254 (2022).
Article CAS PubMed PubMed Central Google Scholar
Stancil, I. T., Michalski, J. E. & Davis-Hall, D. Pulmonary fibrosis distal airway epithelia are dynamically and structurally dysfunctional. Nat. Commun. 12, 4566 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Aros, C. J., Pantoja, C. J. & Gomperts, B. N. Wnt signaling in lung development, regeneration, and disease progression. Commun. Biol. 4, 601 (2021).
Article PubMed PubMed Central Google Scholar
Aros, C. J. et al. Distinct spatiotemporally dynamic wnt-secreting niches regulate proximal airway regeneration and aging. Cell Stem Cell 27, 413–429.e414 (2020).
Article CAS PubMed PubMed Central Google Scholar
Nabhan, A. N., Brownfield, D. G., Harbury, P. B., Krasnow, M. A. & Desai, T. J. Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science 359, 1118–1123 (2018).
Article ADS CAS PubMed PubMed Central Google Scholar
Lynch, T. J. et al. Wnt signaling regulates airway epithelial stem cells in adult murine submucosal glands. Stem Cells 34, 2758–2771 (2016).
Article CAS PubMed Google Scholar
Nusse, R. & Clevers, H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell 169, 985–999 (2017).
Article CAS PubMed Google Scholar
Liu, J. et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct. Target. Ther. 7, 3 (2022).
Article PubMed PubMed Central Google Scholar
Davey, C. F. & Moens, C. B. Planar cell polarity in moving cells: think globally, act locally. Development 144, 187 (2017).
Article CAS PubMed PubMed Central Google Scholar
Lu, X. et al. PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates. Nature 430, 93–98 (2004).
Article ADS CAS PubMed Google Scholar
Martinez, S. et al. The PTK7 and ROR2 protein receptors interact in the vertebrate WNT/planar cell polarity (PCP) pathway. J. Biol. Chem. 290, 30562–30572 (2015).
Article CAS PubMed PubMed Central Google Scholar
Gao, C. & Chen, Y. G. Dishevelled: the hub of Wnt signaling. Cell. Signal. 22, 717–727 (2010).
Article CAS PubMed Google Scholar
Gómez-Orte, E., Sáenz-Narciso, B., Moreno, S. & Cabello, J. Multiple functions of the noncanonical Wnt pathway. Trends Genet. 29, 545–553 (2013).
Article PubMed Google Scholar
Mason, D. E. & Collins, J. M. YAP and TAZ limit cytoskeletal and focal adhesion maturation to enable persistent cell motility. J. Cell Biol. 218, 1369–1389 (2019).
Article CAS PubMed PubMed Central Google Scholar
Totaro, A., Panciera, T. & Piccolo, S. YAP/TAZ upstream signals and downstream responses. Nat. Cell Biol. 20, 888–899 (2018).
Article CAS PubMed PubMed Central Google Scholar
Yun, J. et al. Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling. Nat. Commun. 14, 156 (2023).
Article ADS CAS PubMed PubMed Central Google Scholar
Park, HyunW. et al. Alternative Wnt signaling activates YAP/TAZ. Cell 162, 780–794 (2015).
Article CAS PubMed PubMed Central Google Scholar
Hu, D. J.-K., Yun, J., Elstrott, J. & Jasper, H. Non-canonical Wnt signaling promotes directed migration of intestinal stem cells to sites of injury. Nat. Commun. 12, 7150 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Mitchel, J. A. et al. In primary airway epithelial cells, the unjamming transition is distinct from the epithelial-to-mesenchymal transition. Nat. Commun. 11, 5053 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Akram, K. M., Yates, L. L. & Mongey, R. Live imaging of alveologenesis in precision-cut lung slices reveals dynamic epithelial cell behaviour. Nat. Commun. 10, 1178 (2019).
Article ADS PubMed PubMed Central Google Scholar
Park, J. A. et al. Unjamming and cell shape in the asthmatic airway epithelium. Nat. Mater. 14, 1040–1048 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Haller, S. et al. mTORC1 activation during repeated regeneration impairs somatic stem cell maintenance. Cell Stem Cell 21, 806–818.e805 (2017).
Article CAS PubMed PubMed Central Google Scholar
Engler, A. E. et al. Airway-associated macrophages in homeostasis and repair. Cell Rep. 33, 108553 (2020).
Article CAS PubMed PubMed Central Google Scholar
Borthwick, D. W., Shahbazian, M., Todd Krantz, Q., Dorin, J. R. & Randell, S. H. Evidence for stem-cell niches in the tracheal epithelium. Am. J. Respir. Cell Mol. Biol. 24, 662–670 (2001).
Article CAS PubMed Google Scholar
Cassandras, M., Wang, C. & Kathiriya, J. Gli1(+) mesenchymal stromal cells form a pathological niche to promote airway progenitor metaplasia in the fibrotic lung. Nat. Cell Biol. 22, 1295–1306 (2020).
Article CAS PubMed PubMed Central Google Scholar
Kurche, J. S. et al. Muc5b enhances murine honeycomb-like cyst formation. Am. J. Respir. Cell Mol. Biol. 61, 544–546 (2019).
Article CAS PubMed PubMed Central Google Scholar
Degryse, A. L. et al. Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis. Am. J. Physiol. Lung Cell. Mol. Physiol. 299, L442–L452 (2010).
Article CAS PubMed PubMed Central Google Scholar
MacLean-Fletcher, S. & Pollard, T. D. Mechanism of action of cytochalasin B on actin. Cell 20, 329–341 (1980).
Article CAS PubMed Google Scholar
De Calisto, J., Araya, C., Marchant, L., Riaz, C. F. & Mayor, R. Essential role of non-canonical Wnt signalling in neural crest migration. Development 132, 2587 (2005).
Article PubMed Google Scholar
Phesse, T., Flanagan, D. & Vincan, E. Frizzled7: a promising achilles’ heel for targeting the Wnt receptor complex to treat cancer. Cancers 8, https://doi.org/10.3390/cancers8050050 (2016).
Nile, A. H. et al. A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells. Nat. Chem. Biol. 14, 582–590 (2018).
Article CAS PubMed Google Scholar
Axelrod, J. D. Unipolar membrane association of Dishevelled mediates Frizzled planar cell polarity signaling. Genes Dev. 15, 1182–1187 (2001).
Article CAS PubMed PubMed Central Google Scholar
Park, T. J., Gray, R. S., Sato, A., Habas, R. & Wallingford, J. B. Subcellular localization and signaling properties of dishevelled in developing vertebrate embryos. Curr. Biol. 15, 1039–1044 (2005).
Article CAS PubMed Google Scholar
Liu, J. et al. Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974. Proc. Natl. Acad. Sci. USA 110, 20224 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Ackers, I. & Malgor, R. Interrelationship of canonical and non-canonical Wnt signalling pathways in chronic metabolic diseases. Diab. Vasc. Dis. Res. 15, 3–13 (2018).
Article CAS PubMed Google Scholar
Tadokoro, T. et al. IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells. Proc. Natl. Acad. Sci. USA 111, E3641–E3649 (2014).
Article CAS PubMed PubMed Central Google Scholar
Brechbuhl, H. M. et al. β-catenin dosage is a critical determinant of tracheal basal cell fate determination. Am. J. Pathol. 179, 367–379 (2011).
Article PubMed PubMed Central Google Scholar
Giangreco, A. et al. β-Catenin determines upper airway progenitor cell fate and preinvasive squamous lung cancer progression by modulating epithelial-mesenchymal transition. J. Pathol. 226, 575–587 (2012).
Article CAS PubMed PubMed Central Google Scholar
Tadokoro, T. & Tanaka, K. Dorso-ventral heterogeneity in tracheal basal stem cells. 10, https://doi.org/10.1242/bio.058676 (2021).
Zhou, Y. & Yang, Y. Airway basal cells show regionally distinct potential to undergo metaplastic differentiation. 11, https://doi.org/10.7554/eLife.80083 (2022).
Baarsma, H. A. & Königshoff, M. ‘WNT-er is coming’: WNT signalling in chronic lung diseases. Thorax 72, 746 (2017).
Article CAS PubMed Google Scholar
Königshoff, M. et al. WNT1-inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. J. Clin. Investig. 119, 772–787 (2009).
PubMed PubMed Central Google Scholar
Baarsma, H. A. & Skronska-Wasek, W. Noncanonical WNT-5A signaling impairs endogenous lung repair in COPD. J. Exp. Med. 214, 143–163 (2017).
Article PubMed PubMed Central Google Scholar
Vuga, L. J. et al. WNT5A is a regulator of fibroblast proliferation and resistance to apoptosis. Am. J. Respir. Cell Mol. Biol. 41, 583–589 (2009).
Article CAS PubMed PubMed Central Google Scholar
Königshoff, M. et al. Functional Wnt signaling is increased in idiopathic pulmonary fibrosis. PLoS ONE 3, e2142 (2008).
Article ADS PubMed PubMed Central Google Scholar
Newman, D. R. et al. Expression of WNT5A in idiopathic pulmonary fibrosis and its control by TGF-β and WNT7B in human lung fibroblasts. J. Histochem. Cytochem. 64, 99–111 (2015).
Article PubMed PubMed Central Google Scholar
Eenjes, E. et al. A novel method for expansion and differentiation of mouse tracheal epithelial cells in culture. Sci. Rep. 8, 7349 (2018).
Article ADS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank Drs. Meredith Sagolla, Sarah Gierke, and Max Tauc-Adrian for microscopy assistance, and Drs. Aaron Nile and Rami Hannoush for generation of dFzd7-21.

Author information

Authors and Affiliations

Regenerative Medicine, Genentech Inc., South San Francisco, CA, USA
Daniel Jun-Kit Hu, Xiaoyu Tracy Cai, Jesse Simons, Jina Yun & Heinrich Jasper
Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, IL, USA
Daniel Jun-Kit Hu
Department of Medicine, Loyola University Chicago, Stritch School of Medicine, Maywood, IL, USA
Xiaoyu Tracy Cai
Translational Imaging, Genentech Inc., South San Francisco, CA, USA
Justin Elstrott

Authors

Daniel Jun-Kit Hu
View author publications
Search author on:PubMed Google Scholar
Xiaoyu Tracy Cai
View author publications
Search author on:PubMed Google Scholar
Jesse Simons
View author publications
Search author on:PubMed Google Scholar
Jina Yun
View author publications
Search author on:PubMed Google Scholar
Justin Elstrott
View author publications
Search author on:PubMed Google Scholar
Heinrich Jasper
View author publications
Search author on:PubMed Google Scholar

Contributions

D.J.H. and H.J. conceived the project and wrote the manuscript. D.J.H. performed experiments and analyzed data. X.C. contributed in generating animal protocols, breeding program, polidocanol injury protocols, and figure cartoon, and perform experiments. J.S. assisted in polidocanol injury, tamoxifen injections, trachea dissections, and trachea sectioning. J.Y. contributed in mouse generation. J.E. helped establish the live imaging and laser ablation platform on the 2-photon microscopy system.

Corresponding authors

Correspondence to Daniel Jun-Kit Hu or Heinrich Jasper.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks Debora Sinner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplementary Movie 1

Supplementary Movie 2

Supplementary Movie 3

Supplementary Movie 4

Supplementary Movie 5

Supplementary Movie 6

Supplementary Movie 7

Supplementary Movie 8

Supplementary Movie 9

Supplementary Movie 10

Supplementary Movie 11

Supplementary Movie 12

Supplementary Movie 13

Supplementary Movie 14

Supplementary Movie 15

Supplementary Movie 16

Supplementary Movie 17

Supplementary Movie 18

Reporting Summary

Transparent Peer Review file

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Hu, D.JK., Cai, X.T., Simons, J. et al. Non-canonical Wnt signaling promotes epithelial fluidization in the repairing airway. Nat Commun 16, 4124 (2025). https://doi.org/10.1038/s41467-025-59320-1

Download citation

Received: 10 July 2024
Accepted: 17 April 2025
Published: 03 May 2025
DOI: https://doi.org/10.1038/s41467-025-59320-1

This article is cited by

Mechanistic Insights and Therapeutic Potential of Wnt5a Signaling in Alveolar Epithelial Cell Development and Bronchopulmonary Dysplasia
- Jinglan Huang
- Xin Li
- Jun Tang
Stem Cell Reviews and Reports (2025)