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• W3045351648 abstract "•Tracheal epithelial β-catenin tightly controls proliferation and differentiation•PDGFRα+ lineage comprises Wnt signaling niche necessary for epithelial proliferation•Airway basal stem cell-derived Wnts facilitate differentiation to ciliated cells•Age-related Wnt/β-catenin niche changes drive gland-like epithelial invaginations Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/β-catenin within ABSCs was essential for proliferation post-injury in vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFRα+ lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of β-catenin in young mice induced their formation in vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging. Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/β-catenin within ABSCs was essential for proliferation post-injury in vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFRα+ lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of β-catenin in young mice induced their formation in vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging. The lung is a structurally and functionally intricate organ with several diverse cell types. Appreciable changes in the composition of the epithelial stem cell and mesenchymal cell compartments along the proximo-distal axis of the airway contribute to its striking complexity. At its most proximal portion, the cartilaginous conducting airways play a vital role in host defense that protects mammals from airborne pathogens. This is accomplished by a highly specialized simple pseudostratified mucociliary epithelium that arises from anatomically defined, resident adult airway basal stem cells (ABSCs) marked by Keratin 5 (K5) expression (Cole et al., 2010Cole B.B. Smith R.W. Jenkins K.M. Graham B.B. Reynolds P.R. Reynolds S.D. Tracheal Basal cells: a facultative progenitor cell pool.Am. J. Pathol. 2010; 177: 362-376Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar; Hong et al., 2004Hong K.U. Reynolds S.D. Watkins S. Fuchs E. Stripp B.R. Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium.Am. J. Pathol. 2004; 164: 577-588Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar; Montoro et al., 2018Montoro D.T. Haber A.L. Biton M. Vinarsky V. Lin B. Birket S.E. Yuan F. Chen S. Leung H.M. Villoria J. et al.A revised airway epithelial hierarchy includes CFTR-expressing ionocytes.Nature. 2018; 560: 319-324Crossref PubMed Scopus (334) Google Scholar; Plasschaert et al., 2018Plasschaert L.W. Žilionis R. Choo-Wing R. Savova V. Knehr J. Roma G. Klein A.M. Jaffe A.B. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.Nature. 2018; 560: 377-381Crossref PubMed Scopus (308) Google Scholar; Rock et al., 2009Rock J.R. Onaitis M.W. Rawlins E.L. Lu Y. Clark C.P. Xue Y. Randell S.H. Hogan B.L. Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc. Natl. Acad. Sci. USA. 2009; 106: 12771-12775Crossref PubMed Scopus (843) Google Scholar; Schoch et al., 2004Schoch K.G. Lori A. Burns K.A. Eldred T. Olsen J.C. Randell S.H. A subset of mouse tracheal epithelial basal cells generates large colonies in vitro.Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: L631-L642Crossref PubMed Scopus (125) Google Scholar). In contrast, the non-cartilaginous bronchiolar, alveolar, and bronchioalveolar tissues of the airway have region-specific adult stem cell populations (Barkauskas et al., 2013Barkauskas C.E. Cronce M.J. Rackley C.R. Bowie E.J. Keene D.R. Stripp B.R. Randell S.H. Noble P.W. Hogan B.L. Type 2 alveolar cells are stem cells in adult lung.J. Clin. Invest. 2013; 123: 3025-3036Crossref PubMed Scopus (698) Google Scholar; Desai et al., 2014Desai T.J. Brownfield D.G. Krasnow M.A. Alveolar progenitor and stem cells in lung development, renewal and cancer.Nature. 2014; 507: 190-194Crossref PubMed Scopus (459) Google Scholar; Kim et al., 2005Kim C.F. Jackson E.L. Woolfenden A.E. Lawrence S. Babar I. Vogel S. Crowley D. Bronson R.T. Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer.Cell. 2005; 121: 823-835Abstract Full Text Full Text PDF PubMed Scopus (1715) Google Scholar; Rawlins et al., 2009Rawlins E.L. Okubo T. Xue Y. Brass D.M. Auten R.L. Hasegawa H. Wang F. Hogan B.L. The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium.Cell Stem Cell. 2009; 4: 525-534Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar) capable of self-renewal and differentiation. Following injury, the tracheal repair response occurs in two distinct phases. Denuding of the epithelium triggers an initial proliferative expansion phase in which ABSCs undergo symmetric followed by asymmetric division that gives rise to K8+ early progenitors (Paul et al., 2014Paul M.K. Bisht B. Darmawan D.O. Chiou R. Ha V.L. Wallace W.D. Chon A.T. Hegab A.E. Grogan T. Elashoff D.A. et al.Dynamic changes in intracellular ROS levels regulate airway basal stem cell homeostasis through Nrf2-dependent Notch signaling.Cell Stem Cell. 2014; 15: 199-214Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar; Rock et al., 2009Rock J.R. Onaitis M.W. Rawlins E.L. Lu Y. Clark C.P. Xue Y. Randell S.H. Hogan B.L. Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc. Natl. Acad. Sci. USA. 2009; 106: 12771-12775Crossref PubMed Scopus (843) Google Scholar, Rock et al., 2011Rock J.R. Gao X. Xue Y. Randell S.H. Kong Y.Y. Hogan B.L. Notch-dependent differentiation of adult airway basal stem cells.Cell Stem Cell. 2011; 8: 639-648Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar; Tata et al., 2013Tata P.R. Mou H. Pardo-Saganta A. Zhao R. Prabhu M. Law B.M. Vinarsky V. Cho J.L. Breton S. Sahay A. et al.Dedifferentiation of committed epithelial cells into stem cells in vivo.Nature. 2013; 503: 218-223Crossref PubMed Scopus (376) Google Scholar). A phase of differentiation then proceeds, ultimately leading to repair of the pseudostratified epithelium with the optimal proportion of ABSCs and mucociliary cells. Dysregulated repair can lead to a multitude of pathologies, including mucous metaplasia, ABSC hyperplasia, and stepwise progression to squamous lung cancer (SqLC) (Hogan et al., 2014Hogan B.L. Barkauskas C.E. Chapman H.A. Epstein J.A. Jain R. Hsia C.C. Niklason L. Calle E. Le A. Randell S.H. et al.Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function.Cell Stem Cell. 2014; 15: 123-138Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar; Ooi et al., 2010Ooi A.T. Mah V. Nickerson D.W. Gilbert J.L. Ha V.L. Hegab A.E. Horvath S. Alavi M. Maresh E.L. Chia D. et al.Presence of a putative tumor-initiating progenitor cell population predicts poor prognosis in smokers with non-small cell lung cancer.Cancer Res. 2010; 70: 6639-6648Crossref PubMed Scopus (45) Google Scholar). As such, developing a nuanced understanding of tracheal homeostatic mechanisms is of paramount importance. Conducting airways of the trachea are lined with C-shaped cartilage rings that serve as a structural scaffold, equipping it with a rigidity that prevents its collapse. The subepithelial intercartilaginous zone (ICZ) of the airway harbors diverse cell types including, but not limited to, fibroblasts, endothelial cells, immune cells, and nerves that together have been speculated to comprise an intricate ABSC niche (Borthwick et al., 2001Borthwick D.W. Shahbazian M. Krantz Q.T. Dorin J.R. Randell S.H. Evidence for stem-cell niches in the tracheal epithelium.Am. J. Respir. Cell Mol. Biol. 2001; 24: 662-670Crossref PubMed Scopus (319) Google Scholar; Donne et al., 2015Donne M.L. Lechner A.J. Rock J.R. Evidence for lung epithelial stem cell niches.BMC Dev. Biol. 2015; 15: 32Crossref PubMed Scopus (18) Google Scholar). Previous work observed that the epithelial surface above the ICZ contains a greater density of ciliated cells in comparison with regions above cartilage in the adult rodent trachea (Oliveira et al., 2003Oliveira M.J. Pereira A.S. Guimarães L. Grande N.R. de Sá C.M. Aguas A.P. Zonation of ciliated cells on the epithelium of the rat trachea.Lung. 2003; 181: 275-282Crossref PubMed Scopus (14) Google Scholar; Toskala et al., 2005Toskala E. Smiley-Jewell S.M. Wong V.J. King D. Plopper C.G. Temporal and spatial distribution of ciliogenesis in the tracheobronchial airways of mice.Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 289: L454-L459Crossref PubMed Scopus (44) Google Scholar). Efforts have also identified that the IL-6 from ICZ cells signals onto ABSCs to promote STAT3 activity and subsequently regulate ciliated cell formation from basal cells (Tadokoro et al., 2014Tadokoro T. Wang Y. Barak L.S. Bai Y. Randell S.H. Hogan B.L. IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells.Proc. Natl. Acad. Sci. USA. 2014; 111: E3641-E3649Crossref PubMed Scopus (140) Google Scholar). These findings implicate cell-cell communication between the ICZ and surface airway epithelium (SAE); however, the dynamics of this ICZ niche during injury repair and aging remain unexplored. Recent studies have characterized the active epithelial-mesenchymal crosstalk in murine bronchiolar and alveolar homeostasis, of which Wnt signaling plays an increasingly defined role (Lee et al., 2017Lee J.H. Tammela T. Hofree M. Choi J. Marjanovic N.D. Han S. Canner D. Wu K. Paschini M. Bhang D.H. et al.Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6.Cell. 2017; 170: 1149-1163.e12Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar; Nabhan et al., 2018Nabhan 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. 2018; 359: 1118-1123Crossref PubMed Scopus (231) Google Scholar; Volckaert et al., 2011Volckaert T. Dill E. Campbell A. Tiozzo C. Majka S. Bellusci S. De Langhe S.P. Parabronchial smooth muscle constitutes an airway epithelial stem cell niche in the mouse lung after injury.J. Clin. Invest. 2011; 121: 4409-4419Crossref PubMed Scopus (149) Google Scholar; Zepp et al., 2017Zepp J.A. Zacharias W.J. Frank D.B. Cavanaugh C.A. Zhou S. Morley M.P. Morrisey E.E. Distinct Mesenchymal Lineages and Niches Promote Epithelial Self-Renewal and Myofibrogenesis in the Lung.Cell. 2017; 170: 1134-1148.e10Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). In the trachea, however, the effects of Wnt signaling on proliferation, differentiation, and injury repair are poorly understood. Prior work from others has suggested an importance for β-catenin in basal cell fate decisions (Giangreco et al., 2012Giangreco A. Lu L. Vickers C. Teixeira V.H. Groot K.R. Butler C.R. Ilieva E.V. George P.J. Nicholson A.G. Sage E.K. et al.β-Catenin determines upper airway progenitor cell fate and preinvasive squamous lung cancer progression by modulating epithelial-mesenchymal transition.J. Pathol. 2012; 226: 575-587Crossref PubMed Scopus (48) Google Scholar; Malleske et al., 2018Malleske D.T. Hayes Jr., D. Lallier S.W. Hill C.L. Reynolds S.D. Regulation of Human Airway Epithelial Tissue Stem Cell Differentiation by beta-Catenin, P300, and CBP.Stem Cells. 2018; 36: 1905-1916Crossref PubMed Scopus (11) Google Scholar; Reynolds et al., 2008Reynolds S.D. Zemke A.C. Giangreco A. Brockway B.L. Teisanu R.M. Drake J.A. Mariani T. Di P.Y. Taketo M.M. Stripp B.R. Conditional stabilization of beta-catenin expands the pool of lung stem cells.Stem Cells. 2008; 26: 1337-1346Crossref PubMed Scopus (110) Google Scholar). However, current limitations in our understanding stem from a dearth of studies that dissect the spatiotemporally dynamic role of Wnt/β-catenin signaling during distinct phases of repair, as well as aging, and how this is contextualized within signaling niches in vivo. Here, we employ several transgenic mouse models to dissect the dynamic mechanisms by which Wnt signaling modulates proximal ABSC homeostasis, injury repair, and aging in vivo. We show that canonical Wnt/β-catenin signaling is responsible for the proliferative phase of tracheal repair following injury in vivo and is mediated by phosphorylation of β-catenin protein at tyrosine 489. We reveal that a transiently activated Pdgfrα+ lineage in the ICZ marks a Wnt-producing niche necessary for driving proximal airway epithelial proliferation in vivo. The proximal airway then undergoes a dynamic cellular switch to form an intra-epithelial niche that directs differentiation to ciliated cells in vivo, because K5+ ABSCs secrete Wnt ligand to facilitate their formation. Importantly, the aging proximal airway ICZ niche is marked by the emergence of K5+ ABSC-derived glandular-like epithelial invaginations (GLEIs) that contribute to repair after injury and can be formed by constitutively activating Wnt/β-catenin signaling in the ABSCs of young mice. Our efforts thus demonstrate the spatiotemporal dynamism of Wnt signaling across multiple proximal airway niches that together act as critical regulators of distinct phases of regeneration, as well as aging. At steady state, the mouse proximal airway epithelium has a slow turnover of approximately one ABSC every 7–10 days (Rock et al., 2010Rock 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. 2010; 3: 545-556Crossref PubMed Scopus (403) Google Scholar). As such, to study mechanisms of ABSC self-renewal, we employ an injury model whereby we administer intratracheal (i.t.) polidocanol to slough the proximal airway epithelium, as previously described (Borthwick et al., 2001Borthwick D.W. Shahbazian M. Krantz Q.T. Dorin J.R. Randell S.H. Evidence for stem-cell niches in the tracheal epithelium.Am. J. Respir. Cell Mol. Biol. 2001; 24: 662-670Crossref PubMed Scopus (319) Google Scholar; Paul et al., 2014Paul M.K. Bisht B. Darmawan D.O. Chiou R. Ha V.L. Wallace W.D. Chon A.T. Hegab A.E. Grogan T. Elashoff D.A. et al.Dynamic changes in intracellular ROS levels regulate airway basal stem cell homeostasis through Nrf2-dependent Notch signaling.Cell Stem Cell. 2014; 15: 199-214Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The few remaining ABSCs subsequently undergo an initial phase of symmetric division to repopulate the ABSCs. This is followed by a period of asymmetric division, during which ABSCs give rise to the optimal proportion of differentiated mucociliary cells. Previous work has shown that 48 h post-injury (hpi) is the point of maximal proliferation following injury, with a notable decrease by 72 hpi as differentiation commences (Paul et al., 2014Paul M.K. Bisht B. Darmawan D.O. Chiou R. Ha V.L. Wallace W.D. Chon A.T. Hegab A.E. Grogan T. Elashoff D.A. et al.Dynamic changes in intracellular ROS levels regulate airway basal stem cell homeostasis through Nrf2-dependent Notch signaling.Cell Stem Cell. 2014; 15: 199-214Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Because the role of Wnt signaling remains poorly understood in proximal ABSC homeostasis and injury repair in vivo, we sought to analyze its activity in vivo. Mice were administered i.t. polidocanol and sacrificed at 12, 24, 48, 72, and 96 hpi (Figure 1A). We performed quantitative real-time PCR to determine the mRNA expression of varying Wnt signaling components and known downstream target genes in the epithelial compartment. In comparison with uninjured mouse tracheas, we observed increased mRNA expression of varying Wnt signaling components, including known canonical signaling target genes Axin2 and Ccnd1 (encodes Cyclin D1) (Figure 1B). In situ hybridization studies also demonstrated increased Axin2 mRNA at 48 hpi in the SAE relative to uninjured airways (Figure 1C). We next assessed the spatiotemporal expression of the Wnt/β-catenin signaling pathway components in the normal repairing airway. GSK3β, a known negative regulator of canonical signaling (Liu et al., 2002Liu C. Li Y. Semenov M. Han C. Baeg G.H. Tan Y. Zhang Z. Lin X. He X. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism.Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1516) Google Scholar; Rubinfeld et al., 1996Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly.Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1240) Google Scholar; Yost et al., 1996Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3.Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (974) Google Scholar), was weakly expressed in the uninjured airway epithelium and is most strongly expressed at 72 hpi as proliferation declines following injury (Figure S1A). Further, β-catenin post-translational modifications (PTMs) regulate its subcellular localization and signaling in other contexts (Fang et al., 2007Fang D. Hawke D. Zheng Y. Xia Y. Meisenhelder J. Nika H. Mills G.B. Kobayashi R. Hunter T. Lu Z. Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity.J. Biol. Chem. 2007; 282: 11221-11229Crossref PubMed Scopus (604) Google Scholar; Huber and Weis, 2001Huber A.H. Weis W.I. The structure of the beta-catenin/E-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin.Cell. 2001; 105: 391-402Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar; Rhee et al., 2007Rhee J. Buchan T. Zukerberg L. Lilien J. Balsamo J. Cables links Robo-bound Abl kinase to N-cadherin-bound beta-catenin to mediate Slit-induced modulation of adhesion and transcription.Nat. Cell Biol. 2007; 9: 883-892Crossref PubMed Scopus (113) Google Scholar). To determine whether β-catenin PTMs might be important in the setting of ABSC proliferation following injury, we performed immunofluorescences (IFs) for several known β-catenin phosphorylation sites. We observed that β-catenin phosphorylated at tyrosine 489 (p-β-cateninY489) robustly localized to the nucleus in the repairing SAE relative to uninjured control animals (Figure 1D). This stood in contrast with other phosphorylated forms of β-catenin that primarily remained cytoplasmic or membranous (Figures S1B and S1C). We additionally employed TCF/Lef:H2B/GFP reporter mice to monitor Wnt/β-catenin pathway activity following injury in vivo. In contrast with uninjured airways that displayed minimal reporter activity, mice injured with polidocanol exhibited induced nuclear TCF/Lef activity that was statistically significant (Figures 1E and 1F). These data collectively demonstrate that the canonical Wnt/p-β-cateninY489 signaling axis is activated during repair following injury in vivo. To test whether β-catenin signaling was necessary for repair in vivo, we utilized K5-CreER;Ctnnb1fl/fl transgenic mice, a tamoxifen-inducible system that selectively deletes the β-catenin gene in K5-expressing cells. Mice were administered 75 mg/kg tamoxifen intraperitoneally (i.p.) every 24 h for 5 days, followed by 1 day of rest, to induce Cre-mediated recombination. Mice airways were then injured using i.t. polidocanol and sacrificed at 48 hpi to assess ABSC proliferation (Figure 1G). Effective deletion of β-catenin was first determined by IF in tamoxifen-treated K5-CreER;Ctnnb1fl/fl mice relative to Ctnnb1fl/fl control littermate mice that lacked the K5-CreER transgene (Figure S1D). Further, in comparison with control airways, K5-CreER;Ctnnb1fl/fl mice tracheas displayed scant bromodeoxyuridine (BrdU) uptake and instead had an attenuated epithelium at 48 hpi (Figure 1H). Taken together, these experiments illustrate that canonical Wnt/β-catenin signaling is essential for driving the proliferative phase of proximal airway regeneration following injury in vivo. With knowledge that β-catenin is essential for proliferation in repair, we next sought to determine whether this process was regulated by upstream Wnt ligand secretion. Porcupine is an endoplasmic reticulum (ER)-resident protein known to palmitoylate all 19 mammalian Wnt ligands that are necessary for their secretion (Barrott et al., 2011Barrott J.J. Cash G.M. Smith A.P. Barrow J.R. Murtaugh L.C. Deletion of mouse Porcn blocks Wnt ligand secretion and reveals an ectodermal etiology of human focal dermal hypoplasia/Goltz syndrome.Proc. Natl. Acad. Sci. USA. 2011; 108: 12752-12757Crossref PubMed Scopus (107) Google Scholar; Biechele et al., 2011Biechele S. Cox B.J. Rossant J. Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos.Dev. Biol. 2011; 355: 275-285Crossref PubMed Scopus (94) Google Scholar). As such, Porcupine plays an integral role by functioning as an upstream regulator of the Wnt signaling cascade. To determine whether Wnt secretion regulates airway epithelial proliferation after injury in vivo, we administered wild-type mice either vehicle or small-molecule Porcupine inhibitor, LGK974, prior to polidocanol-induced airway injury as shown in the schematic in Figure 2A. Relative to vehicle-treated mice, LGK974-treated mice exhibited an attenuated SAE with few BrdU+ ABSCs (Figures 2B and 2C), thereby phenocopying the repair response of K5-CreER;Ctnnb1fl/fl mouse tracheas. LGK974-treated mice also displayed reduced TCF/Lef:H2B/GFP reporter activity in comparison with vehicle-treated controls following injury, indicating suppression of the canonical Wnt signaling (Figures S2A and S2B). Furthermore, we observed a statistically significant decrease in the percentage of K5+ ABSCs that harbored nuclear p-β-cateninY489 in mice treated with LGK974 when compared with vehicle-treated mice (Figures 2D and 2E). We additionally treated human ABSCs (hABSCs) in vitro with LGK974 and identified that those treated with Wnt secretion inhibition displayed a significant reduction in the percentage of hABSCs with nuclear p-β-cateninY489 (Figures S2C and S2D). Taken together, these efforts indicate that Porcupine-dependent phosphorylation of β-cateninY489 is necessary for ABSC proliferation. In light of our data that Wnt ligand secretion is necessary for the ABSC proliferative response to injury, we next sought to determine the potential cellular sources of Wnt ligand that mediate this phase of repair. To this end, we performed Porcupine IF staining in the repairing wild-type mouse airway to demarcate Wnt-producing cells within signaling niches (Tammela et al., 2017Tammela T. Sanchez-Rivera F.J. Cetinbas N.M. Wu K. Joshi N.S. Helenius K. Park Y. Azimi R. Kerper N.R. Wesselhoeft R.A. et al.A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma.Nature. 2017; 545: 355-359Crossref PubMed Scopus (146) Google Scholar). Tracheas at 48 hpi harbored Porcupine+ cells in the SAE, as well as subepithelial cells in the ICZ niche, a strong upregulation in comparison with uninjured controls (Figure 3A). Additionally, the Wnt-secreting ICZ niche was dynamic and transiently activated, because 72 hpi airways exhibited Porcupine expression only in the SAE and not in cells of the ICZ (Figure 3A). These data offered two possible distinct cellular mechanisms by which Wnt-mediated proliferation occurs: one in which stem cell-derived Wnt ligand promotes proliferation or a second in which the ICZ niche provides this cue. To dissect whether Wnt secretion from K5+ ABSCs was necessary for repair following injury, we utilized K5-CreER;Porcnfl/fl;ROSAmT/mG mice that harbor selective blockade of Porcupine and Wnt secretion in exclusively K5+ ABSCs. The repairing tracheas of these mice therefore receive only stromal paracrine Wnt contribution. We injured the K5-CreER;Porcnfl/fl;ROSAmT/mG mice and Porcnfl/fl;ROSAmT/mG control littermate mice and analyzed the repair response at varying time points post-injury (Figure 3B). In the absence of tamoxifen treatment, all cells have membrane-targeted tdTomato. Tamoxifen administration therefore allowed for selective Porcupine deletion in K5+ cells and triggered membrane-targeted EGFP in these cells. Effective Porcupine deletion in K5-expressing cells was first confirmed by IF. 81.97% ± 7.18% of K5+ ABSCs in K5-CreER;Porcnfl/fl;ROSAmT/mG mice underwent recombination upon tamoxifen treatment (Figures 3C and S3A). We administered polidocanol via oropharyngeal aspiration in adherence with recently published protocols (Plasschaert et al., 2018Plasschaert L.W. Žilionis R. Choo-Wing R. Savova V. Knehr J. Roma G. Klein A.M. Jaffe A.B. A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte.Nature. 2018; 560: 377-381Crossref PubMed Scopus (308) Google Scholar). We first identified that the repair response timeline is identical to that of mice receiving i.t. polidocanol (data not shown). H&E staining demonstrated that the repairing airways of mouse tracheas with Porcupine deletion in K5+ ABSCs were phenotypically indistinguishable from those of control mice (Figure S3B). Assessment of BrdU incorporation from these mice at 48 hpi showed no statistically significant difference (Figures 3D and 3E). Immunostaining analysis also identified that the 48 hpi K5-CreER;Porcnfl/fl;ROSAmT/mG SAE contained uniformly GFP+ cells, indicating that the repairing airway was comprised of ABSCs devoid of Porcupine and therefore Wnt secretion (Figure S3C). Further, control K5-CreER;ROSAmT/mG and experimental K5-CreER;Porcnfl/fl;ROSAmT/mG mice exhibited no differences in the formation of K8+ early progenitors at 96 hpi (Figures 3F and 3G). Together, these results collectively demonstrate that ABSC-derived Wnt ligand is dispensable for ABSC proliferation and K8+ early progenitor formation. Our data thus far indicated that although Wnt secretion was necessary for ABSC proliferation following injury, this process was not driven by the ABSC lineage. We therefore next reasoned that the dynamic, transiently activated Wnt-secreting ICZ niche was necessary to drive ABSC proliferation following injury. To address this possibility, we administered K5-CreER;Porcnfl/fl;ROSAmT/mG mice either tamoxifen and vehicle to selectively block Wnt secretion from only the ABSC lineage, or tamoxifen and LGK974 to block global Wnt secretion prior to injury (Figure 4A). Analysis of tracheas at 48 hpi illustrated that K5-CreER;Porcnfl/fl;ROSAmT/mG mice treated with LGK974 exhibited an attenuated SAE with a statistically significant decrease in the percentage of proliferating BrdU+ K5+ ABSCs in comparison with those treated with vehicle (Figures 4B and 4C). These data offered the insight that the Wnt-producing ICZ niche is necessary for airway epithelial proliferation following injury. The ICZ has been speculated to serve as a signaling niche for the proximal SAE and is rich in Pdgfrα+ fibroblasts (Borthwick et al., 2001Borthwick D.W. Shahbazian M. Krantz Q.T. Dorin J.R. Randell S.H. Evidence for stem-cell niches in the tracheal epithelium.Am. J. Respir. Cell Mol. Biol. 2001; 24: 662-670Crossref PubMed Scopus (319) Google Scholar; Tadokoro et al., 2014Tadokoro T. Wang Y. Barak L.S. Bai Y. Randell S.H. Hogan B.L. IL-6/STAT3 promotes regeneration of airway ciliated cells from basal stem cells.Proc. Natl. Acad. Sci. USA. 2014; 111: E3641-E3649Crossref PubMed Scopus (140) Google Scholar). To further characterize the Wnt-secreting ICZ compartment during proximal airway regeneration, we utilized tamoxifen-inducible Pdgfrα-CreER;ROSAmT/mG mice to label the Pdgfrα+ lineage prior to injury. We treated these mice with tamoxifen before injury, administered polidocanol, and sacrificed them at 48 hpi to determine whether the Pdgfrα+ lineage in the ICZ secretes Wnt ligand during repair (Figure 4D). We first confirmed that the Pdgfrα+ cell lineage, marked by GFP, localizes to the ICZ (Figure 4E). Further lineage-tracing analysis and IF staining showed that a subset of Pdgfrα+ cells in the ICZ were Porcupine+ at 48 hpi (Figure 4E), while only sparse Pdgfrα+ cells were also Porcupine+ in the uninjured state (∼1%) (Figure 4F). We further quantified at 48 hpi that of the cells that exp" @default.
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• W3045351648 date "2020-09-01" @default.
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• W3045351648 title "Distinct Spatiotemporally Dynamic Wnt-Secreting Niches Regulate Proximal Airway Regeneration and Aging" @default.
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• W3045351648 doi "https://doi.org/10.1016/j.stem.2020.06.019" @default.
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