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Hedgehog actively maintains adult lung quiescence and regulates repair and regeneration


Postnatal tissue quiescence is thought to be a default state in the absence of a proliferative stimulus such as injury. Although previous studies have demonstrated that certain embryonic developmental programs are reactivated aberrantly in adult organs to drive repair and regeneration1,2,3, it is not well understood how quiescence is maintained in organs such as the lung, which displays a remarkably low level of cellular turnover4,5. Here we demonstrate that quiescence in the adult lung is an actively maintained state and is regulated by hedgehog signalling. Epithelial-specific deletion of sonic hedgehog (Shh) during postnatal homeostasis in the murine lung results in a proliferative expansion of the adjacent lung mesenchyme. Hedgehog signalling is initially downregulated during the acute phase of epithelial injury as the mesenchyme proliferates in response, but returns to baseline during injury resolution as quiescence is restored. Activation of hedgehog during acute epithelial injury attenuates the proliferative expansion of the lung mesenchyme, whereas inactivation of hedgehog signalling prevents the restoration of quiescence during injury resolution. Finally, we show that hedgehog also regulates epithelial quiescence and regeneration in response to injury via a mesenchymal feedback mechanism. These results demonstrate that epithelial–mesenchymal interactions coordinated by hedgehog actively maintain postnatal tissue homeostasis, and deregulation of hedgehog during injury leads to aberrant repair and regeneration in the lung.

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Figure 1: The lung epithelium signals to the adjacent mesenchyme via paracrine Hh signalling during normal homeostasis.
Figure 2: Postnatal activation of Hh signalling is required to maintain lung mesenchymal quiescence.
Figure 3: Hh signalling modulates the acute mesenchymal response to epithelial injury.
Figure 4: Hh signalling modulates restoration of quiescence during injury resolution in the lung.
Figure 5: Hh signalling regulates epithelial quiescence via mesenchymal feedback.

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Primary accessions

Gene Expression Omnibus

Data deposits

The Gene Expression Omnibus accession number for the microarray data produced in these studies is GSE68201.


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The authors appreciate the input of M. Kahn, M. Beers and R. Shah in these studies. The authors are grateful to A. Stout and the Department of Cell and Developmental Biology Microscopy Core for help with imaging. These studies were supported by funds from the National Institutes of Health (HL110942, HL100405, HL087825 to E.E.M). T.P. is supported by the American Heart Association Fellow-to-Faculty Transition Award, Actelion ENTELLIGENCE Award, and K08-HL121146.

Author information

Authors and Affiliations



T.P. and E.E.M. designed the overall experimental strategy. T.P. and R.S.K. performed lineage tracing and animal injury experiments. T.P. and D.B.F. performed in vitro BrdU and organoid experiments. T.W. performed right heart catheterization on the animals. S.Z., L.C. and M.M.L. performed histology and immunohistochemistry. T.P., D.B.F., K.S.R., M.P.M. and E.E.M. analysed the data. T.P. and E.E.M. wrote and edited the manuscript with input from all authors.

Corresponding authors

Correspondence to Tien Peng or Edward E. Morrisey.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Characterization of Hh signalling in the lung.

a, A small number of TubbIV+ ciliated cells express GFP in the ShhcreGFP reporter. b, c, Gli1+ Hh activated cells co-label with S100A4, a fibroblast marker. dg, Lineage tracing of Gli1+ Hh activated cells shows little to no co-localization of GFP+ cells with the airway smooth muscle (d, e) or the vascular smooth muscle of the adjacent pulmonary artery (f, g). h, i, The rare exception occurs in the pulmonary vein where Gli1+ Hh activated cells contribute to the venous smooth muscle that is surrounded by the venous myocardium of the proximal pulmonary vein. j, Gli1+ cells also generate myofibroblasts after fibrotic injury such as that induced by bleomycin. k, Lung Gli1+ cells do not contribute to cells of haematopoietic lineage as marked by CD45. AW, airway; V, blood vessel; PV, pulmonary vein. Scale bars, 100 μm. Images representative of 3 animals with 5 sections examined per animal.

Extended Data Figure 2 Conditional deletion of Shh from the adult airway epithelium increases proliferation in the adjacent mesenchyme.

a, The Scgb1a1cre driver predominantly marks the airway epithelium in the adult lung when crossed with the R26RmTmG reporter. b, Whole-lung messenger RNA transcript analysis reveals efficient deletion of Shh transcripts in the Scgb1a1cre:Shhflox/flox animals compared to controls (Shhflox/flox) (n = 4 animals). ch, Deletion of Shh from the airway epithelium resulted in an increased expression of proliferative markers, PCNA and phospho-histone H3 (PH3) in the mesenchyme surrounding the airways in Scgb1a1cre:Shhflox/flox mutants (d, f, h, n = 4 animals) versus controls (Scgb1a1Cre:Shhflox/+) (c, e, g, n = 3 animals). AW, airway; V, blood vessel. Scale bars, 100 μm. *P < 0.05. Error bars, mean ± s.d.

Source data

Extended Data Figure 3 Conditional deletion of Smo from Pdgfrb-derived mesenchyme increases mesenchymal proliferation at the epithelial–mesenchymal interface and vascular remodelling.

ad, Deletion of Smo from Pdgfrb-derived mesenchyme resulted in increased expression of proliferative markers, PCNA and PH3 in the mesenchyme at the epithelial-mesenchymal interface of Pdgfrbcre:Smoflox/flox mutants (b, d) versus controls (Pdgfrb Cre:Smoflox/+) (a, c). ei, Pdgfrbcre:Smoflox/flox:R26RmTmG/+ mutants exhibit increased Ki67+ cells within lineage traced GFP+ cells in the alveoli compared to controls (n = 4 animals per group). j, k, Aged Pdgfrbcre:Smoflox/flox:R26RmTmG/+ mutants (>6 months old) spontaneously develop pulmonary hypertension with increased right ventricular systolic pressure (j, n = 3 animals per group) and right ventricle wall thickness (k, l). Scale bars, 100 μm. *P < 0.05. Error bars, mean ± s.d.

Source data

Extended Data Figure 4 Characterization of isolated lung mesenchyme.

ah, Isolated mesenchymal cells in vitro predominantly express Pdgfra and Pdgfrb (c, d, compared to isotype control a), but not epithelial marker, Epcam (b, compared to isotype control a), endothelial marker, CD31 (f, compared to isotype control e), nor haematopoietic marker, CD45 (h, compared to isotype control g). i, Expression of the constitutively active form of Smo (SmoM2) by 4-OH-tamoxifen induction significantly upregulates Gli1 expression in the isolated lung mesenchyme after 48 h. j, GFP staining of the PdgfraGFP reporter demonstrates that Pdgfra+ cells are expressed broadly in the lung. k, Activation of Smo in isolated lung mesenchymal cells leads to reduced expression of cell cycle progression genes. l, Hh activation of lung mesenchyme with SmoM2 attenuated the proliferation induced by Pdgf-BB ligand in vitro as assayed by BrdU incorporation. mp, s, Expression of activated Pdgfrb(PdgfrbGOF) within Gli1+ cells resulted in their proliferative expansion. qs, Concurrent activation of SmoM2 attenuated the proliferative expansion induced by PdgfrbGOF (n = 3 animals per group). Scale bars, 100 μm. Error bars, mean ± s.d.

Source data

Extended Data Figure 5 Shh expression is decreased with bleomycin and naphthalene injury to the airway.

ag, Repetitive bleomycin injury after one month or single-dose naphthalene injury after three days reduced GFP expression in the airways of the ShhcreGFP reporter compared to controls. Data represent n = 2 animals per group with 5 sections analysed per animal, AW, airway. Scale bars, 100 μm. *P < 0.05.

Source data

Extended Data Figure 6 Hedgehog modulates mesenchymal response to bleomycin injury.

ae, Chronic repetitive injury to the lung epithelium with bleomycin over 4 weeks downregulates Gli1 expression in the mesenchyme adjacent to the airways in Gli1lacZ lungs as noted by histochemical staining for β-galactosidase activity (ad) and as noted using X-gal quantification and qPCR analysis of Shh and Gli1 expression after four weeks of repetitive injury (e, n = 2 animals per group). fi, n, Lineage-traced, Gli1+ Hh activated lung mesenchymal cells undergo proliferative expansion after repetitive bleomycin injury with an increase in Ki67+ mesenchymal cells (n = 4 animals per group). jm, o, Expression of SmoM2 within lineage traced Gli1+ lung mesenchymal cells attenuates the proliferative expansion that normally follows repetitive bleomycin injury (n = 3 animals per group). pv, Gli1 expression remains reduced 2 months after the end of bleomycin treatment (pr, n = 2 animals per group), which might be due to the persistent scarring that is observed after repetitive bleomycin injury (sv). AW, airway; V, blood vessel. Scale bars, 100 μm; *P < 0.05. Error bars, mean ± s.d.

Source data

Extended Data Figure 7 Airway epithelium is able to regenerate in vitro and in vivo.

a, b, d, Scgb1a1+ secretory epithelium is initially depleted 3 days following naphthalene injury while TubbIV+ ciliated epithelium remains relatively intact (n = 3 animals per group). c, d, However, 2 months after the initial naphthalene injury, Scgb1a1+ secretory epithelium repopulates the airway and the ratio of secretory/ciliated cells is restored to levels before injury. e, f, GFP+ bronchial epithelial cells isolated from Scgb1a1cre:R26RmTmG animals were cultured in the presence or absence of isolated lung mesenchymal cells, and only those co-cultured with lung mesenchymal cells were able to form organoids. g, h, Examples of the 3-dimensional structures formed by the bronchial organoids in the presence of lung mesenchyme. ik, Scgb1a1-derived organoids predominantly express markers of secretory airway differentiation, including Scgb1a1 and Nkx2.1 (i, j), while a minority expresses markers of alveolar epithelial lineage including Sftpc (k). ln, Co-culture of lung mesenchyme and bronchial epithelium induces organoid formation (l), which is inhibited in number (m) and colony size (n) with activation of Hh in the mesenchyme. AW, airway. Scale bars, 100 μm. *P < 0.05. n = 3 animals per group for injury time points. Error bars, mean ± s.d. In vitro organoid studies represent technical quadruplicates, with > 140 randomly selected clones analysed for size per group.

Source data

Extended Data Figure 8 Hh signalling mediates both mesenchymal and epithelial quiescence during homeostasis and injury repair in the lung.

The lung epithelium actively maintains mesenchymal quiescence through paracrine Hh signalling, which also regulates a feedback loop to maintain epithelial quiescence. Epithelial injury leads to downregulation of Hh signalling and loss of mesenchymal quiescence, which in turn stimulates epithelial regeneration to replete the airway epithelium until homeostasis is re-established.

Supplementary information

Supplementary Table 1

This table shows GO enrichment analysis for microarray analysis in Extended Data Figure 4. GO analysis was performed on the microarray data obtained from activation of Smo in isolated lung mesenchymal cells. The top 10 (by P value significance) categories identified are shown. (XLSX 9 kb)

Supplementary Table 2

This table contains a list of differentially expressed genes found in the GO category of mitotic nuclear division obtained from activation of Smo in lung mesenchymal cells. (XLSX 23 kb)

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Peng, T., Frank, D., Kadzik, R. et al. Hedgehog actively maintains adult lung quiescence and regulates repair and regeneration. Nature 526, 578–582 (2015).

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