Extended Data Fig. 5: Characterization of Hh- activation during homeostasis and in fibrotic repair.
(a) RNAScope in situ of Shh shows expression in ectopic SOX2 + cells in the alveoli after bleomycin injury. This experiment was repeated independently twice with similar results. (b, c) Histology quantification shows no evidence of KRT5 + metaplasia in Hh-activated animals without injury when compared to controls (n = 3 per group; each data point represents one animal; one-tailed unpaired Student’s t- test). Data are expressed as mean ± SD. (d–g) After bleomycin injury, Hh-activated lungs show similar expansion of Gli1 + cells in distal alveoli compared to controls, a trend towards increased myofibroblasts (SMA + ) differentiation of the Gli1 Lin+ cells in the alveoli, and no difference in ectopic SCGB1A1 + cells in the alveoli (for (e), n = 4 for control, n = 6 for Hh-activated; for (f, g), n = 9 per group; each data point represents one animal; one-tailed unpaired Student’s t-test for (e–g)). Data are expressed as mean ± SD. (h) Model of 3D airway organoid assay using Hh-inducible mesenchyme whereby pretreatment of 4- hydroxytamoxifen (4OHT) induces Hh activation in the mesenchyme as shown by upregulation of Gli1 transcript compared to vehicle (ethanol) (n = 3 per group; each data point represents one biological replicate; one-tailed unpaired Student’s t-test). (i–l) Mesenchymal Hh activation in vitro reduces CFE and organoid size, but increases the expression of Krt5 and number of KRT5 + organoids while reducing Sftpc expression and number of SFTPC + organoids ((i–k) n = 3 per group; (l) n = 3 for Hh-activated, n = 5 for control; each data point represents one well; one-tailed unpaired Student’s t-test for (j–l)). Data are expressed as mean ± SD. Hh = hedgehog, 4OHT = 4-hydroxytamoxifen, AW = airway. Scale bars, 100 μm.
