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. 2023 Jan 24;68(5):523–536. doi: 10.1165/rcmb.2022-0269OC

Figure 5.

Figure 5.

HH inhibition alters key differentiation markers and PDGF pathway targets in Pdgfra-expressing cells. (A) Whole-lung single-cell suspensions of PdgfraEGFP/+ mice, treated with 5E1 or IgG control at E18.5, were sorted for EGFP+ (Pdgfra-expressing) cells. Representative plots of Pdgfra+/+ control lungs (left panel) and PdgfraEGFP/+ lungs (middle panels) show detection of PdgfraDim and PdgfraHigh cell populations. Quantification of PdgfraDim and PdgfraHigh cell populations shows decreased number of total Pdgfra- and PdgfraHigh-expressing cells. (B) RNA-seq analysis of Pdgfra+ sorted cells from P8 PdgfraEGFP/+ lungs, treated with 5E1 or IgG at E18.5, identified 48,216 genes, of which 1,905 were significantly different. (C) Volcano plot of up- and downregulated genes (fold change > 2; adjusted P (Padj) < 0.05). (D) Pathway enrichment analysis using Ingenuity Pathway Analysis shows enrichment in gene sets for PDGF signaling and lung diseases. (E) Effect of HH inhibition on key genes of HH (Gli1, Ptch1, Hhip, and Smo) and PDGF signaling molecules (Pdgfra and Pdgfrb); on markers of Pdgfra-lineage cells: lipofibroblasts (Plin2), myofibroblasts (Acta2, Actg, Wnt5a, Fgf18, and Igf1), and matrix fibroblasts (Col13a1, Vcan, and Fgfra2); and on potential transcriptional targets of PDGF signaling (Klf2, Klf9, Cdh11, and Txnip). Data are normalized mean ± SD. #Adjusted P < 0.001 (t test). Acta2 = actin alpha 2, smooth muscle; Actg = actin gamma 2, smooth muscle; Aspn = asporin; B-H = Benjamini-Hochberg; Cdh11 = cadherin 11; Col13a1 = collagen type XIII alpha 1 chain; ERK = extracellular signal-related kinase; Fgf18 = fibroblast growth factor 18; Fgfra2 = fibroblast growth factor receptor A2; Hhip = hedgehog inhibitory protein; Igf1 = insulin like growth factor 1; Klf = KLF transcription factor; MAPK = mitogen-activated protein kinase; Max = maximum; Plin2 = perilipin 2; RNA-seq = RNA sequencing; Smo = smoothened; SSC-A = side scatter area; Tgfbi = transforming growth factor beta induced; Txnip = thioredoxin interacting protein; Vcan = versican.