Fig 2: Hedgehog/Gli signaling supports tracheal mesenchymal specification. (A) Foxf1 (green) and Sox9 (red) immunostaining of E11.5 foregut transverse sections from control, Foxg1Cre;Gli3TFlag/+, Dermo1Cre;Gli3TFlag/+, Foxg1Cre;Smof/f and Dermo1Cre;Smof/f embryos. Foxg1Cre mutants have fewer Foxf1+ and Sox9+ mesoderm cells compared to Dermo1Cre mutants and control embryos. N=3-5 embryos/genotype. Scale bars: 100 µm. e, esophagus; t, trachea. (B-F) Quantification of E11.5 immunostaining for each genotype showing the total number of cells in the tracheal mesoderm (B, TMes), the ratio of Foxf1+/total TMes cells (C), the ratio of Sox9+/total TMes cells (D), the mitotic index of phospho-histone H3 (pHH3+)/total TMes cells to indicate proliferation (E), and the ratio of cleaved caspase-3 (CC3+)/total TMes cells to indicate apoptosis (F). Histograms are mean±s.e.m., with data points for each individual embryo shown. *P<0.05 (unpaired two-sided Student's t-test with unequal variance). n.s., not significant. N=3-5 embryos/genotype.

Fig 3: Gli3 regulates expression of Wnt pathway components. (A,B) Volcano plot of differentially expressed transcripts in control versus Foxg1Gli3TFlag/+ foreguts, as determined by Log2FC=|1| (P<0.05) at E10.5 (A) and E11.5 (B). Dashed lines indicate FC and P value thresholds. (C) Venn diagram intersecting genes differentially expressed in Foxg1Cre;Gli3TFlag/+ mutants with genes known to be involved in human or mouse tracheal chondrogenesis (Shefchek et al., 2020; Sinner et al., 2019). TM, tracheomalacia-associated genes. (D) Genome browser views of Gli3-3xFlag (GSE133710), Foxf1 (GSE77159) and H3K4me3 (GSE119885) ChiP-seq data, as well as ATAC-seq data (GSE119885) on Foxf1, Sox9, Rspo2 and Notum loci. Gli3 and Foxf1 bind the Foxf1, Sox9 and Notum loci, but only Foxf1 shows direct binding of the Rspo2 locus along with significant ATAC and H3K4me3 peaks, suggesting active transcription. Statistically significant ChIP peaks are underlined in red.

Fig 4: Foxf1 is required for tracheal Sox9 expression. (A) Foxf1 (green) and Sox9 (red) immunostaining of E11.5 control, Foxg1Cre;Foxf1f/f and Dermo1Cre;Foxf1f/f mutants. Foxg1Cre mutants have fewer Foxf1+ and Sox9+ cells compared to controls. Dermo1Cre mutants have some Sox9+ cells in the ventral trachea that co-localize with Foxf1 (arrowhead), suggesting that these cells escaped Cre recombination. (B) Foxf1 and Sox9 immunostaining of E13.5 control and Foxg1Cre;Sox9f/f embryos. Dotted lines indicate the trachea and esophagus. (C-G) Quantification of E11.5 immunostaining for each genotype showing the total number of cells in the tracheal mesoderm (C, TMes), the ratio of Foxf1+/total TMes cells (D), the ratio of Sox9+/total TMes cells (E), the mitotic index of phospho-histone H3 (pHH3+)/total TMes cells to indicate proliferation (F), and the ratio of cleaved caspase-3 (CC3+)/total TMes cells to indicate apoptosis (G). Histograms are mean±s.e.m., with data points for each individual embryo shown. *P<0.05 (unpaired two-sided Student's t-test with unequal variance). n.s., not significant. N=3-5 embryos/genotype. Scale bars: 100 µm. e, esophagus; t, trachea.

Fig 5: Dynamic Sox9 and Foxf1 expression during tracheal chondrocyte specification. (A) Foxf1 (green) and Sox9 (red) immunostaining of control embryos between E10 and E11.5. Foxf1 is initially expressed throughout the lateral plate mesoderm but then downregulated in the ventral tracheal mesoderm relative to the dorsal tracheal mesoderm by E11.5. Sox9 is only found in neural crest cells at E10 but progressively localized to the ventral tracheal mesoderm between E10.5 and E11.5. The inset shows Sox9+/Foxf1+ co-expressing cells at the boundary between the presumptive smooth muscle and chondrocytes. The dotted-yellow lines indicate the epithelial-lined trachea and esophagus lumen. N=3-5 embryos/stage. (B) Immunostaining of LacZ (ß-galactosidase), Foxf1 and Sox9 in E10 and E11.5 Gli1LacZ/+ embryos, showing the direct HH-target Gli1 expressed throughout the mesoderm surrounding the esophageal and tracheal endoderm. Scale bars: 100 µm. e, esophagus; t, trachea.