Loss of airway smooth muscle differentiation led to disrupted cartilage segmentation. (A,B) Whole mount alcian blue staining showed cartilage segmentation defects in MyocdCKO E18.5 tracheas. Scalebar: 50 μm. (C,D) Col2a1 wholemount RNA in situ hybridization revealed reduced chondrocytes in MyocdCKO E14.5 tracheas. Scalebar: 100 μm. (E-J) Immunofluorescent detection of ACTA2 (magenta) and cartilage marker SOX9 (green) expression in transverse sections of tracheas of control and MyocdCKO at E13.5 (E,F), E14.5 (G,H), and E17.5 (I,J). Scalebar: 50 μm for E-H and 100 μm for I,J. “t” indicates trachea and “e” indicates esophagus. (K) Heatmap of RNA-seq data showing groups of significantly downregulated genes, by an adjusted p-value less than 0.05, in the MyocdCKO E18.5 tracheas. (L) qRT-PCR quantification of relative mRNA levels of mechanosensitive genes Runx2, Spp1, Hapln1, and Itgbl1 in control and MyocdCKO tracheas at E18.5. *: p<0.05, **: p<0.005. (M) qRT-PCR quantification of relative mRNA levels of Sox9 and mechanosensitive genes Runx2, Spp1, and Hapln1 in control and MyocdCKO tracheas at E13.5. *: p<0.05. Data are represented as individual points for each biological sample ± SD.