Fig. 6: SHH signaling is essential for generating the convergent mesenchymal force.

a HCR-FISH of PTCH1 in transverse sections of E4.0 chick foreguts with or without in ovo SHH inhibitor (cyclopamine) treatment. b Immunofluorescence of SOX2 and NKX2-1 in transverse sections of E3.75 chick slices with or without in ovo SHH inhibitor treatment. c Stereoscope imaging of a control and a SHH inhibitor-treated GFP chick foregut slice before (magenta) and after (green) surgical removal of the mesenchyme. d Quantification of epithelial neck widths (defined in Fig. 3a) before and after mesenchyme removal (N = 5 biological replicates for control, N = 4 for SHH inhibition). P-values are calculated by a multiple paired t-test. e Comparison of epithelial neck width changes calculated from (d). Data are presented as mean ± SD. P-value is calculated by the Mann-Whitney test. f Distributions of mesenchymal cell orientation in SHH inhibitor-treated slice culture as in Fig. 5l (N = 7 biological replicates). Data are presented as mean ± SD. P-value is calculated by the Chi-square test against uniform distribution (df = 6). g Live imaging of E3.75 GFP chick slices treated with SHH inhibitor in ovo, in the absence (top) or the presence (bottom) of bilateral pressure by a collagen gel trough. h, i Quantification of the neck width (h) and the perimeter-area ratio (i) of SHH inhibitor-treated slices without or with external bilateral force compared with E3.75 controls (as in Fig. 3c, e). Each curve shows an individual sample (N = 5 for SHH inhibitor-only samples, and N = 3 for SHH inhibitor with bilateral pressure). Control data are presented as median ± MAD. Scale bars: 50 µm.