Fig. 8: SHH attracts the foregut mesenchymal cells to deform the epithelium.

a HCR-FISH of PTCH1 in an E3.5 chick foregut slice 24 h after implantation of an Affi-Gel bead loaded with recombinant mouse SHH protein. b Comparison of mesenchymal cell density near the implanted bead (with or without SHH) and the contralateral side. N = 7 biological replicates for control, N = 5 for SHH. P-values are calculated by a paired t-test. c PIV analysis of an E3.75 GFP chick slice with an implanted SHH-bead. The color wheel indicates the directions of PIV vectors. d Live imaging of an SHH-bead-implanted GFP chick slice overlaid with cellular trajectories colored by their average speeds. e Average velocity vectors of individual cell trajectories in the blue and red boxes of (d). Vectors are scaled with the speed and color-coded by their directions according to the color wheel. The dashed circle marks the bead. f Immunofluorescence of GM130 (GOLGA2) in a chick foregut section with a SHH bead. g, h Distributions of mesenchymal cell orientation in slices implanted with control beads (g, N = 4 biological replicates) and SHH beads (h, N = 6 biological replicates). Data are presented as mean ± SD. P-values are calculated by Chi-square test against uniform distribution (df = 3 for Control, df = 4 for SHH bead). i, j In ovo electroporation of plasmids encoding mCherry (i), SHH (i), or HHIP (j) into the right epithelium of the chick foregut. Frontal views of the foreguts by stereo microscopy show the effective electroporation (i). Immunofluorescence of SHH (i) and HCR-FISH of HHIP and PTCH1 (j) confirmed the efficient expression of the constructs. Arrows indicate the asymmetrical deformation of the epithelium induced by SHH overexpression. The dashed lines bisect the foregut lumen as a reference for measuring epithelial distances to the midline. k Comparisons of right (electroporated) and left (non-electroporated) epithelium-to-midline distances with control (mCherry, N = 5 biological replicates), SHH (N = 7 biological replicates), and HHIP (N = 6 biological replicates) electroporation. Data are presented as mean ± SD. P-values are calculated by One-way ANOVA with Dunnett’s test. l Working model of TES initiation. SHH secreted from the foregut epithelium induces the dorsal sub-epithelial mesenchyme’s migration towards the epithelium, generating bilateral compressive force essential to TES. Created in BioRender. Yan, R. (2025) https://BioRender.com/cdwttt9. Scale bars: 50 µm, except those marked on the images.