DEV02346 Supplementary Material

Files in this Data Supplement:

• Supplemental Tables - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Targeting the ENCC. X-Gal-stained whole-mount control samples (A,C,D,E,G) and corresponding sections (B,F,H,I). (A) E10.5 embryo. The black arrow indicates the β-gal activity in the gut. (B) E10.5 foregut. Scattered β-gal⁺ cells are seen in the mesenchyme. (C) E12.5 stomach and midgut are fully colonised byβ-gal⁺ cells. (D) Chains of β-gal⁺ cells are invading the proximal hindgut at the migratory front (red arrow), at E12.5. (E,F) E16.5 small intestine. Myenteric ganglia have formed (black arrow). The black arrowheads show isolated labelled cells in the submucosal zone. (G,H) E16.5 descending colon. Noβ-gal⁺ cell is observed in the submucosal zone. (I) Higher magnification of a E16.5 small intestine transverse section. The broken line represents the limit between the myenteric plexus and the circular muscles. The white arrowhead shows a labelled cell of the myenteric plexus that seems to be migrating through the circular muscle layer toward the submucosal zone. Ab, anterior bud; ep, epithelium; h, heart; m, smooth muscles; pb, posterior bud; sz, submucosal zone. Scale bar: 500 μm in C; 50 μm in F.

• Supplemental Figure 2 -

  Fig. S2. The cohesion of mutant aggregates is mediated by Ca²⁺-dependent mechanisms. Spheroid aggregates formed in the 2D in vitro mutant cultures were incubated with two types of dissociation solutions. (A,C) Short incubation (t<30 minutes). (B,D) Longer incubation (t>45 minutes). (A,B) Low trypsin-EDTA treatment, which perturbs Ca²⁺-dependent adhesion, induces the dissociation of the aggregates. (C,D) Upon trypsin- Ca²⁺ treatment, known to perturb Ca²⁺-independent adhesion, the aggregates keep their compact structure.

• Supplemental Figure 3 -

  Fig. S3. Lamina propria innervation defect in the mutant postnatal small intestine. VIP (A,B), DAPI/VIP (C,D) and Cad6 (E,F) immunostaining on longitudinal sections of control and mutant duodenum villi at P7. White arrowheads in A,E,G,H indicate VIP⁺ neuronal processes innervating the lamina propria in the distal extremity of the villi. (A,C) In the control, VIP⁺ processes can be seen along the entire length of the villus axis. (B,D) In mutant, VIP⁺ processes are absent in most of the distal extremities of the villi. (E,F) Similar defects are observed at P7 with another marker for neuronal processes, Cad6. (G,H) At E16.5, cad-6⁺ neuronal processes reach the distal extremity of the villi, in both control and mutant mice. Scale bar: 1 mm in A-F; 200 μm in G,H.