DEV029942 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Scleraxis expression in avian E9 GI tract. Whole-mount in situ hybridization on an E9 GI tube using a Scleraxis antisense riboprobe. Scleraxis is expressed in two domains identified as intermuscular tendons in the stomach and also in two layers of the caecum.

• Supplemental Figure S2 -

  Fig. S2. Whole-mount in situ hybridization of Fgf7, Fgf10, FgfR1 and FgfR2 on E5 stomachs. At E5, Fgf7, Fgf10 and FgfR2 are exclusively expressed in the gizzard epithelium. By contrast, FgfR1 expression is present in the whole gizzard mesenchyme. Red arrow indicates the mesenchyme, black arrows the epithelium.

• Supplemental Figure S3 -

  Fig. S3. Activation of the FGF signaling pathway is sufficient to induce ectopic Scleraxis expression. Whole-mount in situ hybridization using an antisense Scleraxis riboprobe on wild-type and RCAS-Fgf8 E9 guts. Serial adjacent longitudinal sections of RCAS-Fgf8 E9 proventriculus and caecum analyzed by immunohistochemistry with anti-Gag 3C2 and anti-αSMA antibodies. Ectopic activation of the FGF pathway through Fgf8 misexpression induces ectopic mesenchymal buddings associated with ectopic expression of Scleraxis in the proventriculus (red arrows) and hypertrophic caecum associated with ectopic expression of Scleraxis (red arrows). Sections with RCAS-Fgf8 expressing cells, detected by the 3C2 antibody, are characterized by the reduction or absence of the αSMA-positive domain as compared with the αSMA expression observed in E9 wild-type guts (black arrows).

• Supplemental Figure S4 -

  Fig. S4. Activation of the FGF signaling pathway is sufficient to induce ectopic tendon marker expression in the proventriculus. Serial adjacent longitudinal sections of RCAS-Fgf8 and RCAS-GFP (control) E9 stomachs analyzed by in situ hybridization with specific antisense riboprobes directed against Env, Scleraxis and Type I Collagen. Proventriculus with RCAS-Fgf8 expressing cells, detected by Env, are characterized by the presence of Scleraxis and the tendon marker Type I Collagen. Red arrows indicate ectopic misexpressions (GFP and Fgf8). Pv, proventriculus.

• Supplemental Figure S5 -

  Fig. S5. The effect of Fgf8 misexpression on the gizzard epithelium. Serial adjacent longitudinal sections of RCAS-GFP and RCAS-Fgf8 E9 stomachs analyzed by in situ hybridization with specific antisense riboprobes directed against Env, Shh and Gata4. Ectopic activation of the FGF pathway in the stomach mesenchyme induces stomach morphology defects. This activation has no effect on epithelial Shh expression. However, the expression level of Gata4 in the gizzard epithelium was increased as previously reported and commented by Shin and colleagues (Shin et al., 2006).

  Shin, M., Noji, S., Neubüser, A. and Yasugi, S. (2006). FGF10 is required for cell proliferation and gland formation in the stomach epithelium of the chicken embryo. Dev. Biol. 294, 11-23.

• Supplemental Figure S6 -

  Fig. S6. Whole-mount in situ hybridization of Type XIV Collagen (ColXIV) on E9 stomach. Expression of ColXIV in the E7 hindlimb by whole-mount in situ hybridization using an antisense ColXIV riboprobe. ColXIV is present in the chick limb tendon (black arrows). Expression pattern of ColXIV in the stomach by whole-mount in situ hybridization using an antisense ColXIV riboprobe. ColXIV expression is expressed in the ENS cells (red arrows), but not in the intermuscular tendon of the stomach (black arrow).