DEV023010 Supplementary Material

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. GATA4 is ectopically expressed in the mutant ventral endoderm. Immunostaining analysis on sagittal (A,B) and transversal (C-F) sections. At the 14s stage, decreased GATA4 expression is observed both in WT (A) and mutant (B) ventral hepatic domain (yellow dashed lines). At 20s, GATA4 expression is completely lost in WT (C), whereas it is ectopically expressed in the entire ventral gut endoderm of mutant embryos (D). PDX1 expression is normally excluded from the hepatic domain of WT embryos (E). Few PDX1-positive cells, which coexpress with GATA4, are detected in the ventral endoderm of mutant embryos (F), suggesting a duodenal fate.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Decreased Foxa1/2 expression in 14s mutant ventral endoderm. In situ hybridization analysis at 14s on sagittal sections. The expression of both Foxa1 and Foxa2 is strongly reduced in the presumptive hepatic domain (yellow dashed lines) of mutant embryos (B,D) as compared with WT ventral endoderm (A,C).

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Defective gut regionalisation in the vhnf1^hi2169 mutant. Whole-mount in situ hybridization in lateral view at 24 hpf. Anterior to the left. krox20 (egr2b) or frb35 (egr2a) staining was used to identify rhombomere 5 (r5), which is strongly reduced in vhnf1^hi2169 homozygous embryos. axial (foxa2) and shh are caudally expanded in the mutant gut (B,D) as compared with the wild type (A,C).

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. The hepatic competence defect of vHnf1^−/− embryos is not due to a global disruption of FGF signaling. (A) RT-PCR analysis of hepatic mRNAs in ventral endoderm isolated from vHnf1^+/+ embryos at 2-6s and cultured for 48 hours with DMSO, FGF or SU5402, an FGF receptor inhibitor. Gapdh was used for normalization. As expected, sprouty 2, an FGF pathway target, is strongly decreased when the culture is performed in the presence of SU5402. vHnf1 transcript levels are not influenced either by FGF or pharmacological inhibitors of FGF signaling. (B) Semiquantitative RT-PCR analysis of hepatic mRNAs in ventral endoderm isolated from WT or vHnf1^−/− embryos at 10s. The FGF signaling pathway is not affected in vHnf1^−/− ventral endoderm, as indicated by the normal expression of sprouty 2, Dusp6 (a feedback attenuator of the FGF pathway) and Socs3 (a direct target of vHNF1 and an inhibitor of HGF/EGF signaling and subsequent ERK1/2 phosphorylation).