Fig. 5. vHNF1 is required for hepatic specification of the ventral endoderm. (A,B) Semi-quantitative RT-PCR analysis of RNA from WT and vHnf1^-/- ventral endoderm isolated from 6-8s (A) or 8-10s (B) mouse embryos. Gapdh was used for normalization. Hex, Foxa2 at 6-8s and Hex, Foxa1, Foxa2 at 8-10s are expressed in the hepatic endoderm of vHnf1^-/- mutant embryos. Defective hepatic and ventral pancreatic specification in the mutants is shown by the absence of Alb (A,B) and Ptf1a (B). (C-F) Immunostaining on sagittal sections of 8s control (vHnf1^+/+) (C,E) and vHnf1^-/- (D,F) embryos using vHNF1 (C,D) and HNF4 (E,F) antibodies indicates a similar position of the extraembryonic visceral endoderm (visc.; black arrow; vHNF1- and HNF4-positive) relative to the ventral endoderm (white arrow). Note that the vHNF1 antibody does not detect the weak levels of vHNF1 expression in the ventral endoderm as compared with the visceral endoderm (visc.; black arrow). (G-R) Immunostaining (G,H) and in situ hybridization (I-R) on sagittal sections of 8-10s control (G,I,K,M,O,Q) and vHnf1^-/- (H,J,L,N,P,R) embryos using the lung marker Nkx2.1 (G,H), the thyroid-hepatic marker Hex (I,J) and the lung-tracheopharyngeal marker Irx2 (K,L) show apparently correct foregut endoderm regionalization in mutant embryos. Expression of Foxa1 (M,N), Foxa2 (O,P) and Foxa3 (Q,R) is strongly downregulated in the prehepatic domain of mutant embryos (N,P,R, yellow dashed lines) as compared with WT (M,O,Q). (S) RT-PCR analysis of hepatic RNA from microdissected ventral endoderm of 2-5s control or vHnf1^-/- embryos that has been cultured for 48 hours with bFGF plus heparan sulfate proteoglycan. Normal induction of hepatic specification, as indicated by Alb and Ttr expression in WT explants, is disrupted in the vHnf1^-/- explant cultures.