Fig. 4. vHNF1 is necessary for hepatic bud formation and expansion. (A-F) Whole-mount in situ hybridization analysis at 20-22s. Afp, strongly expressed in the WT mouse liver bud (A, arrow), is absent from the vHnf1^-/- presumptive hepatic bud (arrow, B). Note that Afp, the expression of which in the visceral endoderm depends on vHnf1 (Barbacci et al., 1999), was expressed at normal levels in the visceral endoderm of mutant embryos (arrowhead) because this tissue is derived from 4n WT embryos. Foxa2 is expressed in the WT liver bud and the foregut endoderm (C), but its expression is lost in the presumptive mutant hepatic domain (arrow, D), along with a caudally expanded expression in the gut (arrowhead, D). Shh expression in the foregut endoderm is greatly enhanced in mutant embryos (F), but is excluded from the WT liver bud (E) and presumptive mutant hepatic domain (F). (G-X) In situ hybridization (G,H,K-X) and immunostaining (I,J) analyses on transversal sections at 20-22s. Hex is strongly expressed in the WT liver bud (G), whereas in the mutant only a few Hex-positive cells are detected in the presumptive liver bud (H). Immunostaining in a control embryo shows hepatic bud formation with E-cadherin (Ecadh) expression and hepatoblast delamination into the adjacent STM with disruption of laminin (LN)-positive basal membrane (arrowheads, I). In mutants, the endoderm does not form a pseudostratified epithelium, leading to the absence of a hepatic bud (J). Shh expression is excluded from the presumptive mutant liver bud (L) as in the WT liver bud (K) with a stronger and expanded expression in the lateral foregut. Prox1, which labels hepatoblasts (M), is absent from the vHnf1^-/- ventral endoderm (N). Gata4 is excluded from the ventral endoderm (O) but is ectopically expressed throughout the entire ventral gut of mutant embryos (P). No differences in Gata6 expression are observed between WT and vHnf1^-/- mutant (Q,R). Absence or strongly decreased expression of Foxa1, Foxa2 and Foxa3 is observed in the ventral part of the mutant gut (T,V,X) as compared with the control (S,U,W).