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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.
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), 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).
