Fig. 6. HNF6 controls the expression of HNF1ß. (A,B) Transient downregulation of HNF1ß in the BEC of Hnf6^–/– fetuses. (A) In control livers at E15.5, a strong signal for HNF1ß is detected in the ductal plate, namely in cells located at the interface between the portal mesenchyme and the liver parenchyma (arrow). The inset at higher magnification demonstrates that staining is nuclear. The weak and diffuse staining of the parenchyma corresponds to nonspecific background. Note that the section shown in A is directly adjacent to the section stained for HNF6 shown in Fig. 1B. (B) In Hnf6^–/– livers at E15.5, no HNF1ß nuclear staining is observed. The inset in B shows restoration of HNF1ß expression (arrowheads) in BEC of E17.5 Hnf6^–/– fetuses. (C) Electrophoretic mobility shift assays show that HNF6 can bind two sites in the 5'-flanking sequence of the Hnf1ß gene. Oligonucleotide probes corresponding to each of these sites give a retarded band with wheatgerm extracts programmed for HNF6 (+), but not with unprogrammed extracts (–). (D) Co-transfection experiments demonstrate that HNF6 can stimulate transcription from the Hnf1ß promoter. HEK293 cells were transfected with the indicated amount of HNF6 expression plasmid and with a CAT reporter construct controlled by the indicated fragments of 5'-flanking sequence of the Hnf1ß gene containing (gray bars) or not (white bars) the HNF6-binding sites. To calculate the fold increase in induction by HNF6, the activity of the reporter plasmids in the presence of HNF6 expression vector was normalized for the reporter activity in the presence of empty expression vector (mean values±s.e.m. of at least three experiments). p, liver parenchyma; pm, portal mesenchyme; pv, portal vein. Scale bar: 50 µm.