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The onecut transcription factor HNF6 is required for normal development of the biliary tract

Frédéric Clotman¹, Vincent J. Lannoy^1,*, Michael Reber², Silvia Cereghini², David Cassiman³, Patrick Jacquemin¹, Tania Roskams³, Guy G. Rousseau¹ and Frédéric P. Lemaigre^1,

¹ Hormone and Metabolic Research Unit, Institute of Cellular Pathology and Université catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium
² Unité INSERM 423, Hopital Necker-Enfants Malades, Paris, France
³ Laboratory of Morphology and Molecular Pathology, Katholieke Universiteit Leuven, Leuven, Belgium
^* Present address: Euroscreen, Route de Lennik 802, B-1070 Brussels, Belgium

View larger version (60K): [in a new window]   Fig. 1. HNF6 is expressed in mouse fetal liver and biliary tract. HNF6 expression was assessed by RT-PCR with TATA box-binding protein (Tbp) as a reference (A) or by immunohistochemistry at E15.5 (B-D). (A) In the liver, Hnf6 mRNA is detected throughout the fetal period, although its concentration is lower between E12.5 and E15.5. (B) At E15.5, a strong nuclear staining is observed in the ductal plate, in cells located at the interface between the portal mesenchyme and the liver parenchyma (arrow), and a weaker nuclear signal is detected in hepatocytes (arrowheads). (C) HNF6 is also detected in the epithelium of the gallbladder primordium, but not in the surrounding mesenchyme. (D) The epithelium of the common bile duct shows a strong HNF6 signal, while a weak signal is present in the epithelium of the duodenum. cbd, common bile duct; d, duodenum; gb, gallbladder primordium; m, mesenchyme; p, liver parenchyma; pm, portal mesenchyme; pv, portal vein. Scale bar: 50 µm.

View larger version (142K): [in a new window]   Fig. 2. Lack of HNF6 results in absence of gallbladder, abnormal morphogenesis of the extrahepatic bile ducts and cholestasis. (A) In wild-type adult mice, the gallbladder is seen on the ventral side of the liver and the hepatic ducts merge with the cystic duct to form the common bile duct, which ends up in the duodenum. (B) In Hnf6–/– adult animals, the gallbladder is absent. An enlarged structure (arrow) connects the liver to the duodenum. (C,D) Transverse sections in E10.5 embryos. The sections were stained for Pdx-1 to identify the ventral pancreatic bud (strong staining) and the duodenum (weak staining). Rostral is towards the left, caudal towards the right. (C) In wild-type embryos, the gallbladder primordium, devoid of Pdx-1 staining, is observed between the liver primordium and the ventral pancreatic bud. (D) In Hnf6–/– embryos, the gallbladder primordium is absent. Note that the ventral pancreatic bud is also missing at E10.5, as formation of the pancreatic buds is delayed in Hnf6–/– mice (P. J., G. G. R. and F. P. L., unpublished). (E,F) Fouchet’s staining on liver sections from Hnf6–/– mice at P6. (E) Abnormal bile accumulation between the hepatocytes (arrowheads) is observed. (F) Large areas of bile accumulation surrounded by necrotic parenchyma are also detected. bl, bile lake; cbd, common bile duct; cd, cystic duct; d, duodenum; gb, gallbladder; hd, hepatic duct; lp, liver primordium; p, liver parenchyma; vp, ventral pancreatic bud. Scale bars: 50 µm in C,E (bar in C also applies to D); 100 µm in F.

View larger version (88K): [in a new window]   Fig. 3. Time-course of intrahepatic bile ducts development in control and in Hnf6–/– animals. The biliary tract of Hnf6+/– mice was indistinguishable from that of wild-type mice. Heterozygotes were therefore used as controls together with wild-type fetuses. The biliary epithelial cells (BEC) were visualized on liver sections by cytokeratin (CK) immunostaining. (A,B) At E13.5 in the controls, a few CK-positive cells are found dispersed throughout the liver parenchyma. In Hnf6–/– fetuses, numerous CK-positive cells are observed in the vicinity of the portal vein. These cells extend as cords within the liver parenchyma and a few lumina (arrow) are visible between the cells. (C,D) At E14.5 in the controls, CK-positive cells form a discontinuous layer at the interface between the portal mesenchyme and the liver parenchyma. In Hnf6–/– fetuses, CK-positive cells form irregular duct-like structures (arrow). (E,F) At E15.5 in the controls, BEC form a single continuous layer of cells called the ductal plate around the portal vein. In the Hnf6–/– fetuses, disorganized biliary structures, including duct-like structures (arrows), are observed. These duct-like structures are delineated by a continuous cuboidal epithelium of CK-positive cells. (G,H) At E16.5 in the controls, the ductal plate is partly duplicated and focal dilations (arrowheads) are visible between the two layers of BEC. In some Hnf6–/– livers, large biliary cysts delineated by CK-positive cells and located in the vicinity of the portal vein are observed (note that magnification is lower in H). (I,J) At E17.5 in the controls, the first bile ducts are identified as CK-positive cells surrounding a lumen and incorporated into the portal mesenchyme. Between the bile ducts, CK-positive cells have disappeared from the interface between the portal mesenchyme and the liver parenchyma. In Hnf6–/– fetuses, lumina (asterisks) are still present around the portal vein but the surrounding epithelium is disorganized and partly consists of CK-negative cells. A single layer of CK-positive cells is observed around the portal mesenchyme. (K,L) At P10 in the controls, mature bile ducts are seen within the portal mesenchyme and remnants of the ductal plate ongoing regression (arrowheads) are still visible between the liver parenchyma and the portal mesenchyme. In Hnf6–/– animals, CK-positive cells are located discontinuously around the portal mesenchyme. bc, biliary cyst; bd, bile duct; dp, ductal plate; p, liver parenchyma; pm, portal mesenchyme; pv, portal vein. Scale bar: in A, 50 µm in A-G,I-L; in H, 100 µm.

View larger version (159K): [in a new window]   Fig. 4. Differentiation of biliary epithelial cells (BEC) in control and Hnf6–/– fetuses. (A-D) CK-positive cells and portal mesenchyme express laminin as detected by immunohistochemistry in control and in Hnf6–/– fetuses at E15.5. In Hnf6–/– fetuses, laminin (arrowheads in D) is found at the basal pole of the BEC surrounding the lumen (asterisks in B,D) of duct-like structures. (A,C) and (B,D) show adjacent sections. (E,F) Double immunofluorescence for CK (green) and for the proliferation marker Ki-67 (red) on liver sections. These were analyzed when the number of CK-positive cells was high enough, i.e. at E14.5 in control fetuses and at E13.5 in Hnf6–/– fetuses. Both in control (E) and in Hnf6–/– (F) livers, the CK-positive cells do not express Ki-67, which is detected in CK-negative cells (arrowheads). dp, ductal plate; m, portal mesenchyme; p, liver parenchyma; pv, portal vein. Scale bars: 50 µm in A,E.

View larger version (59K): [in a new window]   Fig. 5. Expression of liver-enriched transcription factors in control (+/+ and +/–) and in Hnf6–/– mice. The expression levels were assessed by semi-quantitative RT-PCR on liver RNA. The data are representative of three independent experiments. (A) At E14.5, the expression levels of Hnf3ß, Hnf3 and Hnf4 are not affected by inactivation of the Hnf6 gene. The expression levels of Hnf1 and Hnf3 are slightly increased in Hnf6–/– fetuses, whereas the two splicing isoforms of Hnf1ß (Hnf1ß-A and Hnf1ß-B) are strongly downregulated. At P3, the expression levels of Hnf3ß, Hnf3 and Hnf4 are not affected, the expression of Hnf1ß has normalized and that of Hnf1 and Hnf3 is still upregulated. (B) At E12.5, the expression of the two splicing isoforms of Hnf1ß is strongly downregulated in the Hnf6–/– livers, as assessed with Tbp as a reference.

View larger version (82K): [in a new window]   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.