Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1ß
Catherine Coffinier1,*,
,
Lionel Gresh1,,
Laurence Fiette2,
François Tronche3,,
Günther Schütz3,
Charles Babinet4,
Marco Pontoglio1,
Moshe Yaniv1,
and
Jacqueline Barra4
1 Unité des Virus Oncogènes-CNRS URA 1644, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France
2 Unité d’Expertise en Histotechnologie et Pathologie, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France
3 Division Molecular Biology of the Cell I, DKFZ, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
4 Unité de Biologie du Développement-CNRS URA 1960, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France
* Present address: Howard Hughes Medical Institute-UCLA, Los Angeles, CA 90095-1662, USA
Present address: Institut de Biologie, CNRS FRE 2401, Collège de France, 11, place Marcelin Berthelot, 75231 Paris Cedex 5, France
Both authors contributed equally to this work
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Fig. 1. Targeted inactivation of the Hnf1ß gene in the liver. (A) Strategy used to generate the conditional allele, featuring the map of the murine locus, the recombinant allele Hnf1ß3lox and the Cre-mediated alleles Hnf1ßflox and Hnf1ßdel. The DNA fragment used as probe for the genomic analysis is indicated as well as the primers used for genotyping. The first exon is featured by a gray box, the selection genes by white boxes, the loxP sites by triangles, and the promoter by an arrow. Restriction sites: Bm BamHI; H, HindIII; P, PmlI; R, EcoRI; X, XbaI. (B,C) Cell specificity of Cre-mediated recombination in newborn AlfpCre; R26R mice. (B) ß-galactosidase staining on vibratome section of liver, showing the specific activation of the lacZ transgene in the hepatic parenchyma (hp) and in the bile ducts (bd) but not in the wall of the portal vein (pv) and of the hepatic arterioles (ha). (C) Thin section through a gallbladder after ß-galactosidase staining. Arrows indicate the staining of fields of the inner epithelium. lu, lumen. (D) Southern blot analysis of Cre-mediated recombination on liver DNA. Liver DNA from mice of either mutant (Hnf1ßlacZ/floxAlfpCre) or control genotypes (Hnf1ßflox/+, Hnf1ßflox/+AlfpCre or Hnf1ßflox/lacZ) were digested by the restriction enzyme HindIII and probed with the KH probe indicated on Fig. 1A. Bands characteristics for the different alleles are indicated on the right. Del* indicates a product of partial digestion specifically observed for the Hnf1ßdel allele. A rate of 80% conversion of the Hnf1ßflox allele into the Hnf1ßdel form was estimated, by semi-quantitative PCR, for the Cre-positive animals. As hepatocytes represent about 70% of liver cells and biliary tree cells approximately 10%, we concluded that Hnf1ß inactivation was achieved in a majority of hepatic cells. (E) Northern blot analysis of 1-month-old mutant and control liver RNA, showing the complete loss of HNF1ß expression after Cre recombination. Total RNA was hybridized with HNF1ß cDNA and after stripping probed for HPRT expression to check for loading. Scale bars: 100 µm.
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Fig. 2. HNF1ß liver-specific mutation results in a severe growth defect and in accumulation of bile components in serum. (A) Picture of 2-month-old control and mutant littermates. (B) Growth curves of a litter resulting from a Hnf1ßflox/flox x Hnf1ßlacZ/+AlfpCre cross. Weight was measured from day 15 after birth. Black squares, control (CO) males (mean of four mice); black circles, control females (mean of five mice); white squares, mutant (M) males (mean of three mice); white circles: a mutant female. Standard deviation for each sample is featured as error bars. (C,D) Biochemical analysis of bilirubin (C) and bile acids (D) levels in mutant (black bars) and control (gray bars) mice. Values are mean of 3-4 mice. All differences between mutant and controls were significant (P<0.05). Standard deviations for each sample are indicted by as error bars.
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Fig. 3. Abnormal extrahepatic biliary epithelium in the mutant mice. (A-C) Extrahepatic biliary tract of 2-month-old control (A) and mutant (B,C) mice. Mutant gallbladders are irregularly shaped. Some mutants show dilated cystic duct. gb, gallbladder; cd, cystic duct; bd, common bile duct; dd, duodenum. Scale bars: 5mm. (D-E) Gallbladder sections for control (D) and mutant (E) animals. In the mutant sample, the normal cuboidal epithelium is replaced in some areas by a stratified squamous epithelium (arrowhead) or mucus-secreting cells (arrow). lu, lumen. Scale bars: 100 µm in D; 150 µm in E.
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Fig. 4. Intrahepatic bile duct status in mutant mice. (A,B) Immunostaining for cytokeratin (CK) on control (CO) and mutant (M) liver sections, isolated two months after birth. (A) Control livers contain a large number of well differentiated bile ducts with a layer of CK-positive cells surrounding a lumen (arrowheads). (B) Mutant livers present a very disorganized biliary system. Most portal tracts do not contain any bile duct or CK-positive cell. Hepatocyte necrosis, inflammation and oval cell proliferation are also seen in mutant livers at this stage. (C,D) Histological analysis of the larger intrahepatic bile ducts on control and mutant animals. Arrows in C indicate two normal bile ducts. The ducts of different size feature a single-layer epithelium with regularly spaced nuclei. By contrast, mutant bile ducts present a disorganized multi-layered epithelium (arrow in D). bd, bile duct; pv, portal vein. Scale bars: 1 mm in A,B; 100 µm in C,D.
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Fig. 5. Lack of arteries in mutant portal tracts. Immunostaining for  smooth muscle actin (SMA) on control (CO) and mutant (M) liver sections, isolated two months after birth. (A,C) Control livers show SMA-positive arteries (arrowheads) located around portal veins. Portal veins (pv) are also stained with the antibody. (B,D) In mutant livers almost all structures stained are veins. Only few large arteries are present. Scale bars: 100 µm.
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Fig. 6. The Hnf1ß mutation affects intrahepatic bile duct development. Immunostaining for cytokeratin (CK) on control (CO) and mutant (M) liver sections. (A-D) E17.5 fetuses. (A,B) Control livers show a well-formed and stained ductal plate, and some bile ducts that are beginning to differentiate (arrows). (C,D) Mutant livers show disorganized ductal plate with irregular duct-like structures not completely surrounded by CK-positive cells (arrowheads). (E-H) P8 livers. (E) Differentiated bile ducts are seen on control mice. Some remnants of the ductal plate are still visible (arrowhead). (F-H) Mutants show no or very few CK-positive cells (arrowhead) (F), ductal plate remnants and abnormal CK-positive structures within the portal mesenchyme (G), or abnormal bile ducts (H). hp, hepatic parenchyma; pv, portal vein; pm, portal mesenchyme; dp, ductal plate; bd, bile ducts. Scale bars: 50 µm.
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Fig. 7. (A,B) HNF1ß is required for the general lipid homeostasis. Biochemical analysis of cholesterol (A) and triglycerides (B) levels in mutant (black bars) and control (gray bars) mice at the age of 1 week (n=8), 2 weeks (n=6) and 8 weeks (n=6). All differences between mutant and controls were significant (P<0.05). There is no significant difference between 2- and 8-week-old mutants. Triglyceride level for 1-week-old mutant is abnormally elevated despite the lack of comparison with the control. Standard deviation for each sample is featured as error bars. (C,D) HNF1ß is required for the expression of the Oatp1 transporter and the fatty acid dehydrogenase VLCAD. (C) Semi-quantitative RT-PCR analysis of liver organic anion transporters expression (Slc21a1/Oatp1; Slc21a5/Oatp2; Slc21a6/Oatp4). Genotypes are indicated on top. HPRT primers were used as a normalizing standard for the RT samples (HPRT) and as a reverse transcription control (HPRT –RT). Gene expression was measured in six mice for each genotype. (D) Northern blot analysis of very long chain acetylCoA dehydrogenase (VLCAD) mRNA levels. HPRT cDNA probe is used as a normalizing standard. Gene expression was measured in four mice for each genotype. Ratios between mutants and controls are shown after HPRT normalization (M/CO).
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© The Company of Biologists Ltd 2002
