1 Unité des Virus Oncogènes-CNRS URA 1644, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France
2 Unité dExpertise 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
Author for correspondence (e-mail: yaniv{at}pasteur.fr)
Accepted 21 January 2002
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SUMMARY |
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Key words: Cre/loxP, Bile duct, Epithelium differentiation, Gallbladder, Lipids
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INTRODUCTION |
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At the adult stage, HNF1ß is strongly expressed throughout the biliary system. It is also expressed in periportal hepatocytes. This expression pattern distinguishes HNF1ß from the closely related HNF1 protein that is uniformly distributed in all hepatocytes, and is expressed at lower levels in BEC (Pontoglio et al., 1996
). The difference in expression of the two genes is interesting in light of the liver zonation phenomenon (Jungermann and Katz, 1982
). Hepatocyte activities are specialized along the porto-central axis; catabolic metabolism occurs in the periportal hepatocytes, whereas anabolic functions are performed by hepatocytes closer to the central vein. Thus, the balance between the two HNF1 activities along the lobules may determine which hepatic genes are activated at a given position.
In addition to its hepatic expression, Hnf1ß (Tcf2 Mouse Genome Informatics) is strongly expressed in several epithelia organized in tubules, such as the pancreatic exocrine ducts and the kidney tubules (De Simone et al., 1991; Lazzaro et al., 1992
). As HNF1ß is expressed from the very onset of formation of such structures, it might play a role during differentiation and organogenesis (Coffinier et al., 1999a
; Ott et al., 1991
). Furthermore, gene inactivation demonstrated an early requirement for Hnf1ß in the differentiation of another epithelium, the visceral endoderm (Barbacci et al., 1999
; Coffinier et al., 1999b
). HNF1ß mutation blocks the differentiation of visceral endoderm and prevents its specialization into the two cell types that are specific for the embryonic and extra-embryonic territories. As visceral endoderm fails to differentiate, HNF1ß-null mice die around the time of gastrulation, preventing further study of the role of HNF1ß during later differentiation events.
To address the role of HNF1ß in liver organogenesis, we generated a conditional allele of the gene and performed its tissue-specific inactivation in the liver using the Cre/loxP system (Gu et al., 1994). The loss of HNF1ß in both hepatocytes and bile ducts resulted in a severe phenotype, including growth retardation and jaundice. Histological analysis of the gallbladder and the larger IHBD revealed epithelial abnormalities. We also observed a strong decrease in the number of the smaller IHBD and a persistence of the ductal plate from which IHBD are formed. These data suggest a requirement for HNF1ß in biliary epithelium formation from the onset of the biliary system development. Biochemical and molecular studies of hepatic markers led to the identification of two HNF1ß specific target genes, Oatp1 (Sla21a1 Mouse Genome Informatics) and Vlcad (Acadvl Mouse Genome Informatics), both of which are involved in lipid metabolism in hepatocytes.
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MATERIALS AND METHODS |
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Gene expression analysis
Northern blot analysis were performed on 30 µg of liver total RNA or 10 µg of kidney total RNA using Hnf1ß and Vlcad cDNA probes, and a Hprt probe to normalize the samples. Expression of the differentiation markers were studied using semiquantitative RT-PCR as described previously (Coffinier et al., 1999b). The PCR cycle number were estimated for each primer pair to assure linear range amplification. The primer sequences are available on request.
Representational difference analysis of cDNA was carried out following a protocol supplied by M. Hubank (Hubank and Schatz, 1994). PolyA mRNA were purified from 2-week-old mutant and control liver total RNA (Dynabeads, Dynal). Double-stranded cDNA was prepared using oligo dT primer (Universal RiboClone cDNA Synthesis System, Promega).
Biochemical and histological analysis
Blood total and conjugated bilirubin concentrations were measured by colorimetry (Sigma 552/553). After overnight fasting, blood triglycerides and cholesterol concentrations were measured enzymatically (Sigma 336 and 352). Blood albumin and biliary acid concentrations were determined by Vebiotel laboratory (94110 Arcueil, France). Organs were fixed in formaldehyde 4% or Bouins fixative, and embedded in paraffin using routine procedures. Sections (5 µm) were stained with Hematoxylin and Eosin.
Immunohistochemical procedure
Livers were frozen in isopentane cooled in a liquid nitrogen bath, and sectioned at 7 µm using a cryostat. Sections were air-dried overnight, fixed in acetone at 4°C for 10 minutes and washed in two changes of Tris-buffered saline and treated with a blocking buffer (TBS/normal goat serum 10%/bovine serum albumin 1%/Triton X-100 0.3%) for 30 minutes. Thereafter, sections were washed in TBS, incubated with primary antibody (rabbit anti-human keratin A0575, Dako or monoclonal anti- smooth muscle actin 1A4, Dako; 1/250 dilution in TBS/normal goat serum 1%/BSA 0.1%/Triton X-100 0.3%) overnight at 4°C, washed again and incubated with secondary antibody (Envision+Peroxidase Rabbit, Dako or anti-mouse IgG, HRP-linked NA931, Amersham; 1/400 dilution) for 30-60 minutes at room temperature. Sections were stained with 3-amino-9-ethylcarbazole (ICN Biochemicals) and counterstained with Hematoxylin.
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RESULTS |
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Liver-specific inactivation of the Hnf1b gene
In the liver, Hnf1ß displays a complex expression pattern. It is strongly expressed in the biliary epithelial cells, and more weakly in the hepatocytes localized at the periphery of the hepatic lobules (Coffinier et al., 1999a). To inactivate the Hnf1ß gene in both cell types, we required a Cre recombinase transgene expressed in these cells or in a common precursor. The AlfpCre transgene contains both albumin and
-fetoprotein regulatory elements, drives Cre recombinase expression in the hepatic bud as early as embryonic day 10 (E10) and targets both cell lineages (Kellendonk et al., 2000
). To extend these initial data, AlfpCre activity was analyzed after crossing the AlfpCre transgenic mice with the ROSA26 Cre reporter line (R26R) carrying a lacZ gene that is activated after Cre-driven recombination (Soriano, 1999
). Liver sections prepared at birth showed specific ß-galactosidase staining in all hepatocytes and in all cells of the IHBD. By contrast no staining was detected in endothelial cells or portal vein mesenchyme (Fig. 1B). The homogenous staining suggested that efficient recombination occurred in both hepatocytes and biliary epithelial cells or more probably in the hepatoblast, the postulated common precursor of both cell types. Furthermore, these data confirm that Cre expression occurred before bile duct morphogenesis, which starts around E15, as differentiated biliary epithelial cells do not express the albumin gene (Shiojiri, 1997
). In contrast to the uniform staining observed in the liver, the gallbladder sections showed only patches of ß-galactosidase staining in the inner epithelium (Fig. 1C). As the gallbladder separates from the liver bud and stops expressing the albumin gene at day E10.5, the patchy staining suggests a shorter exposure to Cre activity, resulting in recombination events occurring only in a few precursor cells.
Liver-specific inactivation of the Hnf1ß gene was achieved by crossing Hnf1ßflox/flox mice with animals carrying one Hnf1ß null allele and an AlfpCre transgene (Hnf1ßlacZ/+AlfpCre) (Coffinier et al., 1999b). One recombination event per cell was sufficient to generate a homozygous null mutation in Hnf1ßlacZ/floxAlfpCre offspring. Pups presenting the four possible genotypes were born in normal Mendelian ratios (data not shown). Recombination efficiency at the Hnf1ß locus was confirmed by Southern blot analysis of liver DNA using a probe distinguishing between all four Hnf1ß alleles: wild type, Hnf1ßlacZ, Hnf1ßflox and Hnf1ßdel (Fig. 1D). To confirm the loss of Hnf1ß expression in Hnf1ßlacZ/floxAlfpCre mice, a northern blot analysis was performed on total liver RNA using a probe specific for Hnf1ß transcript. No Hnf1ß RNA was detected in the mutant (Hnf1ßlacZ/+AlfpCre) livers compared with controls, either Hnf1ßflox/+, Hnf1ßflox/+AlfpCre or Hnf1ßflox/lacZ (Fig. 1E). The loss of the transcript was tissue-specific as equal amounts of Hnf1ß RNA were detected in total kidney RNA from both control and mutant animals (data not shown). Based on AlfpCre activity described above and on both DNA and RNA analysis, we concluded that Cre expression in the mutant mice led to the specific inactivation of the Hnf1ß gene in hepatocytes and IHBD epithelial cells. In addition, our data suggested that partial deletion may have occurred in the gallbladder.
Hnf1ß mutation results in severe growth retardation and jaundice
Liver-specific inactivation of the Hnf1ß gene resulted in a clear phenotype. Hnf1ßlacZ/floxAlfpCre mice showed severe growth retardation, hypertrophy of the liver and chronic jaundice. Although some variations in the growth retardation were observed, the jaundice and the liver enlargement were fully penetrant among the mutant animals.
Body weight differences could be measured as early as day 2 after birth and increased with time (data not shown and Fig. 2A,B). The weight differences between mutant and control littermates were more dramatic by the time of weaning, reaching a growth plateau. In addition to their smaller size, mutants were cachectic with reduced adipose tissue and muscular atrophy. Despite the severity of their phenotype, most mutants survived for several months.
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HNF1ß mutation results in gallbladder epithelial dysplasia and in a paucity of intrahepatic bile ducts
Despite a probably incomplete deletion of the Hnf1ß gene in the gallbladder, as suggested by the data reported in Fig. 1E, its morphology was strongly affected in mutant animals and was characterized by an irregular shape interrupted by several constrictions (Fig. 3A-C). In some extreme cases, the cystic duct was completely dilated and no duct was really identifiable; at the same time, the common bile duct was affected and the boundary between the two ducts could not be determined (Fig. 3C). Histological staining of tissue sections revealed that the inner layer of the gallbladder presented areas with disorganized epithelia in place of a typical cuboidal epithelium (Fig. 3D,E). Some of the abnormal cells presented a morphology reminiscent of mucus-secreting cells (Fig. 3E). Despite these abnormalities, the gallbladders were filled with bile and communicated normally with the liver and the duodenum. We conclude that extrahepatic biliary obstruction was not the cause of cholestasis.
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Hnf1ß is required for lipid metabolism
To further probe the consequence of HNF1ß inactivation in the liver, a panel of biochemical parameters was analyzed in the serum of both mutant and control littermates. General hepatic functions were assessed by measuring the level of serum albumin. No significant difference was detected between the mutants (22.2±2.9 g/l; n=4) and controls (25.7±3.1 g/l; n=4). However, in addition to the accumulation of bilirubin and bile acids described above, the levels of serum cholesterol and triglycerides were found to be dramatically increased, as early as one week after birth (Fig. 7A,B). These levels increased between 1 and 2 weeks of age and then became stabilized. Bile represents a major route for the excretion of organic solutes, such as bilirubin. Furthermore, cholesterol degradation into bile acids and biliary cholesterol secretion are the major pathways to eliminate excess cholesterol (Repa and Mangelsdorf, 2000). Thus, bilirubin, bile acids and cholesterol accumulation observed in mutant mice could result from a reduced bile flow due to IHBD paucity. These accumulations could also be due to the combined effect of a hepatocytic defect and a bile flow defect. Indeed, high levels of triglycerides suggested that hepatocyte metabolism may be affected by Hnf1ß liver-specific deletion.
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Bile traffic involves numerous transporters (Kullak-Ublick et al., 2000; Trauner et al., 1998
). Ntcp (Slc10a1), a Na+-dependent transporter, and the sodium independent transporters, Oatp1, Oatp2 and Oatp4 (Slc21a1, Slc21a5 and Slc21a6), participate in the reabsorption of bile acids from the blood in the sinusoids toward the hepatocytes. They play an important role in allowing the sensing of bile acid levels in the blood by hepatocytes. cMoat (canalicular multiple organic anion transporter; Abcc2 Mouse Genome Informatics) and Bsep (bile salt export pump; Abcb11 Mouse Genome Informatics) deliver, respectively, bilirubin and bile acids from the hepatocytes to the bile ductule. We found that Oatp1 was specifically downregulated in the absence of HNF1ß, whereas the other six transporters remained almost unaffected (Fig. 7C and data not shown). This decrease in Oatp1 expression was observed in 2-week-old mice, suggesting that it could be a direct consequence of HNF1ß deletion in hepatocytes and not a secondary effect of cholestasis. Interestingly, Oatp1 is also a target of HNF1
factor (Shih et al., 2001
). Oatp1 downregulation could also contribute to the bile acid accumulation in the serum, as bile acids will then be less efficiently reabsorbed from blood.
Data resulting from the use of representational difference analysis (RDA) of cDNA (Hubank and Schatz, 1994) of mutant versus control livers led to the identification of another HNF1ß target gene. The transcription of very long chain acetylCoA dehydrogenase (Vlcad), one of the four key enzymes of fatty acid oxidation (Beinert, 1963
), was strongly decreased in the mutant samples (Fig. 7D). The Vlcad promoter (GenBank Accession Number, AJ012054) contains two HNF1 consensus binding sites located at positions 71 and 332, but its expression is not affected in the Hnf1
/ mice (data not shown). Therefore, VLCAD constitutes a HNF1ß specific target gene. A defect in fatty acid oxidation could account for the accumulation of triglycerides observed in the mutant serums. As fatty acid oxidation is performed by hepatocytes, this further indicates that HNF1ß plays specific roles in hepatocyte functions.
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DISCUSSION |
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Replacement of the first exon of HNF1ß with ß-galactosidase followed by in situ staining has demonstrated that this gene is abundantly transcribed in the bile duct epithelium (Coffinier et al., 1999a). Much lower expression was observed in periportal hepatocytes. To inactivate the gene in both cell types, we crossed HNF1ß floxed mice with an AlfpCre mouse. This mouse was shown to express the recombinase in the liver primordium from day 10 of gestation. At this time point, the Cre should be expressed in the hepatoblast that give rise to both hepatocytes and biliary duct cells. Crosses between the AlfpCre mouse and the ROSA26 floxed ß-galactosidase reporter strain, showed that inactivation occurred in both cell types. Furthermore we have seen that partial inactivation occurs in the gallbladder epithelium.
HNF1ß is an essential factor in the intrahepatic bile duct differentiation pathway
Inactivation of HNF1ß in precursors of intrahepatic biliary epithelium led to paucity of interlobular bile ducts and dysplasia of larger hepatic bile ducts epithelia. Immunohistochemical analysis showed that bile duct defects were already present at E17.5, and that ductal plate, from which IHBD develop, abnormally persisted after birth in mutant mice. Paucity of IHBD is thus directly linked to a developmental defect, demonstrating that HNF1ß is essential for normal IHBD morphogenesis during liver formation. Similar features were observed in Hnf6/ mice, demonstrating that both HNF1ß and HNF6 act in a transcription factor network involved in IHBD formation [see accompanying paper (Clotman et al., 2002)]. The results from Clotman et al., suggest that a HNF6
HNF1ß cascade may be an important component of this network. These two complementary studies are the first to identify specific transcription factors involved in the IHBD differentiation program. In addition to the paucity of small IHBD observed in newborn, we observed a dysplasia of larger IHBD and of the gallbladder. The epithelium presented abnormal characteristics with a delocalization of the nuclei from the base of the cell, the organization of multiple layers of cells or the presence of ectopic epithelium. These observations suggest an effect of Hnf1ß inactivation on the maintenance of epithelial differentiation. As previously suggested for the visceral endoderm, HNF1ß could regulate fundamental characters of epithelium identity such as the basal position of the nuclei and the growth in monolayer of cells. Thus, HNF1ß seems to have a dual function in controlling both the differentiation and the proliferation of certain epithelial cells.
Paucity of IHBD correlates, in mutant mice, with a lack of interlobular arteries. As Hnf1ß is not expressed in endothelial cells, this defect is an indirect consequence of the mutation. These findings demonstrate that proper morphogenesis of bile ducts is necessary for arterial vascularization of the liver. Intercellular signals from biliary epithelial cells may act on endothelial or smooth muscle cells differentiation and/or proliferation to establish the correct three-dimensional liver organization.
Relationship with human diseases
Human autosomal dominant mutations in the Hnf1ß gene result in a particular form of type II diabetes called maturity onset diabetes of the young type 5 (MODY5) (Horikawa et al., 1997). In some of the carriers of these mutations, kidney developmental defects, which are associated with internal genital abnormalities, were also observed (Lindner et al., 1999
; Nishigori et al., 1998
). Liver function of individuals with MODY5 was poorly investigated, owing to the low occurrence of cases. However, a liver function exploration in Japanese individuals with MODY5 reported high levels of
-glutamyl transpeptidase, aspartate and alanine amino transferases, indicative of hepatic failure (Iwasaki et al., 1998
). A case of hyperbilirubinemia was also described. These values were significantly higher than normal values and were not observed in individuals with MODY1/HNF4
or MODY3/HNF1
, suggesting that autosomal dominant HNF1ß mutations may, at least in some cases, be specifically associated with liver dysfunction. Whether this liver dysfunction is linked to bile duct defects similar to those observed in mice, remains unknown.
Congenital diseases of IHBD can affect different levels of the biliary tree and can be characterized by dilatation or involution of the bile duct structures. All these diseases seem to have a developmental origin and to result from ductal plate remodeling problems, also called ductal plate malformations (DPM) (Desmet, 1992). The hepatic phenotype of HNF1ß-liver specific inactivation mice resembles, in some aspects, two human syndromes: Alagilles syndrome, which is characterized by a paucity of interlobular bile ducts, and Carolis disease, a congenital non-obstructive dilatation of the larger IHBD. However, the phenotype of our mutant mouse does not perfectly match the description of these congenital diseases. Despite the divergence of phenotypes observed, study of mice lacking Hnf1ß may provide some insights into the early development of DPM disease in humans.
HNF1/HNF1ß duality and hepatic functions
Hnf1 and Hnf1ß were initially characterized as liver-specific genes participating in a network of transcription factors. In vitro studies showed that both proteins bind the same DNA sequences with similar affinities. It has led to a model proposing that the balance between the strong transactivator HNF1
and the weaker HNF1ß constitutes a major element in HNF1 target gene regulation (Baumhueter et al., 1988
; Rey-Campos et al., 1991
; Song et al., 1998
). However, studies of knockout mice have revealed that the molecular basis for this regulation might be more complex. For example, in the Hnf1
/ mutants, the expression of several renal transporters were strongly affected despite the presence of large amounts of HNF1ß in the same renal cells (Pontoglio et al., 2000
), suggesting that HNF1
possesses distinct functions. We present evidence that in hepatocytes, despite the presence of large excess of HNF1
protein, HNF1ß is necessary for activation specific target genes as shown by the decreased transcription of hepatic transporter Oatp1 and Vlcad enzyme in the mutant livers.
Oatp1 participates in the reabsorption of bile acids from the blood. Oatp1 loss of expression could contribute to the accumulation of bile acids in the blood. As Oatp1 is also a target of HNF1 (Shih et al., 2001
), this gene may depend upon transactivation by HNF1
/HNF1ß heterodimers. By contrast, VLCAD, a key enzyme of fatty acid oxidation, is affected by Hnf1ß mutation but not by Hnf1
mutation, suggesting a requirement for HNF1ß homodimers.
In conclusion, our results show that in hepatocytes HNF1ß has a specific role not fully redundant with HNF1. Moreover, metabolic abnormalities observed in the mutants correlate strikingly with the expression of the Hnf1ß gene in the liver. Owing to the functional zonation of hepatocytes in the lobules, the periportal hepatocytes are the major site of bile excretion and of oxidative energetic metabolism, including fatty acid oxidation (Jungermann and Katz, 1982
). As reported in a previous study, HNF1ß is expressed in a subpopulation of hepatocytes located at the lobule periphery, whereas HNF1
has been shown to be expressed in all hepatocytes (Coffinier et al., 1999a
; Pontoglio et al., 1996
). Thus, only the periportal hepatocytes would be affected by the loss of HNF1ß, which correlates with the alterations of biliary acids transport and fatty acid oxidation. We propose that the abnormal HNF1 activity in this subpopulation of cells, which is due to the lack of HNF1ß, resulted in a strong decrease of Oatp1 and VLCAD expression. These observations indicate that HNF1ß may participate to establish functional zonation within the liver by specification of periportal hepatocytes.
Finally, HNF1ß is also expressed in other tubular structures such as pancreatic exocrine ducts and kidney tubules (Coffinier et al., 1999a). Moreover, this expression starts with the onset of differentiation of these structures. The role of HNF1ß in bile duct formation and its specific expression pattern suggests that this gene could also be controlling pancreatic ducts and kidney tubule morphogenesis. HNF1ß could be part of a general developmental program leading to establishment of epithelial tubular structures during organogenesis of several organs. This question may be addressed by studying crosses of the Hnf1ß floxed mouse with other cell-specific Cre-expressing mice.
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ACKNOWLEDGMENTS |
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