In summary…

A number of human diseases are characterized by malformations or functional deficiencies of cells in the liver or pancreas. The aim of the research in our group is to characterize the molecular mechanisms that control development of the liver and pancreas. This will help to understand such diseases and to treat them by pharmacological or cell therapy-based approaches. The present focus is on the study of the role of the Onecut transcription factors, discovered in our laboratory.

The Onecut transcription factors constitute a class of tissue-restricted homeoproteins, with three members in mammals: HNF-6 (OC-1), OC-2 and OC-3 (1-3). The role of HNF-6 in development has been addressed by studying Hnf6 knockout mice generated in the laboratory. The results show that HNF-6 is critical in development for pancreas morphogenesis, for pancreatic endocrine cell differentiation, and for differentiation of biliary cells in the liver (4-9). Some phenotypical aspects of HNF-6 deficient mice mimick human pediatric diseases. The transcriptional regulatory properties of HNF-6 and the mechanisms that control its expression are being studied. In liver, HNF-6 also mediates effects of growth hormone (10), and stimulates transcription of genes coding for enzymes of glucose metabolism, for P450 cytochromes and for secreted proteins. The role of OC-2 and OC-3 is being investigated by the phenotypic analysis of Oc2 and Oc3 knockout mice which have been generated in the laboratory.

The group has established a number of national and international collaborations to investigate the role of Onecut factors in pancreas and liver diseases and to implement its findings in the diagnosis and treatment of biliary diseases and diabetes. It is part of national and international consortia which include the Interuniversity Attraction Pole and the BetaCellTherapy Consortium.

With more details…

Control of liver development by Onecut factors

During liver development, hepatoblasts (the liver precursor cells), differentiate into hepatocytes, which exert the metabolic functions of the liver, and into biliary cells which delineate the bile ducts. We have found that HNF-6 and OC-2 control the segregation of the hepatocytic and biliary lineages in the developing liver (9). Indeed, in mice knockout for both HNF-6 and OC-2, the hepatoblasts fail to differentiate properly into hepatocytes or biliary cells. Instead, the hepatoblasts give rise to hybrid 'hepato-biliary' cells which display characteristics of hepatocytes and biliary cells.

We have shown that the Transforming Growth Factor beta (TGFbeta) is required in liver for differentiation of biliary cells. TGFb signaling is detectable in the liver as a gradient, with high signaling activity near the portal vein, where biliary cells differentiate, and lower signaling activity in the parenchyma, where hepatocytes differentiate (9). We have also found that Onecut factors control the gradient of TGFb signaling (9, 11), and this led to the conclusion that hepatic cell differentiation is controlled by Onecut factors via a modulation of TGFbeta signaling.

In mice in which only the Hnf6 gene is inactivated, hepatic cell differentiation is affected, but less severely than in double HNF-6/OC-2 knockout mice. Biliary differentiation occurs but the bile ducts are abnormal. The mice suffer from cholestasis and display a phenotype that resembles human biliary diseases called "ductal plate malformations", which are related to biliary atresia (6). The Hnf6 knockout mice also lack hepatic artery branches, an anomaly which can aldo be found associated with human biliary diseases (7). Therefore, our findings indicate that HNF-6 is essential for bile duct development.

Abnormal development of the intrahepatic bile ducts in HNF-6 knockout mice. Immunohistochemistry of liver sections 10 days after birth shows in normal mice (left panel) a typical bile duct (bd) delineated by biliary cells (stained red) embedded in mesenchyme. In contrast, biliary cells do not form bile ducts in Hnf6-/- mice (right panel) and remain dispersed as a layer around a branch of the portal vein (pv).

We are currently investigating the pathogeny of human congenital malformations of the liver, as well as the role of HNF-6, OC-2, and TGFb signaling in hepatic differentiation.

Control of endocrine pancreas development by Onecut factors

During pancreas development, cells lining the primitive gut (endoderm) start to express pancreas-specific genes. These pluripotent pancreatic progenitors, then proliferate and differentiate into precursors of the pancreatic endocrine, exocrine or ductal cells, and so contribute to the formation of the pancreas.

We found that HNF-6 is required for differentiation of endocrine precursors, since the pancreas of Hnf6 knockout mice is devoid of islets of Langerhans. HNF-6 controls endocrine development by stimulating the expression of the transcription factor Ngn-3, a factor which is essential for development of endocrine cell precursors (4). The latter finding prompted us to investigate if Onecut factors also control the differentiation of endocrine cells in the gut (enteroendocrine cells), which also depend on Ngn-3. We are addressing this question by analyzing the phenotype of Oc2 and Oc3 knockout mice.

Abnormal development of the endocrine pancreas in HNF-6 knockout mice. Immunohistochemistry of tissue sections four days after birth shows that only a few insulin-producing cells (brown) are found near pancreatic ducts in Hnf6-/- (right panel), instead of being organized in islets as in Hnf6 +/+ littermates (left panel).

Control of pancreatic duct development by Onecut factors

We found that HNF-6 is required for development of the pancreatic ducts (12). Indeed, in Hnf6 knockout mice the ducts are abnormal and form cysts. This is associated with an absence of primary cilia at the apical pole of the ductal cells and with reduced expression of fibrocystin and cystin, two proteins known to control the function and the formation of cilia. Patients deficient in these proteins suffer from polycystic kidney disease, a hereditary disease associated with cysts in kidneys, liver and pancreas. This work is the first to identify a transcription factor that controls pancreatic duct development. It also characterizes HNF-6 as a regulator of cilium formation.

When studying pancreas development we have also described the expression profile of ephrins and of their receptors (13). In addition, using cultured pancreatic explants, we showed that embryonic pancreas can be differentiated in vitro. This explant culture system is now being used to investigate the role of signalling molecules in pancreas development, in particular in the formation of the pancreatic ducts.

Formation of cysts in the pancreatic ducts in Hnf6 knockout mice. Immunofluorescence analysis of tissue sections two days before birth shows that specific segments of the pancreatic ducts develop cysts (asterisks). The ducts are stained with an anti-Mucin-1 antibody (green) and the exocrine acini are stained with an anti-carboxypeptidase A antibody (red). Left panel, wild-type pancreas (+/+); right panel, Hnf6 knockout pancreas (-/-).

Control of endoderm development by Onecut factors

The endoderm is an embryonic cell layer that lines the primitive gut in early embryos and gives rise to the liver and pancreas. To understand how liver and pancreas development is initiated, one needs to characterize the transcription factor network involved, and how it is controlled by extracellular signals secreted by neighboring tissues.

Hnf6 gene regions drive expression of beta-galactosidase in liver and pancreas of transgenic embryos at embryonic day 10.5.

The transcription factor network was analyzed by studying the factors that are regulated by HNF-6 in the endoderm, as well as those that control HNF-6 expression. We have investigated the genes regulated by HNF-6 in the endoderm by studying the gene expression profile in Hnf6 knockout embryos. We showed that HNF-6 controls expression of Pdx-1, a factor critical for pancreas development (5). This project also benefited from a new technique set up in our laboratory, with which a whole embryo is cultured for 24 h after electroporation-mediated gene transfer in the endoderm. This approach allowed us to demonstrate that HNF-6 controls expression of OC-3 in the endoderm (14).

We have also studied the Hnf6 gene regulatory sequences that direct expression of HNF-6 in the endoderm (15). We cloned Hnf6 sequences upstream of the b-gal reporter gene and identified the gene regions that drive expression of HNF-6 in the endoderm and in early liver and pancreas.

Furthermore, using mouse genetic models that have a defective pancreatic mesenchyme or that lack expression of Fibroblast Growth Factor-10 (FGF-10), we showed that FGF-10 secretion by the mesenchyme is essential for pancreas development and for expression of pancreas-specific transcription factors (16).

Taken together, our data define a transcriptional cascade that regulates the initiation of pancreas development in the endoderm.

Conclusions

Our work led to the discovery of the Onecut transcription factors. The characterization of their molecular mode of action, and of their role in development of the endoderm, liver and pancreas, has shed light on how transcription factors are organized in a network that controls cell differentiation and organ formation. Our findings open perspectives for understanding the pathophysiology of liver and pancreatic congenital diseases. The application of our findings to the programmed differentiation of cultured cells should help developing cell therapy of hepatic deficiencies and of pancreatic diseases such as diabetes.

1. Lemaigre FP, Durviaux SM, Truong O, Lannoy VJ, Hsuan JJ, Rousseau GG. Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single cut domain. Proc. Natl. Acad. Sci. (USA) 93, 9460-4, 1996

2. Jacquemin P, Lannoy VJ, Rousseau GG, Lemaigre FP. OC-2, a novel mammalian member of the ONECUT class of homeodomain transcription factors whose function in liver partially overlaps with that of hepatocyte nuclear factor-6. J. Biol. Chem. 274, 2665-71, 1999

3. Vanhorenbeeck V, Jacquemin P, Lemaigre FP, Rousseau GG. OC-3, a novel mammalian member of the ONECUT class of transcription factors. Biochem. Biophys. Res. Commun. 292, 848-54, 2002

4. Jacquemin P, Durviaux SM, Jensen J, Godfraind C, Gradwohl G, Guillemot F, Madsen OD, Carmeliet P, Dewerchin M, Collen D, Rousseau GG, Lemaigre FP. Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3. Mol. Cell. Biol. 20, 4445-4454, 2000

5. Jacquemin P, Lemaigre FP, Rousseau GG. The Onecut transcription factor HNF-6 (OC-1) is required for timely specification of the pancreas and acts upstream of Pdx-1 in the specification cascade. Dev. Biol. 258, 105-116, 2003

6. Clotman F, Lannoy VJ, Reber M, Cereghini S, Cassiman D, Jacquemin P, Roskams T, Rousseau GG, Lemaigre FP. The onecut transcription factor HNF6 is required for normal development of the biliary tract. Development 129, 1819-1828, 2002

7. Clotman F, Libbrecht L, Gresh L, Yaniv M, Roskams T, Rousseau GG, Lemaigre FP.  Hepatic artery malformations associated with a primary defect in intrahepatic bile duct development. J. Hepatol. 39, 686-692, 2003

8. Maestro MA, Boj SF, Luco RF, Pierreux CE, Cabedo J, Servitja JM, German MS, Rousseau GG, Lemaigre FP, Ferrer J. Hnf6 and Tcf2 (MODY5) are linked in a gene network operating in a precursor cell domain of the embryonic pancreas. Hum. Mol. Genet. 12, 3307-3314, 2003

9. Clotman F, JacqueminP, Plumb-RudewiezN,Van der SmissenP, PierreuxCE, DietzHC, CourtoyPJ, RousseauGG, LemaigreFP. Control of liver cell fate decision by a gradient of TGFb signaling modulated by Onecut transcription factors. Genes. Dev. 19, 1849-1854, 2005

10. Lahuna O, Fernandez L, Karlsson H, Maiter D, Lemaigre FP, Rousseau GG, Gustafsson J, Mode A. Expression of hepatocyte nuclear factor 6 in rat liver is sex-dependent and regulated by growth hormone. Proc. Natl. Acad. Sci. (USA) 94, 12309-12313, 1997

11. Plumb-Rudewiez N, Clotman F, Strick-Marchand H Pierreux CE, Weiss MC, Rousseau GG, Lemaigre FP. The transcription factor HNF-6/OC-1 inhibits the stimulation of the HNF-3a/Foxa1 gene by TGFb in mouse liver. Hepatology,40, 1266-1274, 2004.

12. Pierreux CE, Poll AV, Kemp CR, Clotman F, Maestro MA, Cordi S, Ferrer J, Leyns L, Rousseau GG, Lemaigre FP. The transcription factor HNF-6 controls the development of pancreatic ducts in the mouse. Gastroenterology 2006; 130: 532-541, 2006

13. van Eyll JM, Passante L, Pierreux CE, Lemaigre FP, Vanderhaeghen P, Rousseau GG. Eph receptors and their ephrin ligands are expressed in developing mouse pancreas. Gene Expression Patterns, 6, 353-359, 2006

14. Pierreux CE, Vanhorenbeeck V, Jacquemin P, Lemaigre FP, Rousseau GG. The transcription factor HNF-6/0C-1 controls the expression of its paralog OC-3 in developing mouse endoderm. J. Biol. Chem. 279, 51298-51304, 2004

15. Poll AV, Pierreux CE, Lokmane L, Haumaitre C, Achouri Y, Jacquemin P, Rousseau GG, Silvia Cereghini S, Lemaigre FP. A vHNF1/TCF2 – HNF6 cascade regulates the transcription factor network that controls generation of pancreatic precursor cells.Diabetes 55, 61-69, 2006

16. Jacquemin P, Yoshitomi H, Kashima Y, Rousseau GG, Lemaigre FP, Zaret KS. An endothelial-mesenchymal relay pathway regulates early phases of pancreas development. Dev Biol 290: 189-199, 2006