The role of HNF1β in pancreas de�elopment

The transcription factor HNF1β [variant hepatocyte factor one (vHNF1), transcription cell factor two (TCF2) or homologue liver specific factor B (LFB3) (DeSimone et al., 1991; De-martis et al., 1994)], belongs to the group of liver enriched transcription factors known as hepatocyte nuclear factors (HNFs). HNF-group members belong to different transcription factor families, like the homeobox proteins HNF1β and its close relative HNF1α, nuclear receptor HNF4α, forkhead box proteins HNF3α, β and γ (FoxA1-3) and the onecut fam-ily member HNF6 (onecut-1: Cereghini, 1996). All group members were reported to play essential roles during liver specification, as it was also recently demonstrated for HNF1β (Lokmane et al., 2008). As previously described, dominant negative mutations in the HNF1β gene are also linked to diabetic disorder MOD�5 that is characterised by impaired glucose- stimulated insulin secretion and is associated with a severe glomerulocystic kid-ney disease (GCKD; Fajans et al., 2001).

HNF1β was first isolated as albumin promotor binding protein from a rat hepatoma cell line (Cereghini et al., 1988) and demonstrated to be a strong transcriptional activator (DeS-imone et al., 1991). The HNF1β protein contains an N-terminal DNA-binding region that is composed of a dimerisation domain, a POU-like DNA binding motif and a downstream HOMEOBOX motif (Nicosia et al., 1990). Structural analysis of the human homologueStructural analysis of the human homologue

revealed that the homeobox contains a helix- turn- helix motif (HTH), but which differs from other homeobox proteins by an extention of 21 amino acids between the second and third helix (Bach et al., 1991) and which was suggested to stabilize DNA- binding (Fin-ney et al., 1990). The POU- like DNA binding motif is formed by five helices, similar to the POU DNA binding domain, and is therefore also referred as pseudo- POU structure

Figure 1.5 Regulatory factors directing pancreatic lineage specification in Xenopus laevis. A simplifie�

mo�el for the role of the major trans��ription fa��tors an� si��nallin�� pathaways involve� in pan��reati�� li-nea��e �etermination. Cir��les in�i��ate ��ell subtypes. Boxes state trans��ription fa��tors that are require� for

��ell fate �etermination. Arrows in�i��ate �ire��tions of ��ell linea��es that are spe��ifie� a����or�in�� to the ex-presse� marker ��ene �s�. �ifferent arrow len��hts in�i��ate the relative time point of final �ifferentiation

mo-�us after pre��ursor ��ell �etermination. Blunt lines in�i��ate inhibition of parti��ular ��ell linea��es. Question marks in�i��ate instan��es where ��ene �s� in parti��ular linea��e �etermination is not known in Xenopus laevis

�mo�ifie� after Pieler an� Chen, ��006�.

forms were identified, namely HNF1β- B and HNF1β- C (Tronche and �aniv, 1992). In thisHNF1β- B and HNF1β- C (Tronche and �aniv, 1992). In this. In this respect, HNF1β- C that lacks the C- terminal transactivation domain might function as an endogenous dominant negative variant of the transcription factor (Bach and �aniv, 1993).)..

HNF1β and the closely related HNF1α bind to identical DNA binding sites as homo- orHNF1α bind to identical DNA binding sites as homo- orto identical DNA binding sites as homo- or heterodimers (g/aGTTAATNATTAACc/a; Rey-Campos et al.,1991) but it is unclear which protein region is preferred for either interaction. The dimerisation process is promoted by the dimerisation cofactor DCoH. DCoH does not bind to the DNA but stabilizes the struc-ture of the HNF1 dimer, thereby enhancing interaction with nucleic acids (Cerenghini,HNF1 dimer, thereby enhancing interaction with nucleic acids (Cerenghini,(Cerenghini, 1996). Subsequent studies in mice specified its expression profile during development toSubsequent studies in mice specified its expression profile during development to the extraembryonic visceral endoderm and later to the definitive endoderm where it is ex-extraembryonic visceral endoderm and later to the definitive endoderm where it is ex-definitive endoderm where it is ex-pressed in cells of the neural tube and of the foregut epithelium, including the hepatic and pancreatic primordia. Along the gut tube, differential HNF1β expression was detectableβ expression was detectable expression was detectable in the gallbladder, duodenum and both pancreatic anlagen aat stage E8.5. Here,Here, HNF1β expression was detectable in the Pdx1-positive epithelium of the budding pancreatic rudi-ments, where it became restricted to the developing duct cells, excluded from acinar orwhere it became restricted to the developing duct cells, excluded from acinar or endocrine cells, in the branching pancreatic epithelium (E12.5). In adult mice, HNF1β was(E12.5). In adult mice, HNF1β wasIn adult mice, HNF1β was transcribed in the epithelial cells of liver, genital tract, kidney, pancreas and lung (Barbacci et al., 1999; Reber et al., 2001).

HNF1β-deficient mice died before gastrulation due to defective formation of the viscer-al endoderm. This lethviscer-ality was prevented by generation of HNF1β-null embryos with a wildtype (WT) extraembryonic endoderm using tetraploid embryo complementation (Bar-bacci et al., 1999). These teraploid HNF1β deficient mice were completely devoid of ventralHNF1β deficient mice were completely devoid of ventral pancreatic structures and demonstrated a severe hyploplasia of dorsal pancreas (Haumai- (Haumai-tre et al., 2005). Interestingly, inhibited pancreatic development upon HNF1β depletion. Interestingly, inhibited pancreatic development upon HNF1β depletion phenocopied effects caused by impaired expression of the pancreatic progenitor marker Pdx1 (Offield et al., 1996) and Ptf1a/p48 (Kawaguchi et al., 2002). Therefore, data obtaineddata obtained from studies in knockout mice support the idea that the transcription factor HNF1β func-tions as an upstream regulator of Pdx1 and p48. HNF1β-hypomorphic zebrafish mutants showed a similar phenotype regarding pancreatic agenesis, suggesting that HNF1β func-tion during vertebrate development is conserved (Sun and Hopkins, 2001).

In 1994, the Xenopus laevis HNF1β homologue was isolated and its expression traced dur-ing different stages of embryogenesis (Demartis et al., 1994). HNF1β expression was ac-tivated after mid blastula transition (MBT) in ecto- and endoderm as well as later in the mesoderm. In early tadpole stages, HNF1β transcripts were found in the foregut marking the prospective hepatic and pancreatic domain. In adult tissues, HNF1β transcripts were detected abundantly in liver, kidney, digestive tract and lung, but also in the pancreas (De-martis et al., 1994; Vignali et al., 2000).

HNF1β regulates a gene expression cascade essential for differentiation of epithelial cells lining the ducts. In 2002, Clotman et al. revealed that one of the upstream regulators of HNF1β in biliary duct differentiation is the onecut 1 transcription factor HNF6 (Clotman et al., 2002; Coffinier et al., 2002). An additional link between HNF1β and HNF6 was dis-covered regarding endocrine cell differentiation as both were reported as Ngn3 upstreamwere reported as Ngn3 upstream regulators associated with the diabetic disorder MOD�6 (Maestro et al., 2003; Horikawa et al., 1997).

Conversely, it was revealed that the mouse HNF6 gene contained an intronic HNF1β bind-intronic HNF1β bind-ing site with high potency to activate transcription of a reporter constructwith high potency to activate transcription of a reporter construct in vitro. These data located HNF1β upstream of HNF6 in the regulation hierarchy (Poll et al., 2006).