Severe pancreas hypoplasia and multicystic renal ... - Oxford Journals

Human Molecular Genetics, 2006, Vol. 15, No. 15 doi:10.1093/hmg/ddl161 Advance Access published on June 26, 2006

2363–2375

Severe pancreas hypoplasia and multicystic renal dysplasia in two human fetuses carrying novel HNF1b/MODY5 mutations Ce´cile Haumaitre1, Me´lanie Fabre1, Sarah Cormier2, Clarisse Baumann3, Anne-Lise Delezoide2 and Silvia Cereghini1,* 1

Laboratoire de Biologie du de´veloppement, Unite´ Mixte de Recherche 7622, Centre National de la Recherche Scientifique, Universite´ Pierre et Marie Curie, Paris, France, 2Hopital Robert Debre´, Service de Foetopathologie, Paris, France and 3Hopital Robert Debre´, Unite´ de Ge´ne´tique Clinique, Paris, France

Received April 3, 2006; Revised and Accepted June 21, 2006

Heterozygous mutations in the HNF1b /vHNF1/TCF2 gene cause maturity-onset diabetes of the young (MODY5), associated with severe renal disease and abnormal genital tract. Here, we characterize two fetuses, a 27-week male and a 31.5-week female, carrying novel mutations in exons 2 and 7 of HNF1b, respectively. Although these mutations were predicted to have different functional consequences, both fetuses displayed highly similar phenotypes. They presented one of the most severe phenotypes described in HNF1b carriers: bilateral enlarged polycystic kidneys, severe pancreas hypoplasia and abnormal genital tract. Consistent with this, we detected high levels of HNF1b transcripts in 8-week human embryos in the mesonephros and metanephric kidney and in the epithelium of pancreas. Renal histology and immunohistochemistry analyses of mutant fetuses revealed cysts derived from all nephron segments with multilayered epithelia and dysplastic regions, accompanied by a marked increase in the expression of b-catenin and E-cadherin. A significant proportion of cysts still expressed the cystic renal disease proteins, polycystin-1, polycystin-2, fibrocystin and uromodulin, implying that cyst formation may result from a deregulation of cell – cell adhesion and/or the Wnt/b-catenin signaling pathway. Both fetuses exhibited a severe pancreatic hypoplasia with underdeveloped and disorganized acini, together with an absence of ventral pancreatic-derived tissue. b-catenin and E-cadherin were strongly downregulated in the exocrine and endocrine compartments, and the islets lacked the transporter essential for glucose-sensing GLUT2, indicating a b-cell maturation defect. This study provides evidence of differential gene-dosage requirements for HNF1b in normal human kidney and pancreas differentiation and increases our understanding of the etiology of MODY5 disorder.

INTRODUCTION In humans, heterozygous germline mutations in the HNF1b/ vHNF1/TCF2 gene are the cause of one form of maturityonset diabetes of the young, subtype 5 (MODY5), as well as a variety of disorders including congenital abnormalities of the kidney and genital tract, pancreas atrophies, mild liver dysfunction and hyperuricemia. More than 50 heterozygous HNF1b mutations have been identified, including missense, nonsense, frameshift, insertion/deletion and splice site mutations as well as whole gene deletions (1,2). Most of them are familial, but eight are spontaneous and one is a

germline mosaic mutation. Collectively, these studies suggest that MODY5 is more prevalent than previously thought. MODY is a disorder characterized by autosomal dominant inheritance, early-onset type 2 diabetes and impaired glucose-stimulated insulin secretion. Although diabetes is frequently associated with HNF1b mutations, the most consistent clinical feature is severe non-diabetic renal disease. Affected individuals may develop chronic kidney disease and progress to end-stage renal disease requiring transplantation. Because of the prevalence of these two phenotypes in affected families, the term renal cysts and diabetes has been proposed to define

*To whom correspondence should be addressed. Tel: þ33 144272151; Fax: þ33 144273445; Email: [email protected]

# The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

2364

Human Molecular Genetics, 2006, Vol. 15, No. 15

this syndrome. A large spectrum of renal abnormalities has been reported, including few unilateral or bilateral cysts, severe multicystic dysplasia, oligomeganonephronia (3), hypoplastic glomerulocystic kidney disease (4 – 7), solitary or horseshoe kidney (8,9) and atypical familial juvenile hyperuricemic nephropathy (10). Other clinical features include pancreas atrophies (6,11), genital tract malformations and biliary defects (12). The molecular mechanisms by which heterozygous mutations in the HNF1b gene cause this broad spectrum of clinical symptoms remain largely unknown. Likewise, the mechanisms underlying the phenotypic variability among HNF1b-mutant carriers, within and between families, are presently unknown. Indeed, heterozygous mice for an Hnf1b null allele have no phenotype, whereas the homozygous deletion results in early embryonic death because of defective visceral endoderm formation (13,14), thus obscuring its later roles during organogenesis. By rescuing the early lethality of Hnf1b – / – embryos through the generation of mouse diploid and tetraploid chimeras, HNF1b was shown to be required for normal pancreas morphogenesis and regional specification of the gut (15). Besides these essential embryonic roles, Hnf1b-targeted inactivation in liver produces abnormalities in the gall bladder and intrahepatic bile duct morphogenesis (16). Interestingly, renal-specific inactivation of Hnf1b, as well as the kidney-specific overexpression of an HNF1b dominant-negative mutation, were shown to result in the formation of renal cysts and postnatal death (17,18). This is accompanied by a reduced transcription activation of the genes involved in cystogenesis, including Pkd2, Pkhd1 and Umod (17), thus linking the HNF1b transcriptional regulatory network to genes causing polycystic kidney disease. We describe here the kidney and pancreatic phenotype of two fetuses, a 27-week male and a 31.5-week female, carrying a frameshift mutation in exons 2 and 7 of HNF1b, respectively, and whose gestation was terminated following the diagnosis of a fetal form of polycystic renal disease. Both fetuses present the ensemble of the more severe phenotypes described in different HNF1b-mutation carriers, specifically bilateral enlarged polycystic kidneys, severe pancreas hypoplasia and abnormal genital tract. This study, which represents the first comprehensive molecular characterization of human fetal pancreatic and renal tissues with HNF1b mutations, highlights the important role played by HNF1b in human urogenital and pancreas development and provides new insights into the pathology of the MODY5/RCAD syndrome.

RESULTS Clinical and fetopathological report Patient 1. Gestation of a 31-year-old woman was terminated at 27 weeks, following the diagnosis of enlarged polycystic kidneys and abnormal renal function. Postmortem examination demonstrated a male fetus (46, XY) with mild dysmorphic facial features (hypertelorisme and retrognathisme). No other skeletal abnormalities were present. Internal examination confirmed massive enlarged bilateral cystic kidneys (weight 2-fold greater than the normal) and hypoplasia of both the head and the tail of the pancreas. The epididymus and the

rete testis were also hypoplastic with several epididymal cysts (data not shown). Patient 2. Gestation of a 27-year-old woman was terminated at 31.5 weeks, following the diagnosis of enlarged polycystic kidneys and abnormal renal function. Post-mortem examination demonstrated a female fetus (46, XX) with normal cranio-facial features and no skeletal abnormalities. Internal organ examination revealed bilateral enlarged cystic kidneys and pancreas hypoplasia (discussed subsequently), as well as hepatomegalia, uterus didelphys and dysgenic gonads (data not shown). No congenital hepatic fibrosis, biliary dysgenesis, pulmonary hypoplasia, pancreas or liver cysts were found in the two fetuses, thus indicating that they did not exhibit the macroscopic clinical features associated with either autosomal or recessive polycystic kidney disease. We speculated that HNF1b mutations could be responsible for the restricted mutant phenotypes of these fetuses, as multiple bilateral kidney cysts, predominantly glomerular, and renal dysplasia, in association with genital tract abnormalities and pancreas atrophies, are clinical features found in the different MODY5/RCAD-mutation carriers. Identification and functional characterization of two novel frameshift mutations in the HNF1b gene DNA from the two fetuses was screened for HNF1b mutation by direct sequencing, resulting in the identification of two novel heterozygous frameshift mutations. No other mutations were found in the coding regions, intron – exon boundaries and 680 base pairs (bp) promoter sequences. Fetus 1 had the R112fsdelGGATGCTC mutation (further designed as R112fsdel), consisting of an 8 bp deletion in exon 2. This resulted in a frameshift and a termination codon at position 113, in the N-terminal part of the POU-specific domain (POUS), which is essential for DNAbinding specificity (Fig. 1A). This mutation would result in the synthesis of a truncated protein with an intact dimerization domain but lacking part of the POUS domain and the entire POU homeodomain (POUH) as well as the C-terminal transactivation domain. This mutation was a de novo mutation: the parents neither display any phenotype nor carry this mutation. Fetus 2 had the P472fsinsTGCAGCCC mutation (designed as P472fsins), consisting of an 8 bp insertion in the exon 7, leading to a premature stop codon at position 507, instead of 557, and the insertion of 35 novel amino acids at the C-terminus of the transactivation domain of HNF1b (Fig. 1A). Although DNA from the parents was not available, the lack of diabetes and/or renal failure in the parents suggest that P472fsins, as R112fsdel, was a spontaneous de novo mutation. To examine the functional consequences of the R112fsdel and P472fsins mutations, we transiently expressed into HEK293 cells wild-type and mutant proteins and assessed the corresponding transcriptional activity, DNA-binding activity and subcellular localization. As expected, P472fsins, which contained an intact DNA binding and dimerization domains, bound efficiently to the HNF1 probe and heterodimerized with the wild-type protein (Fig. 1B). The P472fsins mutant protein was also expressed at similar levels to the wild-type protein and localized into the nucleus (data not


2365

Figure 1. Functional characterization of two novel frameshift heterozygous mutations in the HNF1bgene. (A) Schematic representation of HNF1bprotein: the dimerization domain (D), the POU-specific domain (POUs), the POU homeodomain (POUH) and the nuclear localization signal (nls) (52) are indicated. (B) EMSAs were performed with labeled PE oligonucleotide and cell extracts overproducing wild-type and/or P472fsins proteins. Arrows show the position of HNF1b and P472fsins homodimeric or heterodimeric complexes between the wild-type and mutant proteins displaying intermediate mobility. S, supershifted complexes with anti-HNF1b (Ab); ns, non-specific. (C) Cells were transiently transfected with Afp-CAT construct and the indicated amounts of mutant and wild-type expression vectors. (D) Cell extracts overproducing wild-type and/or R112fsdel proteins (lanes 1–3) were used. EMSAs (left panel). HNF1b– DNA complex (lane 1), supershifted by the addition of anti-HNF1b (lane 10 ), is not formed with R112fsdel (lane 3). As all lanes contain similar HNF1blevels, the attenuation of HNF1b –DNA complex is probably due to the formation of non-functional wild-type/R112fsdel heterodimers (lane 2). Western blot analysis (right panel). The same extracts shown in the EMSA were used. R112fsdel (12 kDa) and wild-type (60 kDa) proteins are labeled using anti-HNF1b. (E) Cells were transfected with Afp-Cat reporter and a constant amount of HNF1b-expression vector, together with the indicated R112fsdel/HNF1b molar ratios. Normalized CAT activities and standard errors are expressed in percentage of HNF1b activity alone. (F) Immunoflurescence analysis of transiently transfected cells reveals that HNF1b is nuclear, whereas R112fsdel is mainly localized in the cytoplasm. Myc-tagged-HNF1b proteins were visualized by anti-myc (left bottom), whereas both wild-type and mutants proteins were revealed by anti-HNF1b (right bottom).

shown), indicating that this mutation did not affect the stability or the normal folding of the protein and its subsequent translocation to the nucleus. We then evaluate the effect of this mutation on the ability to transactivate an HNF1b-target promoter. Among the already identified target promoters, we chose the alpha-fetoprotein (Afp) promoter, which is transactivated by HNF1b at the highest known levels (6), allowing the detection of weak variations in the transcriptional activity of HNF1b-mutant proteins. As shown in Figure 1C, the P472fsins mutant protein exhibited nearly half of the transactivation activity of the wild-type HNF1b protein. Co-expression of this mutant protein with wild-type HNF1b did not affect its transcriptional activity (data not shown). The R112fsdel mutant protein, which lacks the POUS and POUH, was unable to bind DNA (Fig. 1D). Consistent with previous data (6), a decrease in the specific HNF1b – DNA complex was observed when both mutant and wild-type proteins were co-expressed, suggesting that heterodimers between R112fsdel and wild-type protein were unable to bind DNA. We then examined the effect of this mutant protein on the ability of wild-type HNF1b to potentiate

Afp-reporter activity. A maximal decrease of 30% in the transactivation potential of the wild-type protein was observed in the presence of a 4-fold excess of R112fsdel (Fig. 1E). Immunofluorescence of transiently transfected cells showed that the R112fsdel mutation was located preferentially in the cytoplasm and, at much lower levels, in the nuclei, in agreement with the location of the predicted nuclear localization signal of HNF1b between residues K229 and K237. As previously reported for other truncated HNF1b mutations, when mutant and wild-type proteins were co-expressed, R112fsdel was unable to sequester HNF1b into the cytoplasm by heterodimerization (Fig. 1F). These results suggest that the truncated R112fsdel protein, retaining the N-terminal dimerization domain, might function as a weak dominant-negative mutation, through the formation of non-functional heterodimers between HNF1b and the fraction of mutant protein present in the nucleus. In contrast, the mutation P472fsins, which binds DNA as the wild-type protein, exhibits a decreased transactivation capacity, but it does not interfere with the activity of the wild-type protein under the experimental conditions used.

2366


Embryonic and adult expression of human HNF1b During mouse development, Hnf1b transcripts are present in the neural tube, in the primitive gut and in the hepatic and the dorsal and ventral pancreatic primordia (13,15). During kidney development, Hnf1b is highly expressed in the Wolffian duct and mesonephric tubules, the ureteric bud and the pretubular aggregate derivatives, the comma-shaped and S-shaped bodies, and subsequently in the proximal and distal convoluted tubules and the loop of Henle and medullary collecting ducts (13,19 – 21). The glomeruli, bladder and urethra do not express Hnf1b. In adult mice, Hnf1b transcripts have been detected in the polarized epithelia of the kidney, pancreas, liver, lung and digestive tract. In humans, HNF1b in situ hybridization studies have been reported in the metanephric kidney of a fetus at 91 days of gestation (corresponding to E17 in mouse development) (22). Data on adult tissues are still missing. To examine whether HNF1b transcripts in humans followed a similar tissue distribution compared with rodents, we hybridized a northern blot of poly(A)þ RNA from various human adult tissues using a full-length human probe (Fig. 2A). Consistent with previous data in mice (23), a 2.9 kb transcript was detected predominately in kidney and pancreas and at lower levels in colon, small intestine, prostate and testis. Significant lower levels of HNF1b transcripts were detected in liver, lung and ovary. In adult testis, in addition to the 2.9 kb somatic transcript, we observed two other abundant transcripts of 1.65 and 1.4 kb and one minor of 2.5 kb. Remarkably, a similar pattern of Hnf1b transcripts has been reported in adult mouse testis (23). In situ hybridization analysis of a 56 day gestation human embryo (corresponding to E15 in mouse development) showed high levels of HNF1b transcripts in the metanephric kidney and in epithelium of the pancreas, biliary duct, duodenum and stomach (Fig. 2B and C), whereas the mesenchyme of these organs was negative. We also detected HNF1b transcripts in the lung epithelium and in the mesonephric and mullerian ducts (data not shown). At 25 weeks, this pattern was maintained (data not shown). Thus, the HNF1b expression pattern, which appears strictly conserved from mouse to human, is consistent with the putative role of this transcription factor in epithelial differentiation during early human organogenesis.

Histological and immunohistochemical analyses of mutant fetal kidney Histological examination in MODY5 carriers has essentially been limited to renal biopsies, allowing analysis of restricted regions. The most prominent histological findings described in biopsies from 10-month to 57-year patients were cystic dysplasia (8), enlarged nephrons and oligomeganephronia (3,24), hypoplasic glomerulocystic kidneys (4) and microcysts and glomerular cysts (11). Multiple bilateral cystic kidneys, including occasional glomerular cysts, with no regions of dysplasia were also reported in a 17-week female fetus (25). Histological examination of mutant kidneys of both fetuses revealed bilateral cysts in the whole kidney, both in regions reminiscent of cortex and medulla, with regions of interstitial fibrosis (Fig. 3). The vast majority of glomeruli were cystic.

Figure 2. HNF1b mRNA distribution during human embryonic development and in adult tissues. (A) HNF1b expression sites in human adult tissues. A northern blot of poly(A)þ RNA from various human adult tissues was hybridized using a full-length HNF1b human probe. RNA levels were normalized by the expression of b-actin. (p. leucocytes, peripheral blood leucocytes; s. muscle, skeletal muscle). (B and C) HNF1b transcripts visualized in human embryo. Histological (B) and corresponding in situ hybridization (C) analyses of 56 days of gestation human embryo show high levels of HNF1b transcripts in the metanephric kidney and in the epithelium of several endoderm-derived organs, as indicated by arrows. (bd, biliary duct; p, pancreas; s, stomach; d, duodenum; m, metanephros).

Multiple cysts of different sizes were also present in collecting ducts and distal and proximal tubules, resulting in a completely distorted cortical and medullar structure. Cysts were lined by a single epithelium, which was either columnar or composed of a layer of loosely flattened cells. Many cysts showed regions with a multilayered hyperplastic epithelium (Fig. 4). The region under the capsule in both fetuses was devoid of pretubular aggregates and replaced by a high proportion of glomerular cysts, indicating defective nephrogenesis. In the region reminiscent of medulla, many dysplastic tubules surrounded by typical fibromuscular collars were observed (Fig. 3). To identify the tubular origin of cysts, we analyzed markers of specific nephron segments: the lectin lotus tetragonolobus agglutinin (LTA) for the proximal tubules, NKCC2 (Na – K – Cl cotransporter) for the thick ascending of loops of Henle, AQP2 (aquaporin-2) for the collecting ducts and uromodulin (UMOD) for the thick ascending limbs of loops of Henle and distal convoluted tubules. Although a significant number of cystic structures in the mutant kidneys expressed AQP2, NKCC2, UMOD and, to lesser extent, LTA (Fig. 4A), we observed a global decrease in the number of structures, cystic or not, labeled by these markers. To further define the molecular characteristics of these mutant kidneys, we analyzed the expression of proteins


2367

Figure 3. Renal cystic dysplasia in mutant fetal kidneys. Global histology of a 27-week normal fetal kidney (Control), R112fsdel and P472fsins mutant fetal kidneys reveals that both mutant kidneys are multicystic and dysplastic. Higher magnifications show a lack of the cortex/medulla organization. Cysts are observed in the whole kidney, including proximal and distal tubules, collecting ducts and the vast majority glomeruli. (Cy, Cysts; Gcy, Glomerular cysts). In the medulla, cyst-structures are surrounded by a collar of connective tissue corresponding to primitive mesenchymal structures, a feature of renal cystic dysplasia (g, glomeruli; cd, collecting duct; pt, proximal tubule; dt, distal tubule).

involved in cystic diseases, including polycystin-1 and polycystin-2 encoded by the PKD1 and PKD2 genes and fibrocystin/polyductin encoded by PKHD1 (Fig. 4B). It has been shown previously that the expression of Pkhd1, Pkd2

and Umod is severely affected in Hnf1b-conditionally inactivated mice (17). Surprisingly, we observed in mutant kidneys the expression of polycystin-1, polycystin-2 (26) and Fibrocystin (27) in a significant proportion of cystic structures,

2368


Figure 4. Altered epithelial differentiation and dysregulation of cell–cell adhesion in mutant fetal kidneys. (A) Stainings for LTA, AQP2, NKCC2 and UMOD. Less structures expressing LTA, AQP2, NKCC2 and uromodulin are observed. Some remnant positive immunostained structures, particularly marked by NKCC2, display altered morphology, with hyperplastic epithelia. (B) Stainings for polycystin-1, polycystin-2 and fibrocystin. Polycystin-1, polycystin-2 and fibrocystin-encoded, respectively, by the genes PKD1, PKD2 and PKHD1, are present in cystic and non-cystic structures. (C) Stainings for E-cadherin and b-catenin. Expression of E-cadherin and b-catenin are marked increased in cystic and non-cystic epithelia of mutant kidneys. Note regions of hyperplastic epithelium lining the cysts.

in addition to non-cystic structures. However, similar to the findings with the specific nephron-segment markers, fewer structures expressing these proteins were observed. Together, these analyses indicate that cysts in the mutant fetal kidneys derive from all nephron segments and express proteins encoded by cystic kidney disease genes. The global decrease of structures expressing both the nephron-segment markers and cystoproteins suggests either a primary

developmental defect or a secondary loss of epithelial differentiation. As defects in primary cilia function and/or structure have been recently associated with cystic kidney disease (reviewed in 28), we assessed the morphological integrity of this organelle. Immunostainings with acetylated tubulin, a specific component of the primary cilium, revealed that cystic cells had apparently normal cilia (data not shown).


2369

As alterations in Wnt/b-catenin signaling result in glomerular and multilayered cysts (29,30), similar to those observed in the mutant fetuses, we next examined the expression of b-catenin and E-cadherin (Fig. 4C). b-Catenin is a mutifunctional protein involved in cell proliferation through the Wnt/b-catenin pathway and in cell-adhesion through interaction with E-cadherin/a-catenin to the actin cytoskeleton. Interestingly, we observed high expression of both b-catenin and E-cadherin, with a markedly increase in the cystic epithelia of mutant kidneys. Exaggerated b-catenin expression was observed both in the plasma membrane and in the cytoplasm and may be a cause of increased cell proliferation, leading to early cyst formation in HNF1b-mutant carriers. Besides the fact that the P472fsins mutant kidneys exhibited a higher number of large cysts than R112fdel kidneys (Fig. 3), the analysis of expression of nephron-segment markers, cystic proteins, and of b-catenin and E-cadherin in P472fsins mutant kidneys gave very similar results to those obtained with R112fsdel kidneys (data not shown). Altogether, these analysis show that cysts in the two HNF1b-mutant fetal kidneys derive from multiple cell types and display a dysregulation of cell – cell adhesion together with an altered epithelial differentiation. Persistent expression of HNF1b protein in cystic epithelium of mutant fetal kidneys

Figure 5. Persistent expression of HNF1b protein in the epithelium lining the cysts. Staining for HNF1b in control (27 weeks), R112fsdel and P472fsins mutant kidneys shows high expression in the nephrogenic region (ureteric bud epithelium, S-shaped and comma-shaped bodies) and at lower levels in Bowman capsule, proximal and distal tubules and collecting ducts, as well as in the cyst epithelial nuclei of the R112fsdel and P472fsins mutant fetuses. Note that the epithelium of the cyst (Cy) is characterized by a discontinuity rather than an absence of HNF1b nuclear expression, as evidenced by DAPI nuclear staining of the same section (data not shown). Arrows indicate S-shaped bodies and arrowheads indicate the Bowman capsule. Gcy, Glomerular cyst; Cy, Cyst. Asterisk indicate multilayered epithelia.

Focal cyst formation in autosomal polycystic kidney disease (ADPKD) has been related to the somatic mutation of the residual wild-type allele, a mechanism of cystogenesis known as ‘second hit’ (30). Renal cysts in the R112fsdel and P472fsins heterozygous mutant fetuses often exhibited regions of hyperplastic epithelium similar to those observed upon Hnf1b renal-specific inactivation (17,31), raising the possibility that cyst formation in these fetuses could result from a focal loss of the HNF1b normal allele. Conversely, renal cystic epithelia of both mutant fetuses did not show the expected dramatic downregulation of the proteins encoded by the PKHD1, UMOD and PKD2 genes (Fig. 4), found in Hnf1b-deficient renal tubules, thus suggesting residual wild-type HNF1b expression in cyst-lining epithelia. As a first step to discriminate between these possibilities, we stained normal and mutant fetal kidney sections with a specific anti-HNF1b antibody. In a human control fetal kidney, HNF1b was highly expressed in the nuclei of ureteric bud epithelium and the S-shaped and comma-shaped bodies and at lower levels in the Bowman capsule and in the epithelium of proximal and distal tubules and collecting ducts (Fig. 5), thus displaying an expression pattern that is remarkably similar to that observed in embryonic mouse kidney (data not shown). HNF1b was also detected in the same remnant structures of R112fsdel and P472fsins mutant kidneys and in the cyst-lining epithelium. Notably, nuclear HNF1b expression was consistently observed in cysts lined either by a columnar or squamous dedifferentiated epithelium, as well as in the regions with a multilayered epithelium (Fig. 5). The HNF1b antibody used here, raised against 4 –90 amino acids of the protein, could react with the protein produced by the normal and mutant alleles. Yet, in the case of the R112fsdel mutation, if a protein

2370


was produced from the mutant allele it should be located essentially in the cytoplasm (Fig. 1). Thus, although mutant and normal alleles could encode the HNF1b nuclear protein detected in P472fsins kidneys, the nuclear protein detected in the R112fsdel mutant kidneys is certainly encoded by the normal allele. These results suggest that the HNF1b protein from the wildtype allele continue to be expressed in the epithelium of cystic and non-cystic structures. These levels of HNF1b protein could then explain why the expression of known target genes is not significantly decreased in the mutant kidneys. Histological and immunohistochemical analyses of mutant fetal pancreas Impaired insulin secretion is a fundamental defect in type 2 diabetes, which is found in ~60% of HNF1b-mutation carriers. Dysregulation of insulin secretion could be due to changes in the islet architecture or b-cell mass, defective glucose sensing and metabolism. Pancreas histology of HNF1b-mutant carriers has not yet been reported. Global histological analysis of the R112fsdel mutant pancreatic tissue revealed a severe hypoplasia of both tail and body, which resulted mainly from underdeveloped acini. Acini were significantly smaller and disorganized along with relatively enlarged regions of mesenchyme and zones of fibrosis (Fig. 6). The higher density of the pancreatic duct network visualized by cytokeratin-19 (CK-19) staining (Fig. 6) further highlighted the drastic size reduction of acini. To further define whether the hypoplasia affected both the dorsal and ventral parts of the pancreas, we examined the presence of islets either rich in pancreatic polypeptide (PP)- or glucagon-producing cells. Indeed, in humans, PP-rich islets are found only in the posterior part of the head, which derives from the ventral pancreas bud (32). In contrast, glucagon-rich islets are found in the body, the tail and part of the head, which are dorsal-derived structures (Fig. 7A). Remarkably, very few PP-expressing cells were detected in the entire mutant pancreatic tissue (Fig. 7B). PP staining of the P472fsins mutant pancreas tissue gave identical results (data not shown). These observations provide evidence of an absence of ventral pancreatic-derived tissue in the HNF1b human mutant pancreas, further indicating that the severe pancreas hypoplasia results from both a ventral pancreas agenesis and dorsal pancreas hypoplasia. To examine the islet architecture of mutant pancreatic tissue, we performed immunohistochemical analysis to visualize the expression of the pancreatic hormones, such as insulin, glucagon, and somatostatin, in the major islet cell types, the a, b and d cells, respectively (Fig. 7C –N). We observed no significant decrease in insulin, glucagon and somatostatin expression (Fig. 7C – F). However, the islets of Langerhans appeared slightly disorganized and the density of b-cells was fairly reduced. The exocrine pancreas, although severely hypoplastic, appeared functionally normal as shown by the expression of the digestive hormone carboxypeptidase-A (Fig. 7I –J). Recent studies indicate that pancreas lacking b-catenin are hypoplastic and contain a striking paucity of acinar cells (33 – 35). Interestingly, we found a dramatically reduced expression of b-catenin (Fig. 7K and L) in the mutant pancreas

Figure 6. Severe pancreas hypoplasia in mutant fetuses. Global histology reveals that the R112fsdel pancreas is hypoplastic with a relatively disorganized structure and underdeveloped acini. Acini organization is delimitated by dashed lines, whereas black lines encircle islets. CK-19 immunostaining reveals the arborescence of developing ducts connecting acini via intercalated, intralobular and interlobular ducts. The higher density of the duct network labeled by CK-19 highlights the reduced size of acini in the mutant pancreas. Arrows indicate ducts.

when compared with the control. We also observed very low levels of E-cadherin in the mutant pancreatic tissue (Fig. 7M and N), in agreement with the partial disorganization of both exocrine and endocrine pancreas. Consistent with these observations, E-cadherin has been reported to regulate cell – cell contacts between all pancreatic cells and maintain correct islet topology (36,37). We then examined the expression of markers associated with functional b-cells. Significantly, the glucose transporter 2 (GLUT2), the low-affinity glucose transporter present on b-cell membrane and essential for glucose sensing, was


2371

Figure 7. Defective morphogenesis and b-cell maturation of mutant fetal pancreas. (A and B) Absence of ventral pancreatic-derived tissue in mutant fetuses. Glucagon-rich islets with very few PP-expressing cells characterize the dorsal pancreas-derived tissue, whereas PP-rich islets characterize the ventral part (A). The mutant pancreas shows only islets with high glucagon-expressing cells and very few PP-expressing cells (B). (C–N) Lack of Glut2 expression in b-cells and severe downregulation of cell –cell adhesion molecules in the exocrine and endocrine compartments of mutant fetal pancreas. Immunofluorescence of control (C, E, G, I, K and M) and R112fsdel mutant pancreas (D, F, H, J, L and N) to detect co-expression of insulin and glucagon (C and D), somatostatin and glucagon (E and F), Glut2 and glucagon (G and H) and the expression of carboxypeptidase-A (I and J), b-catenin/Dapi (K and L) and E-cadherin/Dapi (M and N). Endocrine and exocrine cells are present in the R112fsdel pancreas as shown by the presence of insulin, glucagon, somatostatin and carboxypeptidase-A expressing cells, with less organized endocrine cells into the typical structure of mature islets and relatively more scattered distribution of both glucagon and insulin-positive cells (D). The absence of GLUT2 expression in islets (F) indicates that b-cells are not terminally differentiated. Expression of the adhesion molecules b-catenin and E-cadherin is dramatically reduced (compare L with K, and N with M).

undetectable (Fig. 7G and H). Expression of GLUT2 is very likely mediated directly by HNF1b, as functional HNF1-binding sites are present in the GLUT2 promoter (38). Notably, enforced expression of an HNF1a dominant-negative mutation, which can perturb the expression of both HNF1a and b by homo- and heterodimerization, results in reduced expression of GLUT2 and E-cadherin (36,37). Thus, the lack of GLUT2 appears to be a characteristic maturation defect of insulin producing b-cells in mutant pancreas with HNF1b mutations. Altogether these results provide evidence that HNF1b function is required in humans for normal pancreas morphogenesis and terminal differentiation of b-cells.

DISCUSSION We report here the genetic and histological analyses of a 27-week male and a 31.5-week female fetuses, carrying novel heterozygous frameshift mutations, R112fsdel and P472fsins, in the HNF1b gene. Both fetuses exhibited the entire clinical spectrum reported in different HNF1b-mutation carriers: abnormal (male and female) genital tract, bilateral polycystic kidney and an as-yet non-described severe pancreas hypoplasia. They also presented one of the most severe clinical phenotypes described. This provided a unique opportunity to perform a characterization of the clinical spectrum associated with HNF1b mutations. As discussed subsequently, the

2372


confrontation of our data with the results of studies performed in different mouse models provides new insights into the understanding of the disease. The pathological defects of the two mutations described in this study are highly consistent with the expression pattern of HNF1b during early stages of human pancreas and urogenital tract development (Fig. 2 and 5). Although the two mutations identified are predicted to have different functional consequences on the protein, the two fetuses presented a highly similar phenotype, with no clear correlation between the nature of the mutation and the clinical phenotype. This is in fact largely the case for other HNF1b mutations characterized, as no clear genotype – phenotype relationships have been reported. It remains possible that our functional studies do not reflect the precise in vivo consequences of these mutations. The molecular properties of several other previously identified HNF1b mutations have been examined either in transfected cell lines (3,6,39) or in injected Xenopus embryos (40). Although functional assays in the developing Xenopus embryos could define morphogenetic features that were not assessed in cell cultures (40), there are clear limitations to both approaches, as they are based on the effect of transient overexpression of proteins at nonphysiological levels. Notably, the expression of either the wild-type and mutant HNF1b proteins in Xenopus led both to abnormal pronephros development. Therefore, precise in vivo analysis of the properties of HNF1b mutations will require the development of more sophisticated approaches. A further complication in the interpretation of these assays resides on the fact that mutations resulting in premature stop codons may be subject to nonsense-mediated-mRNA decay (NMD), producing haploinsufficiency. A recent study on the susceptibility to NMD of six truncating HNF1b mutations shows that there is a 5’ to 3’ polarity in this process (41). Accordingly, the R112fsdel mutation is predicted to be immune to NMD. However, staining of mutant R112fsdel kidney tissue with an HNF1b antibody detected mainly a nuclear protein, an observation that is consisting with NMD, although low levels of cytoplasmic protein might not be detected. The current understanding of NMD remains incomplete. This mechanism seems to operate with variable efficiency and some expected NMD substrates escape to NMD (42). Thus, the severe clinical phenotypes of the two fetuses characterized here are more readily explained by the action of modifying genes, environmental factors or developmental chance, rather than the specific nature of the mutation. HNF1b and cystic kidney diseases Inherited cystic kidney diseases consist of a large class of disorders characterized by cystic kidneys, including autosomal dominant and autosomal recessive polycystic kidney disease, nephronophtisis and medullary cystic diseases. In most cases, cystic expansion of epithelial-lined renal tubules and collecting ducts from the nephrons leads to kidney fibrosis and end-stage renal failure. Mutations in the PKD1 and PKD2 genes account, respectively, for 90 and 10% of cases of ADPKD (reviewed in 43). Extrarenal manifestations include pancreas and spleen cysts, cardiac valvular abnormalities and intracranial saccular aneurysms. PKHD1 mutations

are associated with autosomal recessive polycystic kidney disease (ARPKD), characterized by the expansion and elongation of collecting tubules into multiple small cysts and by biliary dysgenesis (reviewed in 44). Recently, renal-specific inactivation of Hnf1b in mouse medullar tubules has been shown to led to polycystic kidney disease, accompanied by a reduced transcription activation of several genes involved in cystogenesis, in particular, Pkhd1, Umod and Pkd2 (17). A drastic reduction of the proteins encoded by these genes in the cystic epithelium was also observed. These observations lead to the hypothesis that defective expression of these genes may elicit renal cysts in carriers with autosomal dominant mutations in HNF1b. We show here that in two fetal mutant kidneys, carrying distinct HNF1b mutations, a significant number of cysts still express polycystins-1 and 2, fibrocystin and uromodulin. Large cysts do not express these proteins, nor the differentiation markers LTA, NKCC2 or AQP2, implying defective differentiation, rather than direct transcriptional control of the genes involved cystogenesis. More importantly, the polycystic kidney phenotype of the two HNF1b-mutant fetuses, although apparently similar, is different than that associated with mutations in any of these genes. For instance, hyperplastic regions in the epithelium lining the cysts and cystic dysplasia, which were frequently observed in the two mutant kidneys, are rare in ADPKD. Moreover, in both fetuses the vast majority of glomeruli were cystic, whereas in human ADPKD glomerular cysts are less frequently observed. Extrarenal manifestations were also dissimilar. Neither these fetuses nor other HNF1b-mutation carriers exhibited congenital hepatic fibrosis, pulmonary hypoplasia and liver or pancreatic cysts, often associated with mutations in the PKHD1 or PKD2 genes, despite the fact that HNF1b is expressed in these organs in similar structures. Cyst formation in ADPKD has been related to a second mutation (second hit) in the remaining wild-type copy of the PKD gene (30), a frequent phenomenon observed in several hereditary cancers. However, only a subset of cysts exhibit somatic inactivation of a second PKD allele, raising the question of whether the second hit, rather than being an early event in the initiation of cysts, is a late event arising in a fraction of cysts, in particular, large cysts. With the available mutant fetal kidney sections, we have been unable to determine whether there was a focal HNF1b bi-allelic inactivation in the kidney cysts. We have initially hypothesized that there were two types of cysts: large cysts, in which the HNF1b wild-type allele is inactivated (second hit), as opposed to smaller cysts, which still express the wild-type allele, thus explaining the persistent expression in small cysts of the recently identified targets of HNF1b, PKHD1, PKD2 and UMOD (17). However, we consistently observed the expression of HNF1b protein in the epithelium lining both small and large cysts. Even though these observations do not formally exclude a subtle mutation produced by a somatic second hit, we consider this unlikely. Indeed, if each cyst arose from a discrete second hit, an unusual high rate of somatic mutagenesis would be required to substantiate the large number of cysts observed in the two mutant fetal kidneys. In addition, we did not see a high level of heterogeneity in HNF1b protein levels in different cysts that would be expected if independent second mutation events had occurred. We therefore favor a dose-dependent mechanism,


by which reduced HNF1b expression from the normal allele below a critical level, caused by stochastic fluctuations, led to cyst formation and the other clinical features associated (abnormal genital tract and pancreas hypoplasia). HNF1b protein could be present at enough levels to sustain the expression of the UMOD, PKD2 and PKHD1 genes. As the levels of gene expression appear to exhibit much higher fluctuations in the presence of only one functional allele (45), increased variations in gene expression in HNF1b heterozygous mutant-carriers might trigger cell proliferation in an unpredictable manner. Because immunohistochemical analysis does not allow a quantitative evaluation, it remains possible that a concomitant mild decrease in the expression of these cystic disease genes, not detected in our experiments, could be sufficient to initiate cyst formation. However, deregulation of cell –cell adhesion and/or the Wnt/b-catenin signaling pathway during critical stages of renal tubular development can explain the cystic phenotype in the R112fsdel and P472fsins mutant fetuses. Interestingly and consistent with our results, conditional inactivation of Apc in mouse tubular epithelium, which leads to increased levels of b-catenin, results in early onset of polycystic kidney disease, without changes in the expression of polycystin-1, polycystin-2 or HNF1b (46). Like the two human fetuses, these mice exhibited multiple kidney cysts derived form all nephron segments with both simple and multilayered cysts lined by a hyperplastic epithelium. We noticed that a significant fraction of cysts of mutant fetal kidneys presented regions of multilayered tubular epithelia, suggesting increased proliferation. Interestingly, few noncystic collecting ducts exhibited also regions of hyperproliferation of the epithelium (data not shown). A fraction of cysts of mice with Hnf1b renal-specific inactivation displayed also regions with multilayered epithelia (17). Intriguingly, mitotic alignments in the cystic collecting ducts of this mouse model were recently found completely distorted, thus establishing an interesting correlation between PKD and planar cell polarity (47). Notably, in rescued Hnf1b-deficient mouse embryos, the stomach was enlarged and exhibited similar regions of multilayered epithelium. As in the fetal mutant kidney cysts, part of this pluri-stratified stomach epithelium was delaminated and appeared as clusters of tissue (15). Thus, Hnf1b deficiency results in mice in increased proliferation of distinct epithelial structures, by an as-yet unknown mechanism. Further studies are required to establish whether the increased proliferation and abnormal differentiation of tubular epithelial cells observed in carriers with autosomal mutations in HNF1b result either from decreased functional levels of HNF1b or from a focal HNF1b-deficiency, or from both. Future work will also address whether an early event in the initiation of cyst formation in HNF1b carriers results from alterations of the Wnt/b-catenin signaling and/or cell – cell adhesion, coupled or not with decreased polycystin-2, fibrocystin or uromodulin expression. Pancreas hypoplasia and defective b-cell differentiation In mammals, the pancreas arises from the gut endoderm as dorsal and ventral buds, which fuse together to form a single organ, the duct of the ventral pancreas becoming part of the main pancreatic duct. Growth and morphogenesis of the bud epithelium into the

2373

mesenchyme lead to a ramification of small ductules containing precursor cells of the acini, ducts and islets of Langerhans, the major differentiated tissues of the fully developed organ. The dorsal pancreas that gives rise to the body, the tail and part of the head forms the great mass of the mature organ. The ventral bud forms the posterior part of the head or uncinate process. In mice, Hnf1b is expressed throughout pancreatic development from the onset of bud formation, through the stages of epithelial differentiation, to persist in ductal cells and at low levels in acini and adult b-cells (15,48). In Hnf1b-deficient mouse embryos, a dorsal bud rudiment is formed only transiently to become absent by E13.5, whereas the ventral pancreas is not specified (15). Remarkably and consistent with these observations, the two fetuses exhibit a severe pancreas hypoplasia including a deficiency of ventral pancreatic-derived tissue. Our study indicates that the pancreatic atrophies identified previously in several HNF1b mutations were most certainly caused by a defective morphogenesis of this organ. These findings together with previous studies (6,11) further suggest that pancreas hypoplasia is a more general phenotypic component of this syndrome that has been so far unnoticed, as its detection requires computed tomography. In marked contrast to the high increase of E-cadherin and b-catenin in the cystic epithelium of mutant kidneys, expression of these proteins is extremely reduced in the mutant pancreatic tissue when compared with the control (Fig. 8). Interestingly and consistent with our observations in the mutant pancreas fetuses, recent studies show that b-catenin is required for the robust proliferation of pancreatic progenitors and their differentiation into acinar cells (33 – 35). It is tempting to speculate that the severe pancreas hypoplasia frequently observed in HNF1b-mutation carriers may reflect a perturbation in the Wnt/b-catenin signaling pathway. Remarkably, the transporter GLUT2, a potential direct target of HNF1b (38), was undetectable on b-cell islets of the two mutant fetuses. As Glut2-deficient mice are hyperglycemic and develop diabetes with impaired glucose sensing by pancreatic b-cells (49), these observations may offer a prospective explanation of early onset of diabetes in MODY5 patients. Early loss of GLUT2 in b-cells combined with a gradual decrease in the insulin expression, could be a cause of hyperglycemia in MODY5 patients, which in turn may lead to type II diabetes. In summary, this study increases our understanding of the etiology of the RCAD/MODY5 disorder and the underlying developmental mechanisms. Confrontation of these data with the results of constitutive or conditional generation of null alleles in mice provides evidence of differential gene-dosage requirements for HNF1b in normal human kidney and pancreas differentiation. This information will also be helpful in the identification of novel mutation carriers and should be taken in account in fetopathology diagnosis in cases of suspected ADPKD or ARPKD.

MATERIALS AND METHODS HNF1b-mutation analysis Frozen or paraffin-included fetal tissues and peripheral blood were obtained from family members with informed consent

2374


approved by the Ethics committee of Debre´ Hospital. Genomic DNA from frozen fetal tissue or peripheral blood was used as a template to amplify the proximal promoter (680 bp upstream of the transcription initiation site) and the nine coding exons, including intron – exon boundaries of the HNF1b gene, as described (6). Cell culture, transfection and immunofluorescence conditions Human embryonic kidney HEK293 cells were transiently transfected and b-gal, CAT and immunostaining assays on transfected cells were performed as described (6). As reporter CAT construct we used the Alphafetoprotein (Afp) promoter, which contains two HNF1 sites at 2131 and – 67 nucleotide positions upstream the transcription initiation site.

ACKNOWLEDGEMENTS We kindly thank A.S. Woolf for useful discussions and advice, S. Power for critical reading of the manuscript, C.J. Harris, J.R. Hoyer, R. Sanford, M. Knepper, P. Serup and I. Talianidis for providing key antibodies. We also thank the clinicians and fetopathologists, in particular, G. Bonyha from Debre´ Hospital and M.C. Gubler for initial histological inspection of R112 kidney sections. This work was supported by Association pour la Recherche sur le Cancer (ARC) Contract 3231, Ligue Nationale contre le Cancer (Comite de Paris, RS 06/75 – 48), EuReGene Contract no. LSHG-CT-2004 005 085, Centre national de Recherche Scientifique (CNRS) and Universite´ Pierre et Marie Curie. C.H. is a recipient of PhD fellowship from Ministere National de la Recherche and ARC. Conflict of Interest statement. None declared.

Protein extracts, western blot and electrophoretic gel mobility shift assays Nuclear extracts from transfected cell lines were used, and electrophoretic gel mobility shift assays (EMSA) and western blots were performed as reported (50). A rabbit polyclonal antibody raised in the laboratory against residues 39– 89 of the mouse HNF1b protein was used. Double stranded oligonucleotides used as probe in EMSA experiments correspond to the HNF1-binding site of the albumin promoter (PE) (51). Northern blot analyses MTN multiple tissue northern blots (Human I and Human II; Clontech) were hybridized using the full-length human HNF1b as probe and the Express Hybrid solution (Clontech), as indicated by the manufacturers. In situ hybridization and immunohistochemistry Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. In situ hybrization was performed as previously described (20). For immunohistochemistry, dewaxed slides were subjected to microwave antigen retrieval in 10 mM citrate. For HNF1b immunostainings, a rabbit polyclonal antibody (H85, Santa Cruz) was used; its specificity was tested by pre-incubation with a GST-fusion protein corresponding to the region used as antigen. As primary antibodies, we used guinea pig antibody to insulin (Sigma), rabbit polyclonal antibodies to somatostatin and PP (DAKO), to carboxypeptidase-A (Biogenesis), to Glut2, provided by B. Thorens, to NKCC2 and AQP2, provided by M. Knepper, to Tamm-Horsfall (uromodulin), provided by J.R. Hoyer and to polycystin-1 (LRR 1750) and polycystin-2 (MVN), provided by R. Sandford, and the monoclonal antibodies to glucagon and acetylated tubulin (Sigma), to E-cadherin and b-catenin (BD Biosciences), to Fibrocystin (REC 5A and REC 14B), provided by C.J. Ward, as well as the lectin fluorescein-Lotus tetragonolobus (LTA, Vector laboratories). We used FITC-conjugated and Cyanine 3-conjugated secondary antibodies (Jackson Laboratories) and the Vectastain Elite ABC kit (Vector Laboratories).

REFERENCES 1. Edghill, E.L., Bingham, C., Ellard, S. and Hattersley, A.T. (2006) Mutations in hepatocyte nuclear factor-1beta and their related phenotypes. J. Med. Genet., 43, 84–90. 2. Bellanne-Chantelot, C., Clauin, S., Chauveau, D., Collin, P., Daumont, M., Douillard, C., Dubois-Laforgue, D., Dusselier, L., Gautier, J.F., Jadoul, M. et al. (2005) Large genomic rearrangements in the hepatocyte nuclear factor-1beta (TCF2) gene are the most frequent cause of maturity-onset diabetes of the young type 5. Diabetes, 54, 3126– 3132. 3. Lindner, T.H., Njolstad, P.R., Horikawa, Y., Bostad, L., Bell, G.I. and Sovik, O. (1999) A novel syndrome of diabetes mellitus, renal dysfunction and genital malformation associated with a partial deletion of the pseudo-POU domain of hepatocyte nuclear factor-1beta. Hum. Mol. Genet., 8, 2001–2008. 4. Bingham, C., Bulman, M.P., Ellard, S., Allen, L.I., Lipkin, G.W., Hoff, W.G., Woolf, A.S., Rizzoni, G., Novelli, G., Nicholls, A.J. et al. (2001) Mutations in the hepatocyte nuclear factor-1beta gene are associated with familial hypoplastic glomerulocystic kidney disease. Am. J. Hum. Genet, 68, 219 –224. 5. Mache, C.J., Preisegger, K.H., Kopp, S., Ratschek, M. and Ring, E. (2002) De novo HNF-1 beta gene mutation in familial hypoplastic glomerulocystic kidney disease. Pediatr. Nephrol., 17, 1021–1026. 6. Barbacci, E., Chalkiadaki, A., Masdeu, C., Haumaitre, C., Lokmane, L., Loirat, C., Cloarec, S., Talianidis, I., Bellanne-Chantelot, C. and Cereghini, S. (2004) HNF1beta/TCF2 mutations impair transactivation potential through altered co-regulator recruitment. Hum. Mol. Genet., 13, 3139–3149. 7. Woolf, A.S., Feather, S.A. and Bingham, C. (2002) Recent insights into kidney diseases associated with glomerular cysts. Pediatr. Nephrol., 17, 229–235. 8. Bingham, C., Ellard, S., Cole, T.R., Jones, K.E., Allen, L.I., Goodship, J.A., Goodship, T.H., Bakalinova-Pugh, D., Russell, G.I., Woolf, A.S. et al. (2002) Solitary functioning kidney and diverse genital tract malformations associated with hepatocyte nuclear factor-1beta mutations. Kidney Int., 61, 1243–1251. 9. Carbone, I., Cotellessa, M., Barella, C., Minetti, C., Ghiggeri, G.M., Caridi, G., Perfumo, F. and Lorini, R. (2002) A novel hepatocyte nuclear factor-1beta (MODY-5) gene mutation in an Italian family with renal dysfunctions and early-onset diabetes. Diabetologia, 45, 153 –154. 10. Bingham, C., Ellard, S., van’t Hoff, W.G., Simmonds, H.A., Marinaki, A.M., Badman, M.K., Winocour, P.H., Stride, A., Lockwood, C.R., Nicholls, A.J. et al. (2003) Atypical familial juvenile hyperuricemic nephropathy associated with a hepatocyte nuclear factor-1beta gene mutation. Kidney Int., 63, 1645–1651. 11. Bellanne-Chantelot, C., Chauveau, D., Gautier, J., Dubois-Laforgue, D., Clauin, S., Beaufils, S., Wilhelm, J.M., Boitard, C., Noel, L.H., Velho, G. et al. (2004) Clinical spectrum associated with hepatocyte nuclear factor-1beta mutations. Ann. Intern. Med., 140, 510–517.


12. Kitanaka, S., Miki, Y., Hayashi, Y. and Igarashi, T. (2004) Promoter-specific repression of hepatocyte nuclear factor (HNF)-1 beta and HNF-1 alpha transcriptional activity by an HNF-1 beta missense mutant associated with Type 5 maturity-onset diabetes of the young with hepatic and biliary manifestations. J. Clin. Endocrinol. Metab., 89, 1369–1378. 13. Barbacci, E., Reber, M., Ott, M., Breillat, C., Huetz, F. and Cereghini, S. (1999) Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification. Development, 126, 4795–4805. 14. Coffinier, C., Thepot, D., Babinet, C., Yaniv, M. and Barra, J. (1999) Essential role for the homeoprotein vHNF1/HNF1beta in visceral endoderm differentiation. Development, 126, 4785–4794. 15. Haumaitre, C., Barbacci, E., Jenny, M., Ott, M.O., Gradwohl, G. and Cereghini, S. (2005) Lack of TCF2/vHNF1 in mice leads to pancreas agenesis. Proc. Natl Acad. Sci. USA, 102, 1490–1495. 16. Coffinier, C., Gresh, L., Fiette, L., Tronche, F., Schutz, G., Babinet, C., Pontoglio, M., Yaniv, M. and Barra, J. (2002) Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta. Development, 129, 1829–1838. 17. Gresh, L., Fischer, E., Reimann, A., Tanguy, M., Garbay, S., Shao, X., Hiesberger, T., Fiette, L., Igarashi, P., Yaniv, M. et al. (2004) A transcriptional network in polycystic kidney disease. EMBO J., 23, 1557–1568. 18. Hiesberger, T., Bai, Y., Shao, X., McNally, B.T., Sinclair, A.M., Tian, X., Somlo, S. and Igarashi, P. (2004) Mutation of hepatocyte nuclear factor-1beta inhibits Pkhd1 gene expression and produces renal cysts in mice. J. Clin. Invest., 113, 814–825. 19. Lazzaro, D., De Simone, V., De Magistris, L., Lehtonen, E. and Cortese, R. (1992) LFB1 and LFB3 homeoproteins are sequentially expressed during kidney development. Development, 114, 469 –479. 20. Ott, M.O., Rey-Campos, J., Cereghini, S. and Yaniv, M. (1991) vHNF1 is expressed in epithelial cells of distinct embryonic origin during development and precedes HNF1 expression. Mech. Dev., 36, 47–58. 21. Coffinier, C., Barra, J., Babinet, C. and Yaniv, M. (1999) Expression of the vHNF1/HNF1beta homeoprotein gene during mouse organogenesis. Mech. Dev., 89, 211–213. 22. Kolatsi-Joannou, M., Bingham, C., Ellard, S., Bulman, M.P., Allen, L.I., Hattersley, A.T. and Woolf, A.S. (2001) Hepatocyte nuclear factor-1beta: a new kindred with renal cysts and diabetes and gene expression in normal human development. J. Am. Soc. Nephrol., 12, 2175–2180. 23. Reber, M. and Cereghini, S. (2001) Variant hepatocyte nuclear factor 1 expression in the mouse genital tract. Mech. Dev., 100, 75–78. 24. Sagen, J.V., Bostad, L., Njolstad, P.R. and Sovik, O. (2003) Enlarged nephrons and severe nondiabetic nephropathy in hepatocyte nuclear factor-1beta (HNF-1beta) mutation carriers. Kidney Int., 64, 793– 800. 25. Bingham, C., Ellard, S., Allen, L., Bulman, M., Shepherd, M., Frayling, T., Berry, P.J., Clark, P.M., Lindner, T., Bell, G.I. et al. (2000) Abnormal nephron development associated with a frameshift mutation in the transcription factor hepatocyte nuclear factor-1 beta. Kidney Int., 57, 898 –907. 26. Ong, A.C. (2000) Polycystin expression in the kidney and other tissues: complexity, consensus and controversy. Exp. Nephrol., 8, 208 –214. 27. Ward, C.J., Yuan, D., Masyuk, T.V., Wang, X., Punyashthiti, R., Whelan, S., Bacallao, R., Torra, R., LaRusso, N.F., Torres, V.E. et al. (2003) Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia. Hum. Mol. Genet., 12, 2703–2710. 28. Hildebrandt, F. and Otto, E. (2005) Cilia and centrosomes: a unifying pathogenic concept for cystic kidney disease? Nat. Rev. Genet., 6, 928 –940. 29. Saadi-Kheddouci, S., Berrebi, D., Romagnolo, B., Cluzeaud, F., Peuchmaur, M., Kahn, A., Vandewalle, A. and Perret, C. (2001) Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the beta-catenin gene. Oncogene, 20, 5972–5981. 30. Qian, F., Watnick, T.J., Onuchic, L.F. and Germino, G.G. (1996) The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney disease type I. Cell, 87, 979–987. 31. Gresh, L., Bourachot, B., Reimann, A., Guigas, B., Fiette, L., Garbay, S., Muchardt, C., Hue, L., Pontoglio, M., Yaniv, M. et al. (2005) The SWI/ SNF chromatin-remodeling complex subunit SNF5 is essential for hepatocyte differentiation. EMBO J., 24, 3313–3324. 32. Orci, L., Baetens, D., Ravazzola, M., Stefan, Y. and Malaisse-Lagae, F. (1976) Pancreatic polypeptide and glucagon: non-random distribution in pancreatic islets. Life Sci., 19, 1811– 1815.

2375

33. Dessimoz, J., Bonnard, C., Huelsken, J. and Grapin-Botton, A. (2005) Pancreas-specific deletion of beta-catenin reveals Wnt-dependent and Wnt-independent functions during development. Curr. Biol., 15, 1677–1683. 34. Papadopoulou, S. and Edlund, H. (2005) Attenuated Wnt signaling perturbs pancreatic growth but not pancreatic function. Diabetes, 54, 2844–2851. 35. Murtaugh, L.C., Law, A.C., Dor, Y. and Melton, D.A. (2005) Beta-catenin is essential for pancreatic acinar but not islet development. Development, 132, 4663–4674. 36. Shih, D.Q., Screenan, S., Munoz, K.N., Philipson, L., Pontoglio, M., Yaniv, M., Polonsky, K.S. and Stoffel, M. (2001) Loss of HNF-1alpha function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism. Diabetes, 50, 2472–2480. 37. Yamagata, K., Nammo, T., Moriwaki, M., Ihara, A., Iizuka, K., Yang, Q., Satoh, T., Li, M., Uenaka, R., Okita, K. et al. (2002) Overexpression of dominant-negative mutant hepatocyte nuclear fctor-1 alpha in pancreatic beta-cells causes abnormal islet architecture with decreased expression of E-cadherin, reduced beta-cell proliferation, and diabetes. Diabetes, 51, 114–123. 38. Cha, J.Y., Kim, H., Kim, K.S., Hur, M.W. and Ahn, Y. (2000) Identification of transacting factors responsible for the tissue-specific expression of human glucose transporter type 2 isoform gene. Cooperative role of hepatocyte nuclear factors 1alpha and 3beta. J. Biol. Chem., 275, 18358–18365. 39. Tomura, H., Nishigori, H., Sho, K., Yamagata, K., Inoue, I. and Takeda, J. (1999) Loss-of-function and dominant-negative mechanisms associated with hepatocyte nuclear factor-1beta mutations in familial type 2 diabetes mellitus. J. Biol. Chem., 274, 12975–12978. 40. Bohn, S., Thomas, H., Turan, G., Ellard, S., Bingham, C., Hattersley, A.T. and Ryffel, G.U. (2003) Distinct molecular and morphogenetic properties of mutations in the human HNF1beta gene that lead to defective kidney development. J. Am. Soc. Nephrol., 14, 2033– 2041. 41. Harries, L.W., Ellard, S., Jones, R.W., Hattersley, A.T. and Bingham, C. (2004) Abnormal splicing of hepatocyte nuclear factor-1 beta in the renal cysts and diabetes syndrome. Diabetologia, 47, 932–942. 42. Holbrook, J.A., Neu-Yilik, G., Hentze, M.W. and Kulozik, A.E. (2004) Nonsense-mediated decay approaches the clinic. Nat. Genet., 36, 801–808. 43. Arnaout, M.A. (2001) Molecular genetics and pathogenesis of autosomal dominant polycystic kidney disease. Annu. Rev. Med., 52, 93–123. 44. Igarashi, P. and Somlo, S. (2002) Genetics and pathogenesis of polycystic kidney disease. J. Am. Soc. Nephrol., 13, 2384–2398. 45. Cook, D.L., Gerber, A.N. and Tapscott, S.J. (1998) Modeling stochastic gene expression: implications for haploinsufficiency. Proc. Natl Acad. Sci. USA, 95, 15641–15646. 46. Qian, C.N., Knol, J., Igarashi, P., Lin, F., Zylstra, U., Teh, B.T. and Williams, B.O. (2005) Cystic renal neoplasia following conditional inactivation of apc in mouse renal tubular epithelium. J. Biol. Chem., 280, 3938–3945. 47. Fischer, E., Legue, E., Doyen, A., Nato, F., Nicolas, J.F., Torres, V., Yaniv, M. and Pontoglio, M. (2006) Defective planar cell polarity in polycystic kidney disease. Nat. Genet., 38, 21–23. 48. Maestro, M.A., Boj, S., Luco, R.F., Pierreux, C.E., Cabedo, J., Servitja, J.M., German, M.S., Rousseau, G.G., Lemaigre, F.P. and Ferrer, J. (2003) 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. 49. Guillam, M.T., Hummler, E., Schaerer, E., Yeh, J.I., Birnbaum, M.J., Beermann, F., Schmidt, A., Deriaz, N. and Thorens, B. (1997) Early diabetes and abnormal postnatal pancreatic islet development in mice lacking Glut-2. Nat. Genet., 17, 327 –330. 50. Cereghini, S., Ott, M.O., Power, S. and Maury, M. (1992) Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles. Development, 116, 783 –797. 51. Cereghini, S., Blumenfeld, M. and Yaniv, M. (1988) A liver-specific factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells. Genes Dev., 2, 957 –974. 52. Chi, Y.I., Frantz, J.D., Oh, B.C., Hansen, L., Dhe-Paganon, S. and Shoelson, S.E. (2002) Diabetes mutations delineate an atypical POU domain in HNF-1alpha. Mol. Cell, 10, 1129–1137.