TCF/LEF regulation of the topologically associated domain ADI promotes mESCs to exit the pluripotent ground state.

Mouse embryonic stem cells (mESCs) can be maintained in vitro in defined N2B27 medium supplemented with two chemical inhibitors for GSK3 and MEK (2i) and the cytokine leukemia inhibitory factor (LIF), which act synergistically to promote self-renewal and pluripotency. Here, we find that genetic deletion of the four genes encoding the TCF/LEF transcription factors confers mESCs with the ability to self-renew in N2B27 medium alone. TCF/LEF quadruple knockout (qKO) mESCs display dysregulation of several genes, including Aire, Dnmt3l, and IcosL, located adjacent to each other within a topologically associated domain (TAD). Aire, Dnmt3l, and IcosL appear to be regulated by TCF/LEF in a ß-catenin independent manner. Moreover, downregulation of Aire and Dnmt3l in wild-type mESCs mimics the loss of TCF/LEF and increases mESC survival in the absence of 2iL. Hence, this study identifies TCF/LEF effectors that mediate exit from the pluripotent state.

Profiling Virus-Specific Tcf1+ T Cell Repertoires During Acute and Chronic Viral Infection.

CD8 T cells play a crucial role in providing protection from viral infections. It has recently been established that a subset of CD8 T cells expressing Tcf1 are responsible for sustaining exhausted T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection. Many of these studies, however, have been performed using T cell receptor (TCR) transgenic mice, in which CD8 T cells express a monoclonal TCR specific for the LCMV glycoprotein. To investigate whether the Tcf1+ and Tcf1- repertoires are naturally composed of similar or different clones in wild-type mice exposed to acute or chronic LCMV infection, we performed TCR repertoire sequencing of virus-specific CD8 T cells, including Tcf1+ and Tcf1- populations. Our analysis revealed that the Tcf1+ TCR repertoire is maintained at an equal or higher degree of clonal diversity despite harboring fewer cells. Additionally, within the same animal, there was extensive clonal overlap between the Tcf1+ and Tcf1- repertoires in both chronic and acute LCMV infection. We could further detect these virus-specific clones in longitudinal blood samples earlier in the infection. With respect to common repertoire parameters (clonal overlap, germline gene usage, and clonal expansion), we found minor differences between the virus-specific TCR repertoire of acute and chronic LCMV infection 40 days post infection. Overall, our results indicate that the Tcf1+ population emerging during chronic LCMV infection is not clonally distinct from the Tcf1- population, supporting the notion that the Tcf1+ pool is indeed a fuel for the more exhausted Tcf1- population within the heterogenous repertoire of LCMV-specific CD8 T cells.

Promotion of ß-catenin/Foxo1 signaling ameliorates renal interstitial fibrosis.

Transforming growth factor ß (TGF-ß) is the key cytokine involved in causing fibrosis through cross-talk with major profibrotic pathways. However, inhibition of TGF-ß to prevent fibrosis would also abrogate its anti-inflammatory and wound-healing effects. ß-catenin is a common co-factor in most TGF-ß signaling pathways. ß-catenin binds to T-cell factor (TCF) to activate profibrotic genes and binds to Forkhead box O (Foxo) to promote cell survival under oxidative stress. Using a proximity ligation assay in human kidney biopsies, we found that ß-catenin/Foxo interactions were higher in kidney with little fibrosis, whereas ß-catenin/TCF interactions were upregulated in the kidney of patients with fibrosis. We hypothesised that ß-catenin/Foxo is protective against kidney fibrosis. We found that Foxo1 protected against rhTGF-ß1-induced profibrotic protein expression using a CRISPR/cas9 knockout of Foxo1 or TCF1 in murine kidney tubular epithelial C1.1 cells. Co-administration of TGF-ß with a small molecule inhibitor of ß-catenin/TCF (ICG-001), protected against kidney fibrosis in unilateral ureteral obstruction. Collectively, our human, animal and in vitro findings suggest ß-catenin/Foxo as a therapeutic target in kidney fibrosis.

Improvement of hyperglycemia in a murine model of insulin resistance and high glucose- and inflammasome-mediated IL-1ß expressions in macrophages by silymarin.

Macrophages and inflammasome pathway are involved in high-glucose toxicity and development of insulin resistance. Silymarin (SMR) was known to modulate glucose homeostasis and reduce inflammation. However, it is still unknown whether SMR possess anti-hyperglycemic effects in diabetic-like knockout mice (Hnf-1αkin/-/Ins.cre mice) with insulin resistance and also unclear how SMR regulates LPS induced stress markers and pro-inflammatory cytokines under stresses of high glucose (HG) or NLRP3 inflammasome activation. Current results show that oral administration of SMR (100 mg/kg) reduced hyperglycemia in the mouse model of maturity-onset diabetes of the young type 3-like mice. In cultured macrophages, SMR (5-20 µg/ml) reduces high glucose (HG)-enhanced expressions of inducible nitric oxide synthase, nitric oxide generation stimulated by LPS; however, no effects on COX-2 expressions. The enhanced interleukin-1ß (ΙL-1ß) secretions in the presence of HG or palmitate were also significantly down regulated by SMR in dose-dependent manner in LPS-treated macrophages. Such observations may result from the decreased extracellular signal-regulated kinase 1/2 phosphorylation, while without affecting protein kinase C-α phosphorylation and nuclear factor-κB activation. These findings together show that SMR acts as a protector against HG-related stresses not only by lowering hyperglycemia but also suppressing HG- and inflammasome-mediated IL-1ß expressions to improve insulin resistance.

Hepatocyte nuclear factor 1A deficiency causes hemolytic anemia in mice by altering erythrocyte sphingolipid homeostasis.

The hepatocyte nuclear factor (HNF) family regulates complex networks of metabolism and organ development. Human mutations in its prototypical member HNF1A cause maturity-onset diabetes of the young (MODY) type 3. In this study, we identified an important role for HNF1A in the preservation of erythrocyte membrane integrity, calcium homeostasis, and osmotic resistance through an as-yet unrecognized link of HNF1A to sphingolipid homeostasis. HNF1A-/- mice displayed microcytic hypochromic anemia with reticulocytosis that was partially compensated by avid extramedullary erythropoiesis at all erythroid stages in the spleen thereby excluding erythroid differentiation defects. Morphologically, HNF1A-/- erythrocytes resembled acanthocytes and displayed increased phosphatidylserine exposure, high intracellular calcium, and elevated osmotic fragility. Sphingolipidome analysis by mass spectrometry revealed substantial and tissue-specific sphingolipid disturbances in several tissues including erythrocytes with the accumulation of sphingosine as the most prominent common feature. All HNF1A-/- erythrocyte defects could be simulated by exposure of wild-type (WT) erythrocytes to sphingosine in vitro and attributed in part to sphingosine-induced suppression of the plasma-membrane Ca2+-ATPase activity. Bone marrow transplantation rescued the anemia phenotype in vivo, whereas incubation with HNF1A-/- plasma increased the osmotic fragility of WT erythrocytes in vitro. Our data suggest a non-cell-autonomous erythrocyte defect secondary to the sphingolipid changes caused by HNF1A deficiency. Transcriptional analysis revealed 4 important genes involved in sphingolipid metabolism to be deregulated in HNF1A deficiency: Ormdl1, sphingosine kinase-2, neutral ceramidase, and ceramide synthase-5. The considerable erythrocyte defects in murine HNF1A deficiency encourage clinical studies to explore the hematological consequences of HNF1A deficiency in human MODY3 patients.

Deletion of HNF1α in hepatocytes results in fatty liver-related hepatocellular carcinoma in mice.

Hepatocyte nuclear factor 1α (HNF1α) is a liver-enriched transcription factor that is critical for the maintenance of hepatocyte function. Our previous studies have demonstrated the therapeutic effects of HNF1α on hepatic fibrosis and hepatocellular carcinoma (HCC) in animals. In this study, we created hepatocyte-specific Hnf1α knockout mice using the Cre-loxP recombination system. The knockout mice display increased fatty acid synthesis in the liver. Moreover, these mice spontaneously develop HCC through fatty liver without cirrhosis. Inflammatory cytokines, such as tumor necrosis factor α and IL-6, are upregulated and accompanied by increased phosphorylation of Akt, p-65 and STAT3 in the livers of HNF1α knockout mice. Our findings suggest that HNF1α plays a crucial role in hepatocyte lipid metabolism and hepatocarcinogenesis.

TCF1 deficiency ameliorates autoimmune lymphoproliferative syndrome (ALPS)-like phenotypes of lpr/lpr mice.

Autoimmune lymphoproliferative syndrome (ALPS) is an incurable disease, which is characterized by non-malignant autoimmune lymphoproliferation. TCF1 is a key effector in the canonical Wnt/ß-catenin pathway, regulating the development, activation and function of T cells. In this study, we aimed to explore the potential role of TCF1 in the development of ALPS-like phenotypes of lpr/lpr mice. We acquired TCF1-/- lpr/lpr double mutant mice by crossing TCF1 deficiency mice with lpr/lpr mice. Splenocyte compositions, serum cytokines levels, antidsDNA antibody production and kidney pathology were examined in the TCF1-/- lpr/lpr mice. With these examinations, we revealed that TCF1 deficiency relieved most manifestations of ALPS-like phenotype, which were caused by Fas mutation in TCF1-/- lpr/lpr mice. Splenocyte total numbers and compositions were downregulated to the similar levels with wildtype mice. TE and TEM cells were decreased in TCF1-/- lpr/lpr compared with lpr/lpr mice. The levels of autoantibodies and proinflammatory factors in serum, and the histopathology changes and the relative mRNA levels of proinflammatory factors in kidney all displayed parallel tendency in TCF1-/- lpr/lpr mice. Our study demonstrated that TCF1 deficiency ameliorated the ALPS-like phenotypes of TCF1-/- lpr/lpr mice, which might indicate a potential therapeutic direction for ALPS.

Tubular proteinuria in patients with HNF1α mutations: HNF1α drives endocytosis in the proximal tubule.

Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a-deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of ß2-microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non-MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A.

Exploring the role of the HNF-1αG319S polymorphism in ß cell failure and youth-onset type 2 diabetes: Lessons from MODY and Hnf-1α-deficient animal models.

The prevalence of youth-onset type 2 diabetes (T2D) is rapidly increasing worldwide, disproportionately affecting Indigenous youth with Oji-Cree heritage from central Canada. Candidate gene screening has uncovered a novel and private polymorphism in the Oji-Cree population in the hepatocyte nuclear factor-1 alpha (HNF-1α) gene, where a highly conserved glycine residue at position 319 is changed to a serine (termed HNF-1αG319S or simply G319S). Oji-Cree youth who carry one or two copies of the "S-allele" present at diagnosis with less obesity, reduced indicators of insulin resistance, and lower plasma insulin levels at diagnosis, suggestive of a primary defect in the insulin-secreting ß cells. Few studies on the impact of the HNF-1αG319S variant on ß cell function have been performed to date; however, much can be learned from other clinical phenotypes of HNF-1α-deficiency, including HNF-1α mutations that cause maturity-onset diabetes of the young 3 (MODY3). In addition, evaluation of Hnf-1α-deficient murine models reveals that HNF-1α plays a central role in the regulation of insulin secretion by regulating the expression of key genes involved in ß cell glucose-sensing, mitochondrial function, and the maintenance of the ß cell phenotype in differentiated ß cells. The overall goal of this minireview is to explore the impact of HNF-1α-deficiency on the ß cell to better inform future research into the mechanisms of ß cell dysfunction in Oji-Cree youth with T2D.

Premature expression of Foxp3 in double-negative thymocytes.

Peripheral immune regulation depends on the generation of thymic-derived regulatory T (tTreg) cells to maintain self-tolerance and to counterbalance overshooting immune responses. The expression of the Treg lineage defining transcription factor Foxp3 in developing tTreg cells depends on TCR signaling during the thymic selection process of these T cells. In this study, we surprisingly identify Foxp3+ immature thymocytes at the double-negative (DN) stage in transcription factor 7 (Tcf7)-deficient mice. These Foxp3+ cells did not express a TCR (ß or γδ chains), CD3 or CD5 and therefore these cells were true DN cells. Further investigation of this phenomenon in a transgenic TCR model showed that Foxp3-expressing DN cells could not respond to TCR stimulation in vivo. These data suggest that Foxp3 expression in these DN cells occurred independently of TCR signaling. Interestingly, these Foxp3+ DN cells were located in a transition state between DN1 and DN2 (CD4-CD8-CD3-TCR-CD44highCD25low). Our results indicate that Tcf7 is involved in preventing the premature expression of Foxp3 in DN thymocytes.

Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice.

The transcription factors hepatic nuclear factor (HNF)1α and HNF1ß can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1ß) to examine the effects of knockdown of HNF1α or HNF1ß on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1ß, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (∼ 50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (∼ 1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1ß did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1ß infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1ß, is the primary positive regulator of PCSK9 transcription in mouse liver.

Metabolomics identifies novel Hnf1alpha-dependent physiological pathways in vivo.

Mutations in the HNF1A gene cause maturity-onset diabetes of the young type 3, one of the most common genetic causes of non-insulin-dependent (type 2) diabetes mellitus. Although the whole-body Hnf1a-null mouse recapitulates the low insulin levels and high blood glucose observed in human maturity-onset diabetes of the young type 3 patients, these mice also suffer from Laron dwarfism and aminoaciduria, suggesting a role for hepatocyte nuclear factor 1α (Hnf1α) in pathophysiologies distinct from non-insulin-dependent (type 2) diabetes mellitus. In an effort to identify pathways associated with inactivation of Hnf1α, an ultraperformance liquid chromatography coupled to mass spectrometry-based metabolomics study was conducted on urine samples from wild-type and Hnf1a-null mice. An increase in phenylalanine metabolites is in agreement with the known regulation of the phenylalanine hydroxylase gene by Hnf1α. This metabolomic approach also identified urinary biomarkers for three tissue-specific dysfunctions previously unassociated with Hnf1α function. 1) Elevated indolelactate coupled to decreased xanthurenic acid also indicated defects in the indole and kynurenine pathways of tryptophan metabolism, respectively. 2) An increase in the neutral amino acid proline in the urine of Hnf1a-null mice correlated with loss of renal apical membrane transporters of the Slc6a family. 3) Further investigation into the mechanism of aldosterone increase revealed an overactive adrenal gland in Hnf1a-null mice possibly due to inhibition of negative feedback regulation. Although the phenotype of the Hnf1a-null mouse is complex, metabolomics has opened the door to investigation of several physiological systems in which Hnf1α may be a critical regulatory component.

Loss of hepatocyte-nuclear-factor-1alpha impacts on adult mouse intestinal epithelial cell growth and cell lineages differentiation.

BACKGROUND AND AIMS: Although Hnf1alpha is crucial for pancreas and liver functions, it is believed to play a limited functional role for intestinal epithelial functions. The aim of this study was to assess the consequences of abrogating Hnf1alpha on the maintenance of adult small intestinal epithelial functions. METHODOLOGY/PRINCIPAL FINDINGS: An Hnf1alpha knockout mouse model was used. Assessment of histological abnormalities, crypt epithelial cell proliferation, epithelial barrier, glucose transport and signalling pathways were measured in these animals. Changes in global gene expression were also analyzed. Mice lacking Hnf1alpha displayed increased crypt proliferation and intestinalomegaly as well as a disturbance of intestinal epithelial cell lineages production during adult life. This phenotype was associated with a decrease of the mucosal barrier function and lumen-to-blood glucose delivery. The mammalian target of rapamycin (mTOR) signalling pathway was found to be overly activated in the small intestine of adult Hnf1alpha mutant mice. The intestinal epithelium of Hnf1alpha null mice displayed a reduction of the enteroendocrine cell population. An impact was also observed on proper Paneth cell differentiation with abnormalities in the granule exocytosis pathway. CONCLUSIONS/SIGNIFICANCE: Together, these results unravel a functional role for Hnf1alpha in regulating adult intestinal growth and sustaining the functions of intestinal epithelial cell lineages.

Mitochondrial dysfunction contributes to impaired insulin secretion in INS-1 cells with dominant-negative mutations of HNF-1alpha and in HNF-1alpha-deficient islets.

Maturity Onset Diabetes of the Young-type 3 (MODY-3) has been linked to mutations in the transcription factor hepatic nuclear factor (HNF)-1alpha, resulting in deficiency in glucose-stimulated insulin secretion. In INS-1 cells overexpressing doxycycline-inducible HNF-1alpha dominant-negative (DN-) gene mutations, and islets from Hnf-1alpha knock-out mice, insulin secretion was impaired in response to glucose (15 mm) and other nutrient secretagogues. Decreased rates of insulin secretion in response to glutamine plus leucine and to methyl pyruvate, but not potassium depolarization, indicate defects specific to mitochondrial metabolism. To identify the biochemical mechanisms responsible for impaired insulin secretion, we used (31)P NMR measured mitochondrial ATP synthesis (distinct from glycolytic ATP synthesis) together with oxygen consumption measurements to determine the efficiency of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling was significantly higher in DN-HNF-1alpha cells, such that rates of ATP synthesis were decreased by approximately one-half in response to the secretagogues glucose, glutamine plus leucine, or pyruvate. In addition to closure of the ATP-sensitive K(+) channels with mitochondrial ATP synthesis, mitochondrial production of second messengers through increased anaplerotic flux has been shown to be critical for coupling metabolism to insulin secretion. (13)C-Isotopomer analysis and tandem mass spectrometry measurement of Krebs cycle intermediates revealed a negative impact of DN-HNF-1alpha and Hnf-1alpha knock-out on mitochondrial second messenger production with glucose but not amino acids. Taken together, these results indicate that, in addition to reduced glycolytic flux, uncoupling of mitochondrial oxidative phosphorylation contributes to impaired nutrient-stimulated insulin secretion with either mutations or loss of HNF-1alpha.

Hnf1alpha (MODY3) controls tissue-specific transcriptional programs and exerts opposed effects on cell growth in pancreatic islets and liver.

Heterozygous HNF1A mutations cause pancreatic-islet beta-cell dysfunction and monogenic diabetes (MODY3). Hnf1alpha is known to regulate numerous hepatic genes, yet knowledge of its function in pancreatic islets is more limited. We now show that Hnf1a deficiency in mice leads to highly tissue-specific changes in the expression of genes involved in key functions of both islets and liver. To gain insights into the mechanisms of tissue-specific Hnf1alpha regulation, we integrated expression studies of Hnf1a-deficient mice with identification of direct Hnf1alpha targets. We demonstrate that Hnf1alpha can bind in a tissue-selective manner to genes that are expressed only in liver or islets. We also show that Hnf1alpha is essential only for the transcription of a minor fraction of its direct-target genes. Even among genes that were expressed in both liver and islets, the subset of targets showing functional dependence on Hnf1alpha was highly tissue specific. This was partly explained by the compensatory occupancy by the paralog Hnf1beta at selected genes in Hnf1a-deficient liver. In keeping with these findings, the biological consequences of Hnf1a deficiency were markedly different in islets and liver. Notably, Hnf1a deficiency led to impaired large-T-antigen-induced growth and oncogenesis in beta cells yet enhanced proliferation in hepatocytes. Collectively, these findings show that Hnf1alpha governs broad, highly tissue-specific genetic programs in pancreatic islets and liver and reveal key consequences of Hnf1a deficiency relevant to the pathophysiology of monogenic diabetes.

Partial and whole gene deletion mutations of the GCK and HNF1A genes in maturity-onset diabetes of the young.

AIMS/HYPOTHESIS: Heterozygous mutations of glucokinase (GCK) and hepatocyte nuclear factor-1 alpha (HNF1A; also known as hepatic transcription factor 1 [TCF1]) genes are the most common cause of MODY. Genomic deletions of the HNF1B (also known as TCF2) gene have recently been shown to account for one third of mutations causing renal cysts and diabetes syndrome. We investigated the prevalence of partial and whole gene deletions in UK patients meeting clinical criteria for GCK or HNF-1alpha/-4alpha MODY and in whom no mutation had been identified by sequence analysis. METHODS: A multiplex ligation-dependent probe amplification (MLPA) assay was developed using synthetic oligonucleotide probes for 30 exons of the GCK, HNF1A and HNF4A genes. RESULTS: Partial or whole gene deletions were identified in 1/29 (3.5%) probands using the GCK MLPA assay and 4/60 (6.7%) of probands using the HNF1A/-4A MLPA assay. Four different deletions were detected: GCK exon 2, HNF1A exon 1, HNF1A exons 2 to 10 and HNF1A exons 1 to 10. An additional Danish pedigree with evidence of linkage to HNF1A had a deletion of exons 2 to 10. Testing other family members confirmed co-segregation of the deletion mutations with diabetes in the pedigrees. CONCLUSIONS/INTERPRETATION: Large deletions encompassing whole exons can cause GCK or HNF-1alpha MODY and will not be detected by sequencing. Gene dosage assays, such as MLPA, are a useful adjunct to sequence analysis when a diagnosis of MODY is strongly suspected.

A conditional model reveals that induction of hepatocyte nuclear factor-1alpha in Hnf1alpha-null mutant beta-cells can activate silenced genes postnatally, whereas overexpression is deleterious.

Humans with heterozygous loss-of-function mutations in the hepatocyte nuclear factor-1alpha (HNF1alpha) gene develop beta-cell-deficient diabetes (maturity-onset diabetes of the young type 3), indicating that HNF1alpha gene dosage is critical in beta-cells. However, whether increased HNF1alpha expression might be beneficial or deleterious for beta-cells is unknown. Furthermore, although it is clear that HNF1alpha is required for beta-cell function, it is not known whether this role is cell autonomous or whether there is a restricted developmental time frame for HNF1alpha to elicit gene activation in beta-cells. To address this, we generated a tetracycline-inducible mouse model that transcribes HNF1alpha selectively in beta-cells in either wild-type or Hnf1alpha-null backgrounds. Short-term induction of HNF1alpha in islets from adult Hnf1alpha(-/-) mice that did not express HNF1alpha throughout development resulted in the activation of target genes, indicating that HNF1alpha has beta-cell-autonomous functions that can be rescued postnatally. However, transgenic induction throughout development, which inevitably resulted in supraphysiological levels of HNF1alpha, strikingly caused a severe reduction of cellular proliferation, increased apoptosis, and consequently beta-cell depletion and diabetes. Thus, HNF1alpha is sensitive to both reduced and excessive concentrations in beta-cells. This finding illustrates the paramount importance of using the correct concentration of a beta-cell transcription factor in both gene therapy and artificial differentiation strategies.