Insulin resistance represents a hallmark during the development of type 2 diabetes mellitus and in the pathogenesis of obesity-associated disturbances of glucose and lipid metabolism. MicroRNA (miRNA)-dependent post-transcriptional gene silencing has been recognized recently to control gene expression in disease development and progression, including that of insulin-resistant type 2 diabetes. The deregulation of miRNAs miR-143 (ref. 4), miR-181 (ref. 5), and miR-103 and miR-107 (ref. 6) alters hepatic insulin sensitivity. Here we report that the expression of miR-802 is increased in the liver of two obese mouse models and obese human subjects. Inducible transgenic overexpression of miR-802 in mice causes impaired glucose tolerance and attenuates insulin sensitivity, whereas reduction of miR-802 expression improves glucose tolerance and insulin action. We identify Hnf1b (also known as Tcf2) as a target of miR-802-dependent silencing, and show that short hairpin RNA (shRNA)-mediated reduction of Hnf1b in liver causes glucose intolerance, impairs insulin signalling and promotes hepatic gluconeogenesis. In turn, hepatic overexpression of Hnf1b improves insulin sensitivity in Lepr(db/db) mice. Thus, this study defines a critical role for deregulated expression of miR-802 in the development of obesity-associated impairment of glucose metabolism through targeting of Hnf1b, and assigns Hnf1b an unexpected role in the control of hepatic insulin sensitivity.

Heterozygous mutations in the human POU-homeobox TCF2 (vHNF1, HNF1␤) gene are associated with maturity-onset diabetes of the young, type 5, and abnormal urogenital tract development. Recently, pancreas atrophies have been reported in several maturity-onset diabetes of the young type 5 patients, suggesting that TCF2 is required not only for adult pancreas function but also for its normal development. Tcf2-deficient mice die before gastrulation because of defective visceral endoderm formation. To investigate the role of this factor in pancreas development, we rescued this early lethality by tetraploid aggregation. We show that TCF2 has an essential function in the first steps of pancreas development, correlated with its expression domain that demarcates the entire pancreatic buds from the earliest stages. Lack of TCF2 results in pancreas agenesis by embryonic day 13.5. At earlier stages, only a dorsal bud rudiment forms transiently and expresses the transcription factors Ipf1 and Hlxb9 but lacks the key transcription factor involved in the acquisition of a pancreatic fate, Ptf1a, as well as all endocrine precursor cells. Regional specification of the gut also is perturbed in Tcf2 ؊/؊ embryos as manifested by ectopic expression of Shh and lack of Ihh and Ipf1 in the posterior stomach and duodenum. Our results highlight the requirement of Tcf2 for ensuring both accurate expression of key regulator molecules in the stomach-duodenal epithelium and proper acquisition of the pancreatic fate. This study provides further insights into early molecular events controlling pancreas development and may contribute to the development of cell-replacement strategies for diabetes.diabetes MODY5 ͉ homeodomain transcription factor ͉ pancreas development ͉ gut regionalization ͉ tetraploid aggregation I n mammals, the pancreas emerges as ventral and dorsal evaginations from the foregut-midgut junction that subsequently fused to form a complex organ. The signaling molecule Sonic Hedgehog (SHH) demarcates a molecular boundary between the prepancreatic endoderm and adjacent stomach and duodenal anlagen and exerts an inhibitory action on pancreas development (1-3). Genetic studies in mice have identified a hierarchical regulatory network involved in pancreas morphogenesis, with significant and sequential differences between ventral and dorsal pancreas. In the mouse, the dorsal bud appears at embryonic day 9.5 (E9.5) concomitantly with the first differentiated glucagon-producing cells. The homeobox gene Ipf1(Pdx1) is expressed before and during this budding, and all pancreatic cell types derive from IPF1 ϩ progenitors (4, 5). However, in Ipf1-deficient mice, pancreas development is arrested after budding (6, 7), implying that other factors promote pancreas specification. Recently, the transcription factor Ptf1a (P48) has been shown to be essential for the acquisition of a pancreatic fate by undifferentiated ventral foregut endoderm, being required for the specification of the ventral pancreas and robust outgrowth of the dorsal bud. In its a...

During pancreas development, transcription factors play critical roles in exocrine and endocrine differentiation. Transcriptional regulation in eukaryotes occurs within chromatin and is influenced by posttranslational histone modifications (e.g., acetylation) involving histone deacetylases (HDACs). Here, we show that HDAC expression and activity are developmentally regulated in the embryonic rat pancreas. We discovered that pancreatic treatment with different HDAC inhibitors (HDACi) modified the timing and determination of pancreatic cell fate. HDACi modified the exocrine lineage via abolition and enhancement of acinar and ductal differentiation, respectively. Importantly, HDACi treatment promoted the NGN3 proendocrine lineage, leading to an increased pool of endocrine progenitors and modified endocrine subtype lineage choices. Interestingly, treatments with trichostatin A and sodium butyrate, two inhibitors of both class I and class II HDACs, enhanced the pool of ␤ cells. These results highlight the roles of HDACs at key points in exocrine and endocrine differentiation. They show the powerful use of HDACi to switch pancreatic cell determination and amplify specific cellular subtypes, with potential applications in cell replacement therapies in diabetes.

The transmembrane semaphorin protein Sema6A is broadly expressed in the developing nervous system. Sema6A repels several classes of developing axons in vitro and contributes to thalamocortical axon guidance in vivo. Here we show that during cerebellum development, Sema6A is selectively expressed by postmitotic granule cells during their tangential migration in the deep external granule cell layer, but not during their radial migration. In Sema6A-deficient mice, many granule cells remain ectopic in the molecular layer where they differentiate and are contacted by mossy fibers. The analysis of ectopic granule cell morphology in Sema6a-/- mice, and of granule cell migration and neurite outgrowth in cerebellar explants, suggests that Sema6A controls the initiation of granule cell radial migration, probably through a modulation of nuclear and/or soma translocation. Finally, the analysis of mouse chimeras suggests that this function of Sema6A is primarily non-cell-autonomous.

Heterozygous mutations in the HNF1beta/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 HNF1beta, 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 HNF1beta carriers: bilateral enlarged polycystic kidneys, severe pancreas hypoplasia and abnormal genital tract. Consistent with this, we detected high levels of HNF1beta 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 beta-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/beta-catenin signaling pathway. Both fetuses exhibited a severe pancreatic hypoplasia with underdeveloped and disorganized acini, together with an absence of ventral pancreatic-derived tissue. beta-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 beta-cell maturation defect. This study provides evidence of differential gene-dosage requirements for HNF1beta in normal human kidney and pancreas differentiation and increases our understanding of the etiology of MODY5 disorder.

Mouse liver induction occurs via the acquisition of ventral endoderm competence to respond to inductive signals from adjacent mesoderm, followed by hepatic specification. Little is known about the regulatory circuit involved in these processes. Through the analysis of vHnf1 (Hnf1b)-deficient embryos, generated by tetraploid embryo complementation, we demonstrate that lack of vHNF1 leads to defective hepatic bud formation and abnormal gut regionalization. Thickening of the ventral hepatic endoderm and expression of known hepatic genes do not occur. At earlier stages, hepatic specification of vHnf1 -/-ventral endoderm is disrupted. More importantly, mutant ventral endoderm cultured in vitro loses its responsiveness to inductive FGF signals and fails to induce the hepatic-specification genes albumin and transthyretin. Analysis of liver induction in zebrafish indicates a conserved role of vHNF1 in vertebrates. Our results reveal the crucial role of vHNF1 at the earliest steps of liver induction: the acquisition of endoderm competence and the hepatic specification.

OBJECTIVEClass IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development.RESEARCH DESIGN AND METHODSWe took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments.RESULTSWe show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine β- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing β-cells in Hdac5−/− and Hdac9−/− mice and an increased pool of somatostatin-producing δ-cells in Hdac4−/− and Hdac5−/− mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing β-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper β-and δ-cell differentiation and amplifies endocrine β- and δ-cells.CONCLUSIONSWe conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic β/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for β-cell differentiation-based therapies.

Heterozygous mutations in the human HNF1B gene are associated with maturity-onset diabetes of the young type 5 (MODY5) and pancreas hypoplasia. In mouse, Hnf1b heterozygous mutants do not exhibit any phenotype, whereas the homozygous deletion in the entire epiblast leads to pancreas agenesis associated with abnormal gut regionalization. Here, we examine the specific role of Hnf1b during pancreas development, using constitutive and inducible conditional inactivation approaches at key developmental stages. Hnf1b early deletion leads to a reduced pool of pancreatic multipotent progenitor cells (MPCs) due to decreased proliferation and increased apoptosis. Lack of Hnf1b either during the first or the secondary transitions is associated with cystic ducts. Ductal cells exhibit aberrant polarity and decreased expression of several cystic disease genes, some of which we identified as novel Hnf1b targets. Notably, we show that Glis3, a transcription factor involved in duct morphogenesis and endocrine cell development, is downstream Hnf1b. In addition, a loss and abnormal differentiation of acinar cells are observed. Strikingly, inactivation of Hnf1b at different time points results in the absence of Ngn3 + endocrine precursors throughout embryogenesis. We further show that Hnf1b occupies novel Ngn3 putative regulatory sequences in vivo. Thus, Hnf1b plays a crucial role in the regulatory networks that control pancreatic MPC expansion, acinar cell identity, duct morphogenesis and generation of endocrine precursors. Our results uncover an unappreciated requirement of Hnf1b in endocrine cell specification and suggest a mechanistic explanation of diabetes onset in individuals with MODY5.