Silvia Cereghini

Obesity-induced overexpression of miR-802 impairs glucose metabolism through silencing of Hnf1b

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 Leprdb/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.

Crucial role of vHNF1 in vertebrate hepatic specification

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.

HNF1B controls proximal-intermediate nephron segment identity in vertebrates by regulating Notch signalling components and Irx1/2

The nephron is a highly specialised segmented structure that provides essential filtration and resorption renal functions. It arises by formation of a polarised renal vesicle that differentiates into a comma-shaped body and then a regionalised S-shaped body (SSB), with the main prospective segments mapped to discrete domains. The regulatory circuits involved in initial nephron patterning are poorly understood. We report here that HNF1B, a transcription factor known to be involved in ureteric bud branching and initiation of nephrogenesis, has an additional role in segment fate acquisition. Hnf1b conditional inactivation in murine nephron progenitors results in rudimentary nephrons comprising a glomerulus connected to the collecting system by a short tubule displaying distal fates. Renal vesicles develop and polarise normally but fail to progress to correctly patterned SSBs. Major defects are evident at late SSBs, with altered morphology, reduction of a proximo-medial subdomain and increased apoptosis. This is preceded by strong downregulation of the Notch pathway components Lfng, Dll1 and Jag1 and the Irx1/2 factors, which are potential regulators of proximal and Henles loop segment fates. Moreover, HNF1B is recruited to the regulatory sequences of most of these genes. Overexpression of a HNF1B dominant-negative construct in Xenopus embryos causes downregulation specifically of proximal and intermediate pronephric segment markers. These results show that HNF1B is required for the acquisition of a proximo-intermediate segment fate in vertebrates, thus uncovering a previously unappreciated function of a novel SSB subcompartment in global nephron segmentation and further differentiation.

vHNF1 functions in distinct regulatory circuits to control ureteric bud branching and early nephrogenesis

Mouse metanephric kidney development begins with the induction of the ureteric bud (UB) from the caudal portion of the Wolffian duct by metanephric mesenchymal signals. While the UB undergoes branching morphogenesis to generate the entire urinary collecting system and the ureter, factors secreted by the UB tips induce surrounding mesenchymal cells to convert into epithelia and form the nephrons, the functional units of the kidney. Epithelial branching morphogenesis and nephrogenesis are therefore tightly orchestrated; defects in either of these processes lead to severe kidney phenotypes ranging from hypoplasia to complete aplasia. However, the underlying regulatory networks have been only partially elucidated. Here, we identify the transcription factor vHNF1 (HNF1β) as a crucial regulator of these early developmental events. Initially involved in timing outgrowth of the UB and subsequent branching, vHNF1 is also required for nephric duct epithelial maintenance, Müllerian duct formation and early nephrogenesis. Mosaic analyses further suggest a cell-autonomous requirement for vHNF1 in the acquisition of a specialized tip domain and branching morphogenesis. vHNF1 exerts these intricate functions at least in part through the direct control of key regulatory molecules involved in different aspects of early kidney development. Notably, vHNF1 acting directly upstream of Wnt9b appears to orchestrate Wnt signaling action in the mesenchymal-epithelial transitions underlying the initiation of nephrogenesis. These results demonstrate that vHNF1 is an essential transcriptional regulator that, in addition to the known later functions in normal duct morphogenesis, plays a crucial role during the earliest stages of urogenital development and provide novel insights into the regulatory circuits controlling events.

Isolation and characterization of a third isoform of human hepatocyte nuclear factor 4

Hepatocyte nuclear factor 4 (HNF-4) is an essential positive regulator of a large number of liver-specific genes. We report here the isolation of three HNF-4 isoforms from a human liver cDNA library. hHNF-4A and hHNF-4B, differing by the insertion of 10 amino acids in the C-terminal region, have been previously identified in mouse, rat and human liver. The novel isoform, hHNF-4C, is identical to hHNF-4A and B in the regions encompassing the DNA-binding and dimerization domains, but contains a different C-terminal domain. Similar to the other isoforms, hHNF-4C is produced in a limited number of tissues and represents 2.6-13% of the total hHNF-4 mRNA population, depending on the cell type. The chromosomal origin of all three isoforms has been localized to human chromosome 20. hHNF-4C can form heterodimers with hHNF-4A and B in vitro, and exhibits similar transactivation potential as hHNF-4A or B in transient transfection assays, suggesting that the divergent C-terminal region is not part of the transactivation domain.

Hnf1b controls pancreas morphogenesis and the generation of Ngn3+ endocrine progenitors

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. Summary: Mice with conditional depletion of the transcription factor Hnf1b, whose mutation is associated with maturity-onset diabetes of the young in humans, show multiple defects in pancreas development.

Variant Hepatocyte Nuclear Factor 1 expression in the mouse genital tract

Variant Hepatocyte Nuclear Factor 1 (vHNF1/HNF1beta) is a homeodomain-containing transcription factor first expressed in the primitive endoderm and its derivatives, the visceral and parietal endoderm. It is subsequently expressed in epithelial cells of different organs, including the primitive gut and derivatives (liver, pancreas, lung), the kidney, and transiently, in the neural tube. We report here new data concerning vHnf1 expression in the mouse genital tract, using both RNA analyses and our vHnf1 heterozygous mutant mouse line, in which the first coding exon of the vHnf1 gene is replaced by the NLSLacZ reporter gene. Both beta-galactosidase activity and vHnf1 transcripts are detected in epididymus, vas deferens, seminal vesicle, prostate, uterus and oviduct. RNA analysis and in situ hybridization studies demonstrate that vHnf1 transcripts are restricted to the germinal cells of the testis. Unexpectedly, no beta-galactosidase activity is detected in the testis. We further show that, in addition to the somatic transcript, two more abundant vHnf1 transcript variants, which lack exons 1-4, appear in this organ after meiosis.

Krox20 hindbrain cis-regulatory landscape: interplay between multiple long-range initiation and autoregulatory elements.

The vertebrate hindbrain is subject to a transient segmentation process leading to the formation of seven or eight metameric territories termed rhombomeres (r). This segmentation provides the basis for the subsequent establishment of hindbrain neuronal organization and participates in the patterning of the neural crest involved in craniofacial development. The zinc-finger gene Krox20 is expressed in r3 and r5, and encodes a transcription factor that plays a key role in hindbrain segmentation, coordinating segment formation, specification of odd- and even-numbered rhombomeres, and cell segregation between adjacent segments, through the regulation of numerous downstream genes. In order to further elucidate the genetic network underlying hindbrain segmentation, we have undertaken the analysis of the cis-regulatory sequences governing Krox20 expression. We have found that the control of Krox20 transcription relies on three very long-range (200 kb) enhancer elements (A, B and C) that are conserved between chick, mouse and human genomes. Elements B and C are activated at the earliest stage of Krox20 expression in r5 and r3-r5, respectively, and do not require the Krox20 protein. These elements are likely to function as initiators of Krox20 expression. Element B contains a binding site for the transcription factor vHNF1, the mutation of which abolishes its activity, suggesting that vHNF1 is a direct initiator of Krox20 expression in r5. Element A contains Krox20-binding sites, which are required, together with the Krox20 protein, for its activity. This element therefore allows the establishment of a direct positive autoregulatory loop, which takes the relay of the initiator elements and maintains Krox20 expression. Together, our studies provide a basis for a model of the molecular mechanisms controlling Krox20 expression in the developing hindbrain and neural crest.

Functions of HNF1 family members in differentiation of the visceral endoderm cell lineage

The two members of the hepatocyte nuclear factor 1 (HNF1) transcription factor family, HNF1 and variant HNF1 (vHNF1), show a strong homology in their atypical POU-homeodomain and dimerization domain but differ in their transactivation domains. Moreover, two vHNF1 isoforms generated by alternative splicing are present in all tissues expressing this gene. vHnf1-deficient mouse embryos die soon after implantation due to defective visceral endoderm formation, an extraembryonic tissue essential for development and survival of the embryo proper. In contrast, invalidation of Hnf1, which is expressed at later developmental stages than vHnf1, does not lead to embryonic lethality or developmental defects. To examine the specific or potential equivalent functions of vHNF1 isoforms and HNF1 during the process of visceral endoderm differentiation, we stably reexpressed these factors in vHnf1-deficient embryonic stem cells. Analysis of these embryonic stem cells upon differentiation into embryoid bodies shows that vHNF1 isoforms exhibit specific behaviors depending on particular target genes and cooperate in the establishment of a functional visceral endoderm. Furthermore, forced expression of HNF1 in vHnf1-deficient embryonic stem cells fully restores the formation of a mature visceral endoderm with the correct expression profile of early and late markers of this lineage. Thus, in this context, HNF1 functionally replaces both vHNF1 isoforms, suggesting that the different developmental functions of these transcription factors are mainly due to the acquisition of novel expression patterns.

vHnf1 Regulates Specification of Caudal Rhombomere Identity in the Chick Hindbrain

The homeobox‐containing gene variant hepatocyte nuclear factor‐1 (vHnf1) has recently been shown to be involved in zebrafish caudal hindbrain specification, notably in the activation of MafB and Krox20 expression. We have explored this regulatory network in the chick by in ovo electroporation in the neural tube. We show that misexpression of vHnf1 confers caudal identity to more anterior regions of the hindbrain. Ectopic expression of mvHnf1 leads to ectopic activation of MafB and Krox20, and downregulation of Hoxb1 in rhombomere 4. Unexpectedly, mvhnf1 strongly upregulates Fgf3 expression throughout the hindbrain, in both a cell‐autonomous and a non‐cell‐autonomous manner. Blockade of FGF signaling correlates with a selective loss of MafB and Krox20 expression, without affecting the expression of vHnf1, Fgf3, or Hoxb1. Based on these observations, we propose that in chick, as in zebrafish, vHnf1 acts with FGF to promote caudal hindbrain identity by activating MafB and Krox20 expression. However, our data suggest differences in the vHnf1 downstream cascade in different vertebrates. Developmental Dynamics 234:567–576, 2005.