Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors have been classified into five families. Described in this study is the mammalian prototype of a sixth family, which has therefore been called hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans (Lemaigre, 1996).
Hepatocyte nuclear factor-6 (HNF-6) contains a single cut domain and a homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. Two isoforms of HNF-6 are described that differ by the linker that separates these domains. Both isoforms stimulate transcription. The affinity of HNF-6alpha and HNF-6beta for DNA differs, depending on the target sequence. Binding of HNF-6 to DNA involves the cut domain and the homeodomain, but the latter was not required for binding to a subset of sites. Mutations of the F48M50 dyad that do not affect DNA binding reduce the transcriptional stimulation of constructs that do not require the homeodomain for DNA binding, but these mutations did not affect the stimulation of constructs that do require the homeodomain. Comparative trees of mammalian, Drosophila, and Caenorhabditis elegans proteins show that HNF-6 defines a new class, called ONECUT, of homeodomain proteins. C. elegans proteins of this class bind to HNF-6 DNA targets. Thus, depending on their sequence, these targets determine for HNF-6 at least two modes of DNA binding, which depend on the homeodomain and on the linker that separates it from the cut domain, and two modes of transcriptional stimulation, which depend on the homeodomain (Lannoy, 1998).
Transcription factors of the Onecut class, whose prototype is hepatocyte nuclear factor (HNF)-6, are characterized by the presence of a single cut domain and by a peculiar homeodomain. Human OC-2, the second mammalian member of this class, has been identified and characterized. The OC-2 gene is located on human chromosome 18. The distribution of OC-2 mRNA in humans is tissue-restricted, the strongest expression being detected in the liver and skin. The amino acid sequence of OC-2 contains several regions of high similarity to HNF-6. The recognition properties of OC-2 for binding sites present in regulatory regions of liver-expressed genes differ from, but overlap with, those of HNF-6. Like HNF-6, OC-2 stimulates transcription of the HNF-3beta gene in transient transfection experiments, suggesting that OC-2 participates in the network of transcription factors required for liver differentiation and metabolism (Jacquemin, 1999).
Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a new class of cut homeodomain transcription factors. During mouse development, HNF-6 is expressed in the epithelial cells that are precursors of the exocrine and endocrine pancreatic cells. The role of HNF-6 in pancreas differentiation has been investigated by inactivating its gene in the mouse. In hnf6-/- embryos, the exocrine pancreas appears to be normal but endocrine cell differentiation is impaired. The expression of Neurogenin 3 (Ngn-3), a transcription factor that is essential for determination of endocrine cell precursors, is almost abolished. Consistent with this, HNF-6 binds to and stimulates the ngn3 gene promoter. At birth, only a few endocrine cells are found and the islets of Langerhans are missing. Later, the number of endocrine cells increases and islets appear. However, the architecture of the islets is perturbed, and their beta cells are deficient in glucose transporter 2 expression. Adult hnf6-/- mice are diabetic. Taken together, these data demonstrate that HNF-6 controls pancreatic endocrine differentiation at the precursor stage and HNF-6 is identified as the first positive regulator of the proendocrine gene ngn3 in the pancreas. The data also suggest that HNF-6 is a candidate gene for diabetes mellitus in humans (Jacquemin, 2000).
Transcription factors of the ONECUT class, whose prototype is HNF-6, contain a single cut domain and a divergent homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. The cut domain is required for DNA binding. The homeodomain is required either for DNA binding or for transcriptional stimulation, depending on the target gene. Transcriptional stimulation by the homeodomain involves the F48M50 dyad. How HNF-6 stimulates transcription has been investigated. Transcriptionally active domains of HNF-6 have been identified that are conserved among members of the ONECUT class and it has been shown that the cut domain of HNF-6 participates in DNA binding and, via a LXXLL motif, in transcriptional stimulation. On a target gene to which HNF-6 binds without a requirement for the homeodomain, transcriptional stimulation involves an interaction of HNF-6 with the coactivator CREB-binding protein (CBP). This interaction depends both on the LXXLL motif of the cut domain and on the F48M50 dyad of the homeodomain. On a target gene for which the homeodomain is required for DNA binding, but not for transcriptional stimulation, HNF-6 interacts with the coactivator p300/CBP-associated factor but not with CBP. These data show that a transcription factor can act via different, sequence-specific, mechanisms that combine distinct modes of DNA binding with the use of different coactivators (Lannoy, 2000).
Genes encoding a novel group of homeodomain transcription factors, ONECUT class homeodomain proteins, have been isolated from vertebrates and insects. Among them, vertebrate HNF-6 is expressed in hepatocytes and the central nervous system during embryogenesis. Although the functions of HNF-6 in hepatocytes have been well studied, the functions of HNF-6 in the central nervous system have remained unknown. In this study, HrHNF-6, which encodes a new ONECUT class homeodomain protein, has been isolated from an ascidian, Halocynthia roretzi. HrHNF-6 mRNA was expressed exclusively in neural cells, just posterior to the expression of Hroth (the ascidian homolog of vertebrate Otx) during embryogenesis. One of the functions of HrHNF-6 in neural cells is the activation of the expression of HrTBB2, the ascidian beta-tubulin gene. Another is the restriction of the expression of HrPax-258 (which is expressed in the neural tube), suggesting that HrHNF-6 functions in the specification of the neural tube. These results indicate that HrHNF-6 functions in the differentiation and regional specification of neural cells during ascidian embryogenesis (Sasakura, 2001).
Double-staining in situ hybridization had revealed that HrPax-258 is expressed just next to one of the HrHNF-6-positive zones. Moreover, ectopic expression of HrHNF-6 markedly reduces the expression of HrPax-258 in the neural plate. These results suggest that HrHNF-6 func tions in the specification of the neural tube by restricting the HrPax-258 expression. Because HrHNF-6-EnR is associated with strong activity to repress the HrPax-258 expression in the neural plate and epidermis, HrHNF-6 itself likely represses the transcription of HrPax-258. Since HNF-6 has been reported to be a transcriptional activator, the activity of HrHNF-6 as a repressor is surprising. It is possible that HrHNF-6 uses an unknown cofactor, and the Engrailed repressor domain mimics its function. HrPax-258 was expressed as early as the neural plate stage, in one bilateral pair of cells in the neural plate. The timing of this expression is very close to that of HrHNF-6. HrHNF-6 may regulate HrPax-258 from the beginning of its expression. HrHNF-6 has only a very weak effect on the expression of HrPax-258 in the epidermal lineage cells. This suggests that the expression of HrPax-258 is regulated differently in the neural plate and epidermis, through different cis elements and trans acting factors that bind to the cis elements. HrHNF-6 regulates primarily the expression from the cis element corresponding to the neural plate expression. When the amount of HrHNF-6 mRNA injected is increased or when HrHNF-6-EnR mRNA is injected, HrPax-258 expression in the epidermis is reduced, suggesting that HrHNF-6 also regulates the expression of HrPax-258 in the epidermis in a weak fashion (Sasakura, 2001).
HrPax-258 is thought to resemble the ancestral gene of mammal Pax-2, Pax-5 and Pax-8. Mouse Pax-2 and Pax-5 are expressed at the midbrain-hindbrain boundary (MHB), and function in midbrain formation. Mutation at the Pax-2 locus in mice results in a defect of MHB and subsequent defects in midbrain and cerebellum. In chick, the ectopic expression of Pax-2 or Pax-5 at the mesencephalon induces the ectopic midbrain to form at that position. In zebrafish, a mutation in the pax-b gene affects the formation of MHB. The dominance of Pax-2 in brain regionalization implies that there must be mechanisms that restrict the expression of Pax-2 at the MHB. There have been two reports that mammalian HNF-6 is expressed in the brain. Whether HNF-6 functions in the regulation of Pax-2, -5, -8 transcription in vertebrate brain of interest and should be investigated (Sasakura, 2001).
Ectopic expression of HrHNF-6 does not affect the expression of HrPax-37 or Hroth. These two genes may be regulated through mechanisms independent of HrHNF-6. Both HrPax-37 and Hroth are expressed in cells of neural lineage before HrHNF-6 is expressed; HrPax-37 is expressed in gastrula embryos in six bilateral pairs of cells that are destined to form the dorsal part of the neural tube, and Hroth is expressed in precursors of the anterior neuroectoderm and of mesoendoderm at the 110-cell stage. These results were consistent with HrHNF-6 expression being independent of HrPax-37 and Hroth expression. Elucidating how Hroth and HrHNF-6 are differentially expressed may provide an insight into ascidian neural tissue construction (Sasakura, 2001).
During liver development, hepatoblasts differentiate into hepatocytes or biliary epithelial cells (BEC). The BEC delineate the intrahepatic and extrahepatic bile ducts, and the gallbladder. The transcription factors that control the development of the biliary tract are unknown. Previous work has shown that the onecut transcription factor HNF6 is expressed in hepatoblasts and in the gallbladder primordium. HNF6 is also expressed in the BEC of the developing intrahepatic bile ducts, and its involvement in biliary tract development was investigated by analyzing the phenotype of Hnf6(-/-) mice. In these mice, the gallbladder is absent, the extrahepatic bile ducts are abnormal and the development of the intrahepatic bile ducts is perturbed in the prenatal period. The morphology of the intrahepatic bile ducts is identical to that seen in mice whose Hnf1beta gene has been conditionally inactivated in the liver. HNF1beta expression is downregulated in the intrahepatic bile ducts of Hnf6(-/-) mice during development. Furthermore, HNF6 can stimulate the Hnf1beta promoter. It is concluded that HNF6 is essential for differentiation and morphogenesis of the biliary tract and that intrahepatic bile duct development is controlled by a HNF6-->HNF1beta cascade (Clotman, 2002).
A complete cDNA of a novel zebrafish gene named onecut has been isolated; this gene encodes a protein of 446 amino acids with a Cut domain (73 amino acid residues) and a homeodomain. The Cut domain of zebrafish Onecut is highly similar to those in mammalian hepatocyte nuclear factor-6 and Drosophila Onecut, sharing 90% and 88% amino acid identity, respectively. The expression of zebrafish onecut is restricted to neuronal cells, being first detected in trigeminal ganglia neurons at the end of gastrulation. By the 1-somite stage, onecut expression begins in primary neurons of the lateral stripes in the neural plate, and appears in neuronal cells of the medial stripes at the 2-somite stage. By the 4-somite stage, onecut expression expands to the intermediate stripes and to subsets of neuronal cells in the midbrain and hindbrain. Subsequently, onecut expression intensifies in the lateral region of midbrain and hindbrain, yet no onecut-positive cells are seen in the telencephalon. By 24hpf, onecut transcripts remains abundant in the spinal cord but are no longer detectable in differentiated Rohon-Beard sensory neurons. The expression of onecut is greatly increased in the neural mutant mindbomb, while being decreased in narrowminded (Hong, 2002).
The pancreas derives from cells in the ventral and dorsal foregut endoderm that express the transcription factor Pdx-1. These specified cells give rise to the precursors of the endocrine, ductal, and exocrine pancreatic cells. The identification of transcription factors that regulate the onset of Pdx-1 expression is therefore essential to understand pancreas development. No such factor that acts both in the ventral and in the dorsal endoderm is known. The Onecut transcription factor HNF-6 has been shown to promote differentiation of the endocrine cell precursors in which it stimulates expression of the proendocrine gene Ngn-3. By analyzing the phenotype of HNF-6 null mice, HNF-6 has been found to control an earlier step in pancreas development. Indeed, the pancreas of Hnf6-/- mice is hypoplastic. This does not result from decreased proliferation or from increased apoptosis, but from retarded pancreatic specification of endodermal cells. The onset of Pdx-1 expression is delayed both in the ventral and in the dorsal endoderm, leading to a reduction in the number of endodermal cells expressing Pdx-1 at the time of pancreatic budding. In normal embryos, HNF-6 is detected in the endoderm prior to the expression of Pdx-1. Moreover, HNF-6 can directly stimulate the Pdx1 promoter. These data therefore identify HNF-6 as the first factor known to control Pdx-1 expression both in the ventral and in the dorsal endoderm. It is concluded that HNF-6 controls the timing of pancreas specification and that HNF-6 acts upstream of Pdx-1 in this developmental process. Together with the known role of HNF-6 in pancreatic endocrine cell differentiation, these data point to HNF-6 as a key regulator of pancreas development (Jacquemin, 2003).
Mouse genetic models have helped to identify transcription factors that are expressed by hemopoietic cells and control their differentiation into lymphoid cells. However, little is known on transcription factors that are involved in this process, but are expressed in nonhemopoietic cells of the microenvironment. Inactivation of the gene coding for hepatocyte nuclear factor-6 (HNF-6) in mice has been shown to lead to B lymphopenia in the bone marrow and spleen. This phenotype disappears shortly after birth when fetal B lymphopoiesis is no longer active, pointing to a defect in fetal liver. Indeed, the number of B cells is decreased in this organ as well. An analysis of B cell developmental markers in fetal liver cells showed that B lymphopoiesis is impaired just beyond the pre-pro B cell stage. Hemopoietic cells from hnf6(-/-) fetal liver can reconstitute the lymphoid system when injected into scid mice. Because parenchymal cells, but not hemopoietic cells, express hnf6 in normal liver, it is concluded that HNF-6 controls B lymphopoiesis in fetal liver and that HNF-6 exerts this control indirectly by acting in parenchymal cells. The involvement of genes known to exert such an indirect control in the B cell defect of hnf6(-/-) fetuses, was ruled out by expression analysis, including microarrays, and by in vivo rescue experiments. This work identifies HNF-6 as the first noncell-intrinsic transcription factor known to control B lymphopoiesis specifically in fetal liver (Bouzin, 2003).
The hepatocyte nuclear factor (HNF) 4alpha gene possesses two promoters, proximal P1 and distal P2, whose use results in HNF4alpha1 and HNF4alpha7 transcripts, respectively. Both isoforms are expressed in the embryonic liver, whereas HNF4alpha1 is almost exclusively in the adult liver. A 516-bp fragment, encompassing a DNase I-hypersensitive site associated with P2 activity that is still retained in adult liver, contains functional HNF1 and HNF6 binding sites and confers full promoter activity in transient transfections. A critical role of the Onecut factors in P2 regulation has been demonstrated using site-directed mutagenesis and embryos doubly deficient for HNF6 and OC-2 that show reduced hepatic HNF4alpha7 transcript levels. Transient transgenesis shows that a 4-kb promoter region is sufficient to drive expression of a reporter gene in the stomach, intestine, and pancreas, but not the liver, for which additional activating sequences may be required. Quantitative PCR analysis has revealed that throughout liver development HNF4alpha7 transcripts are lower than those of HNF4alpha1. HNF4alpha1 represses P2 activity in transfection assays and as deduced from an increase in P2-derived transcript levels in recombinant mice in which HNF4alpha1 has been deleted and replaced by HNF4alpha7. It is concluded that although HNF6/OC-2 and perhaps HNF1 activate the P2 promoter in the embryo, increasing HNF4alpha1 expression throughout development causes a switch to essentially exclusive P1 promoter activity in the adult liver (Briancon, 2004).
The Strongylocentrotus purpuratus hnf6 (Sphnf6) gene encodes a new member of the ONECUT family of transcription factors. The expression of hnf6 in the developing embryo is triphasic, and loss-of-function analysis shows that the Hnf6 protein is a transcription factor that has multiple distinct roles in sea urchin development. hnf6 is expressed maternally, and before gastrulation its transcripts are distributed globally. Early in development, its expression is required for the activation of PMC differentiation genes such as sm50, pm27, and msp130, but not for the activation of any known PMC regulatory genes, for example, alx, ets1, pmar1, or tbrain. Micromere transplantation experiments show that the gene is not involved in early micromere signaling. Early hnf6 expression is also required for expression of the mesodermal regulator gatac. The second known role of hnf6 is its participation after gastrulation in the oral ectoderm gene regulatory network (GRN), in which its expression is essential for the maintenance of the state of oral ectoderm specification. The third role is in the neurogenic ciliated band, which is foreshadowed exactly by a trapezoidal band of hnf6 expression at the border of the oral ectoderm and where it continues to be expressed through the end of embryogenesis. Neither oral ectoderm regulatory functions nor ciliated band formation occur normally in the absence of hnf6 expression (Otim, 2004).
During development, the endoderm gives rise to several organs, including the pancreas and liver. This differentiation process requires spatial and temporal regulation of gene expression in the endoderm by a network of tissue-specific transcription factors whose elucidation is far from complete. These factors include the Onecut protein hepatocyte nuclear factor-6 (HNF-6), which controls pancreas and liver development as shown in Hnf6 knock-out embryos. In mammals, HNF-6 has two paralogs, Onecut-2 (OC-2) and OC-3, whose patterns of expression in the adult overlap with that of HNF-6. In the present work, the expression profile was examined of the three Onecut factors in the developing mouse endoderm. HNF-6, OC-2, and OC-3 are expressed sequentially, which defines new steps in endoderm differentiation. By analyzing Hnf6 knock-out embryos it was found that HNF-6 is required for expression of the Oc3 gene in the endoderm. OC-3 colocalizes with HNF-6 in the endoderm and in embryonic pancreas and liver. Based on transfection, chromatin immunoprecipitation, and whole embryo electroporation experiments, HNF-6 has been shown to bind to and stimulate the expression of the Oc3 gene. This study identifies a regulatory cascade between two paralogous transcription factors, sheds new light on the interpretation of the Hnf6 knock-out phenotype, and broadens the transcription factors network operating during development of the endoderm, liver, and pancreas (Pierreux, 2004).
Targeted disruption of the onecut transcription factor, hnf-6, alters mammalian biliary system development. A related zebrafish cDNA expressed in the developing liver has been identified that is a functional ortholog of mammalian hnf-6. Antisense-mediated knockdown of zebrafish hnf-6 perturbs development of the intrahepatic biliary system. Knockdown of zebrafish hnf-6 alters expression of vhnf1 and the zebrafish orthologs of other mammalian genes regulated by hnf-6. Coinjection of mRNA encoding zebrafish vhnf1 rescues the biliary phenotype of hnf-6 morphants. These experiments strongly suggest that hnf-6 and vhnf1 function within an evolutionarily conserved pathway that regulates biliary development. Forced expression of either hnf-6 or vhnf1 also produces biliary phenotypes. Altered bile duct development in both loss- and gain-of-function experiments suggests that zebrafish biliary cells are sensitive to the dosage of hnf-6-mediated gene transcription (Matthews, 2004).
During liver development, hepatocytes and biliary cells differentiate from common progenitors called hepatoblasts. The factors that control hepatoblast fate decision are unknown. A gradient of activin/TGFß signaling controls hepatoblast differentiation. High activin/TGFß signaling is required near the portal vein for differentiation of biliary cells. The Onecut transcription factors HNF-6 and OC-2 inhibit activin/TGFß signaling in the parenchyma, and this allows normal hepatocyte differentiation. In the absence of Onecut factors, the shape of the activin/TGFß gradient is perturbed and the hepatoblasts differentiate into hybrid cells that display characteristics of both hepatocytes and biliary cells. Thus, a gradient of activin/TGFß signaling modulated by Onecut factors is required to segregate the hepatocytic and the biliary lineages (Clotman, 2005 ).
The present work shows that a gradient of activin/ TGFß signaling, shaped by Onecut transcription factors, controls cell lineage decision during liver development. Such a gradient is expected to result from the integration of local concentrations of active ligands, antagonists, and receptors, with the expression levels of the activin/TGFß signaling mediators. The expression pattern of some components of the activin/TGFß pathway has been described in fetal liver, but these data are not sufficient to explain how the gradient is formed. It was found that a perturbation of the TGFß gradient is associated with perturbed expression of tbrII, follistatin, and alpha2-macroglobulin in the single Hnf6–/– or Oc2–/– knockouts and in double Hnf6/Oc2 knockouts. The sum of these defects in each single or double knockout correlates with the intensity of gradient perturbation. This suggests that Onecut factors control the shape of the gradient, at least in part by modulating the expression of tbrII, follistatin, and alpha2-macroglobulin. The induction of the biliary differentiation program in hepatoblasts located at a distance from the portal mesenchyme in the Hnf6/Oc2–/– livers indicates that a direct interaction between hepatoblasts and the portal mesenchyme, which is thought to be mediated by the Notch pathway, is not required to induce biliary differentiation. The Notch pathway may act in parallel to or downstream from the activin/TGFß signaling to support biliary differentiation induced by activin and TGFß, or to repress the hepatocytic differentiation program in biliary cells (Clotman, 2005).
The developmental mechanisms uncovered in this study have implications for the identification of growth factors and transcription factors required to generate differentiated cells for cell therapy of liver diseases. In addition, they may help identify the etiology of human congenital biliary diseases, such as biliary atresia. These diseases are associated with developmental anomalies similar to the ductal plate malformations found in Hnf6–/– or Oc2–/– knockout livers. Therefore, it is proposed that the Onecut factors and TGFß signaling components are new candidates to study the etiopathogeny of congenital biliary diseases (Clotman, 2005).
Somatosensory information from the face is transmitted to the brain by trigeminal sensory neurons. Whether neurons innervating distinct areas of the face possess molecular differences has been an open question. This study identified a set of genes differentially expressed along the dorsoventral axis of the embryonic mouse trigeminal ganglion and thus can be considered trigeminal positional identity markers. Interestingly, establishing some of the spatial patterns requires signals from the developing face. Bone morphogenetic protein 4 (BMP4) was identified as one of these target-derived factors; spatially defined retrograde BMP signaling controls the differential gene expressions in trigeminal neurons through both Smad4-independent and Smad4-dependent pathways. Mice lacking one of the BMP4-regulated transcription factors, Onecut2 (OC2), have defects in the trigeminal central projections representing the whiskers. These results provide molecular evidence for both spatial patterning and retrograde regulation of gene expression in sensory neurons during the development of the somatosensory map (Hodge, 2007).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.