InteractiveFly: GeneBrief

onecut: Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Drosophila onecut homeobox gene encodes a protein product with one cut domain (see Drosophila Cut for more information on cut domain function) and one homeodomain. Onecut can bind to similar DNA sequences with the same high specificity and affinity as Onecut proteins from other species through the highly conserved cut domain and homeodomain. Interestingly, the cut domain alone can mediate DNA-binding, but the homeodomain cannot. However, depending upon the promoter context, cooperative interactions are observed between the two domains to confer high DNA-binding affinity and specificity. Onecut appears to be a moderate transcriptional activator and functions as a nuclear protein in neuronal tissues of both the CNS and PNS during development and in the adult. In the eye, Onecut expression is independent of glass, a transcriptional regulator of R cell differentiation. Taken together, these results suggest a role for Onecut in the regulation of some aspects of neural differentiation or maintenance. In support of this notion, overexpression of a putative dominant negative form of Onecut during eye development does not affect early cell fate specification, but severely affects photoreceptor differentiation (Nguyen, 2000).

A filter DNA-binding screen was carried out to identify transcriptional regulators of rhodopsin (rh) gene expression using a Drosophila adult retina cDNA expression library with defined cis-acting regulatory sequences from the rh promoters as probes. Among the genes identified was a novel gene, termed A1 (Fortini, 1991 and references therein). A blast search of the subsequently isolated full length cDNA encoded sequence revealed two highly conserved regions in the carboxyl half of the conceptual protein: a single cut domain followed by a homeodomain. These motifs characterize a rapidly expanding group of homeodomain proteins known as the Onecut proteins (Lannoy, 1998). The founding member is hepatocyte nuclear factor-6 (HNF-6), which was originally identified among a group of nuclear factors required for liver gene expression (Lemaigre, 1996; Samadani, 1996). No homolog of HNF-6 has yet been identified in the fly, thereby, making Onecut the only known Drosophila Onecut family member (Nguyen, 2000).

The bipartite DNA-binding domains of Onecut and of other cut-homeodomain members illustrate a successful strategy for assembling multiple DNA-binding domains that function independently or cooperatively with the homeodomain to achieve a greater level of specificity or to enhance DNA-binding affinity. The cut domain of Onecut alone can clearly mediate high affinity DNA-binding in a sequence-specific manner. The homeodomain, however, is incapable of DNA-binding by itself. This is also true for the HNF-6 homeodomain as shown by mutant proteins that have either a deleted or mutated homeodomain but are still capable of binding (Lannoy, 1998). Likewise, among the Cut proteins, the homeodomain of mammalian CDP/Cux displays very low DNA-binding affinity and generally binds in a non-specific manner. Therefore, the Onecut homeodomain has no or relatively very low DNA-binding affinity. This may reflect sequence differences in the third DNA-recognition helix of the homeodomain. For example, the Onecut members have the defining F48 and M50 residues that are atypical divergences from the classical homeodomain (Nguyen, 2000).

However, the apparent inability to bind does not rule out a role for the homeodomain in Onecut DNA-binding. It is evident that the cut domain alone is not sufficient for binding, but depends on the presence of the homeodomain for the DNA-binding activity. Changes in DNA-binding affinity are also influenced by the homeodomain. This indicates a significant involvement of cooperative interactions between the cut domain and the homeodomain in determining Onecut DNA-binding activity. A similar case is seen for the POU-class homeodomain proteins. The POU homeodomain alone is insufficient for high affinity DNA-binding, but requires the POU-specific domain for effective interaction with target sites. Analogously, the Paired domain and the Paired homeodomain can function as independent DNA-binding domains, where the latter can operate through dimerization. However, the Paired domain and the homeodomain can also cooperate together to specify DNA-binding activity. Thus, cooperative interaction between bipartite DNA-binding domains appears to be an important mechanism for achieving higher DNA-binding affinity and sequence specificity, and is not exclusive to the cut-homeo domain proteins (Nguyen, 2000).

The indirect role of the homeodomain in Drosophila Onecut binding also points to the possibility that the homeodomain may participate in protein-protein interaction with other transcription factors to effect target specificity and transcriptional activity. One line of evidence in support of this is the observation that mutations in the F48M50 dyad of homeodomain of Onecut homolog HNF-6 do not abolish binding, but affect transcriptional activity in a target-dependent manner (Lannoy, 1998). For target sites that do not utilize the homeodomain for binding, the homeodomain may be involved in promoting transcriptional activation either directly or indirectly by recruiting other factors. In the case of Onecut, its weak transactivation activity may suggest a potential interaction with additional activators, Glass or Eyeless for example, in order to promote high levels of transcriptional activation (Nguyen, 2000).

Ectopic expression of onecut in early eye development appears insufficient to specify photoreceptor cell fate. Although Onecut is expressed in the nervous system throughout development, the developing eye was used as a sensitive assay for examining onecut function. Two independent UAS-onecut transgenic lines were generated that allow overexpression of full-length onecut protein using the Gal4/UAS binary expression system. The GMR-Gal4 driver was used to induce Onecut expression in all cells behind the morphogenetic furrow, and the sev-Gal4 driver was used for expression in a subset of cells that includes the R-cell and the cone cell precursors. If the expression of Onecut were sufficient to specify the photoreceptor cell fate, the formation of extra R cells would be expected in GMR-Gal4/UAS-D-onecut flies or the neural transformation of the cone cells in the sev-Gal4/UAS-onecut flies. However, eye development was normal in both lines examined. This is consistent with the idea that onecut is not involved in establishing R cell identity, but is required for some aspects of neural differentiation that occur subsequent to the specification of the R cell fate (Nguyen, 2000).

Thus, as with Glass, ubiquitous expression or expression of Onecut in the non-neuronal cells, such as cone cell precursors, has no apparent effect on eye development. Intriguingly, genes such as Chaoptin, which are directly regulated by Glass and are normally restricted to the R cells, do not respond to ectopic Glass expression in non-photoreceptors. Thus, Glass is necessary for the differentiation of the R cells but not sufficient to drive neural differentiation in the non-photoreceptor cells. This may indeed be the case for ectopically expressed Onecut. Two possibilities may explain these observations: (1) the R cells may have developed along a different developmental history, as compared to the non-photoreceptor cells, thus endowing them with other factors necessary for the initiation and maintenance of neural differentiation in response to regulatory molecules such as Onecut and Glass; (2) although Glass is expressed in all cells behind the morphogenetic furrow in the eye disc, Glass- dependent transcription is only restricted to the R cells. This suggests the existence of some inhibitory function in the non-photoreceptors. Interestingly, this restriction has been shown to be sufficiently directed by the conserved ATTG motif in the Rhodopsin1 promoter (Rh1PE). When the ATTG is mutated, Glass activity is no longer R cell-specific but is found in all cells. An inhibitor could bind to this ATTG to block Glass activity in the non-photoreceptors. If this inhibitor is ubiquitously expressed, an R cell-specific positive factor must exist to prevent this inhibition of Glass-mediated transcriptional activation. Given its ability to bind to the Rh1PE, its affinity for the ATTG motif of this promoter element, and expression in the photoreceptors, Onecut is a likely candidate for this R-cell specific positive factor (Nguyen, 2000).

Without a loss-of-function mutation in onecut it is difficult to investigate its role in neural development. In an attempt to address this issue, a dominant negative approach was tried, by fusing the DNA-binding domain of Onecut to the repressor domain of Engrailed and expressing the fusion protein under the control of the UAS promoter (UAS-EnCH). This approach has been used successfully to reveal the possible functions of transcriptional activators during development. Such fusion protein is expected to compete with endogenous wild type protein for target genes and to interfere with gene expression by active repression. When expressed in all cells behind the morphogenetic furrow during eye development as driven by GMR-Gal4, the EnCH fusion protein causes a dramatic reduction of the adult eye, giving it a rough, glossy, and flattened appearance. A histological section of the mutant eye reveals an apparent lack of R cells and a remnant of support cells such as pigment cells and cone cells. However, cryosections stained for the neuronal marker Elav, suggest that photoreceptor nuclei are still present, but that the retina does not fully extend as in wild type eyes. Morphologically, the optic lobes are reduced in size (Nguyen, 2000).

To determine when the initial developmental defect occurs, GMR-Gal4/+;UAS-EnCH/+ third instar larval eye discs were stained with an antibody to Elav, an early neuronal cell marker, and with an antibody to Cut, a marker for non-neuronal cone cells. The result shows that the EnCH protein does not affect early cell fate specification events, as pattern formation occurs normally for both R cells and cone cells. Abnormal R cell differentiation must have occurred during later stages of eye development. This observation is remarkably reminiscent of the loss-of-function glass mutants, in which early cell fate determination proceeds normally, but a defect in R cell differentiation leads to the subsequent degeneration of the retina (Nguyen, 2000 and references therein).

To gain insight on how the EnCH fusion protein may affect R cell differentiation, candidate genes that are known to be required for proper R cell differentiation were examined. Unlike glass mutants, the EnCH fusion protein does not affect the expression of Chaoptin, an R cell-specific protein that is under glass regulation. The expression of the homeodomain gene, orthodenticle (otd), also is not affected as determined by the expression of a lacZ-reporter gene under the control of an eye-specific otd enhancer. Surprisingly, a Rh1-promoter lacZ transgene does not respond to overexpression of the EnCH fusion protein and neither does a Rh1PE-lacZ reporter line. The response of a Rh4-lacZ transgene to the EnCH fusion protein was examined. In this case, occasionally only a few cells express the lacZ reporter. It cannot be distinguished whether this is due to an absence of R7 cells or that these cells are more sensitive to the EnCH fusion protein. Nevertheless, these findings are still consistent with the idea that onecut and other regulators such as glass may control different aspects of R cell differentiation by impinging on different target genes that are required during late stages of eye development (Nguyen, 2000).

With a distinct nuclear expression in neuronal cells throughout development and in the adult, Onecut is likely to play a role in regulating neural differentiation or maintenance by controlling neural-specific gene expression. In the eye, one candidate target gene is rhodopsin, as suggested by specific DNA-binding in vitro to two cis-acting elements of Rhodopsin1: RCSI and Rh1PE. Interestingly, the RCSI element is conserved not only in the opsin genes of many different species but also in the promoters of many R cell-specific genes, and is required for their expression in photoreceptors. Therefore, onecut might regulate other R cell-specific genes containing RCSI-like binding sites in addition to rhodopsin for proper R cell differentiation (Nguyen, 2000).

In addition to Onecut, several other transcription factors have been shown to be required for R-specific gene expression. For example, orthodenticle appears to be required for late differentiation of R cells. The loss of otd function causes a reduction in rh gene expression and abnormal morphogenesis of photoreceptor rhabdomeres. In addition to its role in initiating eye development, the Pax-6/eyeless gene has been implicated in regulating late differentiation events during Drosophila eye development by directly regulating target genes such as the Rh1 gene. Therefore, Onecut, Glass, and additional transcription factors may form a cross-regulatory network that coordinates retina-specific gene expression during Drosophila eye development (Nguyen, 2000).

The striking conservation of the cut domain and homeodomain among the Onecut genes suggests that their functions in sequence-specific DNA-binding and transcriptional regulation are conserved during the course of evolution. However, the genes or classes of genes they regulate may be entirely different. For example, the mammalian Onecut proteins are expressed in numerous tissues such as endodermal and neural derivatives (Landry, 1997; Rausa, 1997). In contrast, Onecut has exclusive neuronal expression. Interestingly, similar to Onecut, HNF-6 is also expressed in the retina, which first appears at embryonic stage E17 and implicates a potential role in photoreceptor development (Landry, 1997). The observation that Drosophila Onecut and the mammalian Onecut proteins share common expression in neuronal tissues may point to an ancestral site of action of the Onecut proteins (Nguyen, 2000).

In C. elegans there are five Onecut members (Lannoy, 1998). Interestingly, Ceh-21 and Ceh-39 are capable of binding to HNF-6 binding sites (Lannoy, 1998), which suggests that the C. elegans genes may function similarly to Drosophila Onecut and the mammalian Onecut proteins. The Ceh-38 gene has been reported to be expressed in multiple tissues throughout development like the mammalian genes, with particular expression in endodermal derivatives and in many types of neurons. The C17H12 gene perhaps represents the most ancestral member within this lineage since it is more like Onecut as indicated by sequence homology and by the possession of an STP box. Finally, since all but C17H12 show much greater divergence among the Onecut proteins, it will be interesting to see whether these genes will have overlapping expression patterns, like the HNF-6 and OC- 2, or have restricted expression pattern like onecut (Nguyen, 2000 and references therein).

REGULATION

Transcriptional Regulation

Glass is a zinc-finger transcription factor that is required for the differentiation of all photoreceptor cells. It is expressed in all cells behind the morphogenetic furrow; however, glass-dependent gene transcription is restricted to only the R cells. Since onecut probably participates in late differentiation events and is expressed exclusively in all R cells, it was of interest to investigate if its expression in the developing eye is dependent on glass. Immunostaining of third instar larval eye discs from a glass mutant, with Onecut antibodies reveals that onecut expression is not affected. It is interesting to note that the expression of several other photoreceptor-specific genes that are required for proper differentiation of R cells, for example, Orthodenticle (Otd), a homeodomain protein, and Calphotin, a calcium-channel protein, are also independent of Glass. This observation suggests that glass may regulate only some aspects of neuronal differentiation in the eye. Indeed, in null glass mutants, the expression of some neural-specific antigens such as those recognized by monoclonal antibody 22C10 and anti-HRP antibody is not affected. Thus, onecut is not downstream of glass, but may act in a parallel regulatory pathway in the control of photoreceptor cell differentiation (Nguyen, 2000).

Characterization of DNA-binding properties of Onecut

Electrophoretic mobility gel shift assays (EMSAs) were carried out to address the DNA-binding properties of Onecut. For EMSA, the putative DNA-binding domain of Onecut synthesized and purified as the carboxyl half of the protein (aa 731-1081), which includes the cut domain and homeodomain fused to GST (GST-CHD). Binding was tested to the RCSI elements, a sequence element found in all rhodopsin promoters, from the rh2, rh3, and rh4 genes, whose sequences are similar but not identical (RCSIs from rh1 and rh4 are the same) (Fortini, 1991). All three RCSI probes bind strongly to GST-CHD. Increasing the amount of unlabeled double-stranded oligonucleotides effectively competes and reduces these binding interactions, demonstrating that Onecut specifically recognizes these RCSI promoter elements (Nguyen, 2000).

There are two lines of evidence to suggest that Onecut may have similar DNA-binding properties as Onecut proteins from other species: (1) the amino acid sequences of the cut domain and homeodomain are highly conserved; (2) although much more divergent than the mammalian and Drosophila Onecut proteins, both of the C. elegans proteins, Ceh-39 and Ceh-21, have been shown to bind to mammalian target DNA sequences (Lannoy, 1998). In light of these findings, the ability of Drosophila Onecut to bind to previously described HNF-6 probes (Lannoy, 1998; Lemaigre, 1996; Rausa, 1997; Samadani, 1996) was tested. The sequences are derived from the promoters of the TTR (transthyretin), HNF-3beta, and PFK-2 (6-phosphofructo-2-kinase) genes. GST-CHD fusion protein binds equally well to all three mammalian promoter sequences and can be competed off with the corresponding unlabeled oligonucleotides. As a further demonstration of the specificity of this interaction, a mutant HNF-3beta probe was found to be unable to form any DNA-protein complexes with GST-CHD, nor was it able to compete with the wild type probe in the binding reaction. Thus, in vitro, the cut-homeobox DNA-binding domain of Drosophila Onecut behaves very similar to other Onecut proteins in recognizing the same set of target binding sites (Nguyen, 2000).

This raises an interesting question: is there a common recognition sequence within these cis-acting sequences that confers DNA-binding specificity to Drosophila Onecut and other Onecut proteins? Previous studies on HNF-6 have suggested a consensus sequence from an alignment of all oligonucleotide sequences that bound (Lannoy, 1998; Lemaigre, 1996; Samadani, 1996). An obvious core motif ATTG is shared by both the Drosophila and mammalian sequences used in this study. Two lines of evidence lend support to this finding: (1) oligonucleotide sequences that do not bind to Drosophila Onecut do not contain the tetranucleotide sequence, as is the case for the RUS4A promoter element from the rh4 gene; (2) when changes are made to nucleotides within the ATTG motif, as in the mutant HNF-3beta probe, DNA-binding activity is completely abolished (i.e. mutant probe cannot compete with wild type probe for binding. It is interesting to note that some of the oligonucleotide sequences also contain multiple ATTG sequences and/or the general homeobox recognition sequence ATTA (Nguyen, 2000).

rh gene expression is regulated by multiple cis-acting regulatory elements (Fortini, 1991). Upstream of the Rh1 RCSI sequence lies a proximal enhancer Rh1PE that is bound by Glass, a zinc-finger transcription factor required for rh1 gene expression in photoreceptor cells (R1-6). Within the Rh1PE sequence there is an evolutionarily conserved ATTG tetra-nucleotide immediately downstream of a sequence protected by Glass binding. It was of interest to determine if Onecut binds to this rh1 enhancer element. Onecut (GST-CHD) does indeed bind strongly to the Rh1PE sequence, which includes a single ATTG repeat. This interaction is specific, as demonstrated by competition experiments with an unlabeled ds-oligonucleotide. However, when the ATTG core sequence is mutated (Rh1PE m1) DNA-binding is not completely abolished. This is in contrast to the ATTG mutation in the mutant HNF-3beta probe, which results in a complete loss of binding. This conflicting result suggests that there may be additional sequences required to potentiate the function of the ATTG in the Rh1PE. To substantiate this idea, a mutant probe was generated in the 5' flanking tetranucleotide (Rh1PE m2) and a double mutant probe encompassing the entire eight base pairs (Rh1PE m3). Surprisingly, mutations in the 5' flanking region reduce binding to a greater extent than mutations in the ATTG sequence. The double mutation greatly abolishes DNA-binding activity of both GST-CHD and GST-CD. Thus, the results point to additional flanking sequences within Rh1PE that are required for Onecut binding, and suggest that the sequence context surrounding the ATTG motif render a significant contribution to high DNA-binding affinity and specificity (Nguyen, 2000).

To test if the individual cut domain and homeodomain of Onecut can bind to DNA, fusion proteins GST-CD and GST-HD were used for EMSA analysis. Surprisingly, the homeodomain alone is unable to bind to either the Drosophila Rh1PE element or to the mammalian TTR, HNF-3beta, and PFK-2 binding sites. In contrast, evidence is provided that the cut domain alone can indeed bind to some target sequences with high affinity, such as the Rh1PE element, and the promoter elements from the TTR and HNF-3beta genes. However, the cut domain fails to form a complex with a binding site derived from the PFK-2 promoter. Interestingly, however, the GST-CHD (cut domain and homeodomain together) does bind to this site. This suggests that the DNA-binding activity of the cut domain, in this sequence context, is dependent on the presence of the homeodomain. The addition of the individual domains together in the binding reaction does not promote the formation of any ternary DNA-protein complexes. Taken together, these results imply the existence of cooperative cis-interaction between the homeodomain and the cut domain to effect DNA-binding (Nguyen, 2000).

The role of the homeodomain can be further illustrated by its influence on the behavior of the cut domain interaction with some target sequences. When mutations (m1) are introduced within the ATTG core sequence, the cut domain alone, surprisingly, binds much stronger in comparison to the intact cut-homeodomain fusion protein. The affinities of the cut domain for the TTR and HNF-3beta probes are decreased when compared to binding by GST-CHD, particularly for the HNF-3beta probe. Thus, the presence of the homeodomain clearly regulates some aspects of the cut domain DNA-binding affinity and specificity (Nguyen, 2000).

Thus, an alignment of the oligonucleotide sequences used in electrophoretic mobility gel shift assay (EMSA) analysis reveals an ATTG core motif that is common to all probes that bind to Onecut. This motif is included in a consensus sequence, WTATTGATTW (where W is A/T), previously defined for HNF-6 binding (Lannoy, 1998; Lemaigre, 1996; Samadani, 1996). In some cases, there is a strong requirement for this tetranucleotide since binding is completely abolished when this sequence is mutated. However, in another sequence context there appears to be additional sequence requirement in addition to the ATTG core motif. For example, mutant Rhodopsin1 promoter (RH1PE) probes with an intact ATTG show a dramatic reduction in binding to GST-CHD, but are still highly effective in binding to the cut domain alone. Only when both the flanking sequences and the ATTG core are changed is the DNA-binding abolished. This suggests that in addition to the ATTG core, the flanking sequences in Rh1PE also contribute significantly to the DNA-binding activity of Onecut. Indeed, a mutation in a flanking nucleotide of the ATTG motif has been identified in the HNF-6 binding site within the Type I protein C promoter that reduces HNF-6 binding and abolishes transactivation of a reporter gene in tissue culture (Spek, 1998). Likewise, the cut repeats of the Cut proteins could also bind to other sequences, as well as the major CCAAT binding site (Nguyen, 2000).

The observations regarding binding characteristics of the mutant Rh1PE probes may suggest an interesting possibility that Onecut binding may require an initial docking event after which the cut domain would scan for the ATTG motif. The homeodomain may play a role in regulating these processes and stabilizing the interaction. In this view, mutations in the flanking sequences but not in the ATTG could affect the initial docking of Onecut. Mutations in the ATTG motif but not in the flanking sequences would still allow for docking (Nguyen, 2000).

The presence of two DNA-binding motifs in the Onecut protein and its nuclear localization provide strong evidence that Onecut transcriptionally regulates gene expression. Since the mammalian Onecut proteins, HNF-6 and OC-2, have been shown to be transcriptional activators (Jacquemin, 1999; Lannoy, 1998; Lemaigre, 1996; Rausa, 1997), it was of interest to determine the transcriptional property of Onecut in the Drosophila Schneider S2 cell line by transient transfection assays. A onecut expression vector was constructed under the control of the copia LTR (pDOC-Copia), which provides constitutive expression; also prepared was a luciferase reporter construct driven by a Drosophila minimal hs43 promoter (pLuc-hs43) with or without six tandem copies of the Rh1PE enhancer element (pLuc-6XRh1PE). During the course of the experiment, it was found that the S2 cell line appears to express a low level of endogenous Onecut, since transfection of the pLuc- 6XRh1PE reporter vector alone gives about a 3-fold induction of luciferase activity over the empty pLuc-hs43 vector. Cotransfection of the onecut expression vector pDOC-Copia with either pLuc-hs43 or the pLuc-6XRh1PE reporter vector gives an approximately 2.5-fold or 5-fold induction, respectively, of reporter gene activity over the basal level. This level of induction is comparable to those reported for HNF-6, which can induce a 3-4-fold level of reporter gene activity under the control of the PFK- 2 L promoter in hepatoma FTO-2B cells (Lemaigre, 1996). It is interesting to note that the pDOC-Copia vector carrying the minimal hs43 promoter also generates some induction of luciferase activity. A closer inspection of the hs43 promoter reveals sequences containing the ATTG motif that may favor low affinity binding to Onecut. Nevertheless, under the transfection assay condition used, Onecut appears to function as a moderate transcriptional activator (Nguyen, 2000).

DEVELOPMENTAL BIOLOGY

To gain insight into the developmental role of onecut, its expression pattern was examined to determine where it might function. For this purpose, mouse polyclonal antibodies were raised against two non-overlapping peptide regions at the N-terminus of Onecut. The earliest expression of Onecut protein is detected during early embryogenesis within the central nervous system (CNS) at the time correlated with neuroblast formation (stage 8-9 embryos). In late stage 15 embryos, Onecut is expressed in both the CNS and the peripheral nervous system (PNS). During larval development, onecut is expressed in photoreceptor (R) cells of the third instar imaginal eye disc. An antibody to a neural-specific protein Elav was used to confirm this expression pattern. Double-antibody staining with anti-Elav shows that the onset of Onecut expression follows that of Elav. This suggests that the R cell precursors have already been committed to a neural developmental pathway before Onecut is expressed, which would imply a role for Onecut in subsequent stages of neural cell differentiation and that it does not function in the determination of the R cell fate (Nguyen, 2000).

During pupal development, the expression of Onecut is also restricted to neuronal cells. This includes the 8 R cells of the ommatidial clusters and a single cell that is most likely the neuronal cell of the nerve bristle group, which is shared by three surrounding ommatidial clusters. In the adult head, Onecut is expressed in all differentiated photoreceptors as well as in the nuclei of brain neurons in the lamina, medulla, and many cells of the lobula complex. Interestingly, studies on the Drosophila Eph receptor tyrosine kinase (dek) gene, which lies upstream of onecut, reveal a neural-specific enhancer within a 520 bp EcoRI- HindIII fragment located 300 bp from the start site of onecut and 2.2 kb from the dek start site (Scully, 1999). This enhancer may indeed be responsible for both onecut and dek expression in the nervous system. Similar to prokaryotes, multi-gene regulation by a common regulatory unit does exist in eukaryotes, particularly for genes that are functionally related. In this case, onecut and dek may function in some aspects of neuronal development. Consistent with this idea, genetic analysis of the mammalian Eph receptors points to a role in axon pathfinding, while dek is expressed in growth cones and axons of embryonic interneurons, and in larval R cells (Scully, 1999).

Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression

The mechanism that specifies olfactory sensory neurons to express only one odorant receptor (OR) from a large repertoire is critical for odor discrimination but poorly understood. This study describes the first comprehensive analysis of OR expression regulation in Drosophila. A systematic, RNAi-mediated knock down of most of the predicted transcription factors identified an essential function of acj6, E93, Fer1, onecut, sim, xbp1, and zf30c in the regulation of more than 30 ORs. These regulatory factors are differentially expressed in antennal sensory neuron classes and specifically required for the adult expression of ORs. A systematic analysis reveals not only that combinations of these seven factors are necessary for receptor gene expression but also a prominent role for transcriptional repression in preventing ectopic receptor expression. Such regulation is supported by bioinformatics and OR promoter analyses, which uncovered a common promoter structure with distal repressive and proximal activating regions. Thus, these data provide insight into how combinatorial activation and repression can allow a small number of transcription factors to specify a large repertoire of neuron classes in the olfactory system (Jafari, 2012).

How many OR selector genes are required to uniquely express one OR in each OSN class? Seven OR selector genes were identified, but given the limitations of RNAi, it is likely that there are a total of at least ten critical TFs to specify all OSN classes. Even this probably low estimate generates a rather high number of TFs considering that Drosophila antennae have 34 OSN classes that express ORs. Theoretically the number of TFs needed for a binary combinatorial code to generate 34 unique outcomes is six (2⁶ = 64). Seven TFs can in theory separate 2⁷ = 128 combinations, and ten TFs designate more than 1,000 combinations, suggesting a large number of unused combinations. This surplus of combinations may be due to the inherent randomness of evolution and the impossibility of creating a streamlined code by chance. Another possibility for this large number is the need for a high degree of fidelity, with little or no ectopic OR expression tolerable for proper functioning of the olfactory system. Extrapolation of these observations to the regulatory requirements of the mammalian olfactory system indicates that at least 200-300 TFs would be required to provide a regulatory system that controls >1,000 mammalian ORs, a daunting number. Therefore, it is reasonable to suspect that the stochastic OR selection mechanism found in vertebrates was added during evolution to accommodate the heavy increase in regulatory costs resulting from an expanded number of OR genes (Jafari, 2012).

To date very few TFs have been found to be restricted to small neuronal populations in neuroepithelia or in the developing brain in general. This situation has motivated the suggestion that combinatorial TF regulation defines broad expression patterns of molecules such as neurotransmitters, but is insufficient to generate the large number of neuron classes in, for example, the olfactory system. Similarly, all seven selector genes in this study are expressed across the antenna but still are required for the expression of some few ORs. How can widely expressed TFs then produce restricted expression patterns? Two explanations have been formulated. First, promoter analysis suggests that the OSN class specificity is in part due to repression. Most ORs have a proximal regulatory region next to the gene that is sufficient for expression in OSNs but requires repression from more distal regions for the spatial restriction to each OSN class. In this model, the expression of the TFs that produce OR expression does not need to be particularly specific as long as they are counteracted by repressive factors. Second, the identified TFs can both activate and repress OR expression dependent on the location of the binding site or by the available cofactors. Dual use of the TFs might increase their regulatory power and as a likely consequence the number of TFs required for OR expression to be reduced. It is therefore suggested that specification of large numbers of neuron classes in the olfactory system and likely in the nervous system, require two layers of combinatorial coding, one layer of terminal selector genes that produce expression and a layer of repressors that restrict the expression to each class (Jafari, 2012).

EVOLUTIONARY HOMOLOGS

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).

Within the developing pancreas Hepatic Nuclear Factor 6 (HNF6) directly activates the pro-endocrine transcription factor, Ngn3. HNF6 and Ngn3 are each essential for endocrine differentiation and HNF6 is also required for embryonic duct development. Most HNF6^−/− animals die as neonates, making it difficult to study later aspects of HNF6 function. This study describes, using conditional gene inactivation, that HNF6 has specific functions at different developmental stages in different pancreatic lineages. Loss of HNF6 from Ngn3-expressing cells (HNF6^Δendo) resulted in fewer multipotent progenitor cells entering the endocrine lineage, but had no effect on β cell terminal differentiation. Early, pancreas-wide HNF6 inactivation (HNF6^Δpanc) resulted in endocrine and ductal defects similar to those described for HNF6 global inactivation. However, all HNF6^Δpanc animals survived to adulthood. HNF6^Δpanc pancreata displayed increased ductal cell proliferation and metaplasia, as well as characteristics of pancreatitis, including up-regulation of CTGF, MMP7, and p8/Nupr1. Pancreatitis was most likely caused by defects in ductal primary cilia. In addition, expression of Prox1, a known regulator of pancreas development, was decreased in HNF6^Δpanc pancreata. These data confirm that HNF6 has both early and late functions in the developing pancreas and is essential for maintenance of Ngn3 expression and proper pancreatic duct morphology (Zhang, 2009).

Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification

During development, spinal motoneurons (MNs) diversify into a variety of subtypes that are specifically dedicated to the motor control of particular sets of skeletal muscles or visceral organs. MN diversification depends on the coordinated action of several transcriptional regulators including the LIM-HD factor Isl1, which is crucial for MN survival and fate determination. However, how these regulators cooperate to establish each MN subtype remains poorly understood. Using phenotypic analyses of single or compound mutant mouse embryos combined with gain-of-function experiments in chick embryonic spinal cord, this study demonstrates that the transcriptional activators of the Onecut family critically regulate MN subtype diversification during spinal cord development. Evidence that Onecut factors directly stimulate Isl1 expression in specific MN subtypes and are therefore required to maintain Isl1 production at the time of MN diversification. In the absence of Onecut factors, major alterations are observed in MN fate decision characterized by the conversion of somatic to visceral MNs at the thoracic levels of the spinal cord and of medial to lateral MNs in the motor columns that innervate the limbs. Furthermore, Sip1 (Zeb2) was identified as a novel developmental regulator of visceral MN differentiation. Taken together, these data elucidate a comprehensive model wherein Onecut factors control multiple aspects of MN subtype diversification. They also shed light on the late roles of Isl1 in MN fate decision (Roy, 2012).

REFERENCES

Search PubMed for articles about Drosophila onecut

Bouzin, C., et al. (2003). The onecut transcription factor hepatocyte nuclear factor-6 controls B lymphopoiesis in fetal liver. J. Immunol. 171(3): 1297-303. 12874218

Briancon, N., et al. (2004). Expression of the alpha7 isoform of hepatocyte nuclear factor (HNF) 4 is activated by HNF6/OC-2 and HNF1 and repressed by HNF4alpha1 in the liver. J. Biol. Chem. 279(32): 33398-408. 15159395

Clotman, F., et al. (2005). Control of liver cell fate decision by a gradient of TGFbeta signaling modulated by Onecut transcription factors. Genes Dev. 19: 1849-1854. 16103213

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Hodge, L. K., et al. (2007). Retrograde BMP signaling regulates trigeminal sensory neuron identities and the formation of precise face maps. Neuron 55(4): 572-86. PubMed citation: 17698011

Jacquemin, P., Lannoy, V. J., Rousseau, G. G. and Lemaigre, F. P. (1999). OC-2, a novel mammalian member of the Onecut class of homeodomain transcription factors whose function in liver partially overlaps with that of hepatocyte nuclear factor-6. J. Biol. Chem. 274: 2665-2671. 9915796

Jacquemin, P., et al. (2000). Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3. Mol. Cell. Biol. 20(12): 4445-54. 10825208

Jacquemin, P., Lemaigre, F. P. and Rousseau, G. G. (2003). The Onecut transcription factor HNF-6 (OC-1) is required for timely specification of the pancreas and acts upstream of Pdx-1 in the specification cascade. Dev. Biol. 258: 105-116. 12781686

Jafari, S., et al. (2012). Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression. PLoS Biol. 10(3): e1001280. PubMed Citation: 22427741

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Lannoy, V. J., Rodolosse, A., Pierreux, C. E., Rousseau, G. G. and Lemaigre, F. P. (2000). Transcriptional stimulation by HNF-6: Target-specific recruitment of either CBP or p/CAF. J. Biol. Chem. 275: 22098-22103. 10811635

Lemaigre, F. P., et al. (1996). Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single cut domain. Proc. Natl. Acad. Sci. 93: 9460-9464. 8790352

Matthews, R. P., Lorent, K., Russo, P. and Pack, M. (2004). The zebrafish onecut gene hnf-6 functions in an evolutionarily conserved genetic pathway that regulates vertebrate biliary development. Dev. Biol. 274(2): 245-59. 15385156

Nguyen, D. N. T., Rohrbaugh, M. and Lai, Z.-C. (2000). The Drosophila homolog of Onecut homeodomain proteins is a neural-specific transcriptional activator with a potential role in regulating neural differentiation. Mech. Dev. 97: 57-72. 11025207

Otim, O., Amore, G., Minokawa, T., McClay, D. R. and Davidson, E. H. (2004). SpHnf6, a transcription factor that executes multiple functions in sea urchin embryogenesis. Dev. Biol. 273(2): 226-43. 15328009

Pierreux, C. E., et al. (2004). The transcription factor hepatocyte nuclear factor-6/Onecut-1 controls the expression of its paralog Onecut-3 in developing mouse endoderm. J. Biol. Chem. 279(49): 51298-304. 15381696

Rausa, F., et al. (1997). The cut-homeodomain transcriptional activator HNF-6 is coexpressed with its target gene HNF-3beta in the developing murine liver and pancreas. Dev. Biol. 192: 228-246. 9441664

Roy, A., Francius, C., Rousso, D. L., Seuntjens, E., Debruyn, J., Luxenhofer, G., Huber, A. B., Huylebroeck, D., Novitch, B. G. and Clotman, F. (2012). Onecut transcription factors act upstream of Isl1 to regulate spinal motoneuron diversification. Development 139: 3109-3119. PubMed ID: 22833130

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Sasakura, Y. and Makabe, K. W. (2001). A gene encoding a new ONECUT class homeodomain protein in the ascidian Halocynthia roretzi functions in the differentiation and specification of neural cells in ascidian embryogenesis Mech. Dev. 104: 37-48. 11404078

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Zhang, H., et al. (2009). Multiple, temporal-specific roles for HNF6 in pancreatic endocrine and ductal differentiation. Mech. Dev. 126: 958-973. PubMed Citation: 19766716

date revised: 10 December 2012

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