BarH1 and BarH2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene Name - BarH1 and BarH2

Synonyms -

Cytological map position - 15F9/16A1

Function - transcription factors

Keyword(s) - homeotic - neural selector gene

Symbol - B-H1 and B-H2

FlyBase IDs:FBgn0011758 & FBgn0004854

Genetic map position -

Classification - homeodomain

Cellular location - nuclear



BarH1 NCBI links: Precomputed BLAST | Entrez Gene

BarH2 NCBI links: Precomputed BLAST | Entrez Gene | UniGene


BIOLOGICAL OVERVIEW

Recent literature
Li, Q., Barish, S., Okuwa, S., Maciejewski, A., Brandt, A. T., Reinhold, D., Jones, C. D. and Volkan, P. C. (2016). A functionally conserved gene regulatory network module governing olfactory neuron diversity. PLoS Genet 12: e1005780. PubMed ID: 26765103
Summary:
Sensory neuron diversity is required for organisms to decipher complex environmental cues. In Drosophila, the olfactory environment is detected by 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes. Each sensilla subtype houses stereotypically clustered 1-4 ORN identities that arise through asymmetric divisions from a single multipotent sensory organ precursor (SOP). How each class of SOPs acquires a unique differentiation potential that accounts for ORN diversity is unknown. Previously, it was reported that a critical component of SOP diversification program, Rotund (Rn), increases ORN diversity by generating novel developmental trajectories from existing precursors within each independent sensilla type lineages. This study shows that Rn, along with BarH1/H2 (Bar), Bric-a-brac/ (Bab), Apterous (Ap) and Dachshund (Dac), constitutes a transcription factor (TF) network that patterns the developing olfactory tissue. This network was previously shown to pattern the segmentation of the leg, which suggests that this network is functionally conserved. In antennal imaginal discs, precursors with diverse ORN differentiation potentials are selected from concentric rings defined by unique combinations of these TFs along the proximodistal axis of the developing antennal disc. The combinatorial code that demarcates each precursor field is set up by cross-regulatory interactions among different factors within the network. Modifications of this network lead to predictable changes in the diversity of sensilla subtypes and ORN pools. In light of these data, a molecular map is proposed that defines each unique SOP fate. These results highlight the importance of the early prepatterning gene regulatory network as a modulator of SOP and terminally differentiated ORN diversity. Finally, this model illustrates how conserved developmental strategies are used to generate neuronal diversity.

Miller, D. E., Cook, K. R., Yeganeh Kazemi, N., Smith, C. B., Cockrell, A. J., Hawley, R. S. and Bergman, C. M. (2016). Rare recombination events generate sequence diversity among balancer chromosomes in Drosophila melanogaster. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 26903656
Summary:
Multiply inverted balancer chromosomes that suppress exchange with their homologs are an essential part of the Drosophila melanogaster genetic toolkit. Despite their widespread use, the organization of balancer chromosomes has not been characterized at the molecular level, and the degree of sequence variation among copies of balancer chromosomes is unknown. To map inversion breakpoints and study potential diversity in descendants of a structurally identical balancer chromosome, a panel of laboratory stocks containing the most widely used X chromosome balancer, First Multiple 7 (FM7) were sequenced. The locations of FM7 breakpoints were mapped to precise euchromatic coordinates, and the flanking sequence of breakpoints were identified in heterochromatic regions. Analysis of SNP variation revealed megabase-scale blocks of sequence divergence among currently used FM7 stocks. Evidence is presented that this divergence arose through rare double-crossover events that replaced a female-sterile allele of the singed gene (snX2) on FM7c with a sequence from balanced chromosomes. It is proposed that although double-crossover events are rare in individual crosses, many FM7c chromosomes in the Bloomington Drosophila Stock Center have lost snX2 by this mechanism on a historical timescale. Finally, the original allele of the Bar gene (B1) that is carried on FM7 was characterized, and the hypothesis was validated that the origin and subsequent reversion of the B1 duplication are mediated by unequal exchange. These results reject a simple nonrecombining, clonal mode for the laboratory evolution of balancer chromosomes and have implications for how balancer chromosomes should be used in the design and interpretation of genetic experiments in Drosophila.

BarH1 and BarH2 are a pair of homeobox proteins involved in the development of the external sensilli (parts of the peripheral nervous system). These sense organs are responsive to chemical stimulation. BarH1 and BarH2 proteins are also expressed in subsets of neurons in several head segments and the developing brain. In the eye, BarH1/BarH2 proteins are expressed in a subset of photoreceptors.

In the peripheral nervous system BarH1/BarH2 expression is highest in precursors of the chemically sensitive campaniform sensilla and almost absent in precurors of the mechanically sensitive trichord sensilla. Deletion of BarH1/BarH2 in the PNS leads to a homeotic change in these organs with consequent conversion from campaniform-like sensilla to trichord sensilla. Overexpression yields the opposite result, that is, the conversion from chemical to mechanical sensilla. The strongest expression of BarH1/Bar H2 is in the thecogen cells, glial support cells for external sensory neurons. Do these cells participate in the change of fate? Current understanding is not complete. Knockout mutations of BarH2 have been generated. Such flies are perfectly viable. A double mutation in both loci is necessary to affect the transformation of sensory organs described above (Higashijima, 1992b).

Regulation of expression is likely to be complex. Since cut is vital to the develoment of the peripheral nervous system, a question arises: are the Bar genes downstream of cut in external sense organs? They are not under cut control in the brain or eye, since cut is not expressed there. Fuller understanding of basic BarH1/BarH2 expression will provide insight into the process that determines alternative fates in neurons.

The Bar homeobox genes also function as latitudinal prepattern genes in the developing Drosophila notum. In Drosophila notum, the expression of achaete-scute proneural genes and bristle formation have been shown to be regulated by putative prepattern genes expressed longitudinally. The two Bar locus genes may belong to a different class of prepattern genes expressed latitudinally: it is suggested that the developing notum consists of subdomans patterned like checkerboard squares, each subdomain governed by a different combination of prepattern genes. BarH1 and BarH2 are coexpressed in the anterior-most notal region and regulate the formation of microchaetae within the region of BarH1/BarH2 expression through activation of achaete-scute. Presutural macrochaetae formation also requires Bar gene activity. Bar gene expression is restricted in dorsal and posterior regions by Decapentaplegic signaling, while the ventral limit of the expression domain of Bar genes is determined by wingless, whose expression is under the control of Decapentaplegic signaling (M. Sato, 1999).

The Drosophila notum is considered genetically divided into several longitudinal, side by side, domains whose boundaries are determined by pannier, wingless and iroquois expression (listed respectively from medial to lateral). To further clarify relative locations of pnr, wg and iro expression areas, third-instar larval and pupal future notum were stained with various combinations of molecular markers. In larval and pupal future notum, pnr-Gal4 is expressed medially and iro-lacZ laterally. pnr-Gal4 and iro-lacZ domains partially overlap one another, and wg-lacZ (or Wg) expression is noted in the pnr-iro overlapping region and its immediate neighbors. Bar homeobox genes may belong to an additional class of notal subdivision genes. Staining for BarH1 indicates that BarH1 is expressed latitudinally (anterior vs. posterior) in the anterior-most region of future notum and postnotum. BarH1 expression begins at early to mid third instar. Anti-Ac antibody staining and neur-lacZ expression indicates PS macrochaetae are situated in the vicinity of posterior-ventral corners of the anterior BarH1 expression domain. BarH1 and BarH2 are referred to as Bar collectively and the anterior portion of the prescutum or its precursor expressing Bar is referred to as Bar prescutum. The Bar expression domain overlaps that of pnr, wg and iro. Bar expression similar to that in wing discs is observed in haltere discs (M. Sato, 1999).

For clarification of Bar's possible roles in notal development, FLP/FRT-mediated mosaic analysis was undertaken. Few microchaetae are generated in mutant clones within the prescutum. PS macrochaetae formation takes place only when PS macrochaetae formation sites are not included within mjtant clones. In flies hemizygous or homozygous for a deficency in the chromosomal region of Bar, the expression of BarH1 in the medial Bar prescutum is totally absent, implying that this particular deficiency uncovers a Bar enhancer specific to the medial Bar prescutum. In these deficiency flies, microchaetae are lost medially in the anterior three quarters of the prescutum. PS macrochaetae are lost 60% of the time. Loss of BarH1 expression and the microchaetae-less phenotype in the medial Bar prescutum are also detected in another deficiency, uncovering only BarH2 and its 3' Bar enhancer sequences. In these deficiency lines, there is no PS macrochaetae formation. Taken together, these results suggest that Bar genes are essential for bristle formation in the Bar prescutum. Should Bar homeobox genes be redundant in function and involved in bristle formation in the Bar prescutum, bristle defects would certainly be rescued by Bar targeted expression. These considerations were confirmed through the use of the GAL4/UAS system. Loss of microchaetae but not PS macrochaetae is virtually restored by the targeted expression of Bar genes. BarH1 and BarH2 would appear functionally redundant to each other and essential for microchaetae formation in the medial Bar prescutum and PS macrochaetae formation. While searching for Bar enhancers, two notum enhancers have been identified. S8 is found responsible for Bar expression in the medial Bar prescutum. A second enhancer, B4.5, is capable of driving reporter gene (lacZ) expression in the lateral Bar prescutum in wild-type background (M. Sato, 1999).

To assess the capability of Bar for inducing microchaetae formation, hs-BarH1 or hs-BarH2 transgenes were heat-induced during larval or pupal development and the same results were obtained for each. Ectopic microchaetae are generated in the scutellum, wing blades and head capsule. In the scutellum (normally possessing no microchaetae) 30- 50 ectopic microchaetae are generated by a heat-shock at 6 hours APF (after puparium formation), but few ectopic microchaetae are formed by heat shock before 2 hours APF or after 12 hours APF. In contrast to ectopic microchaetae formation in neurogenic mutations, extra microchaetae induced by hs-Bar are unclustered, suggesting that Bar acts as an activator of proneural genes. Staining for Ac shows that pupal notum-specific Ac expression begins in characteristic regions at 6 hours APF, peaking at 8 hours APF and eventually disappearing at 12 hours APF. These Ac-positive regions appear to correspond to microchaetae proneural regions since microchaetae SOP formation starts at 8 hours APF. Macrochaetae SOP are formed during third instar. In the Bar prescutum, the area of Ac expression is seen to overlap that of Bar. Sc expression is quite similar, if not identical, to Ac expression. Thus, a study was made to find whether Bar is capable of acting as an activator of ac-sc to control microchaetae formation. Ac expression is almost entirely absent from the Bar enhancer deficency medial Bar prescutum, where no microchaetae formation takes place. Following induction of hs-Bar, not only ectopic microchaetae formation but strong Ac (and Sc) expression is apparent in the scutellum. hs-Bar dependent microchaetae formation is significantly suppressed in ac-sc hypomorphic mutant backgrounds, while bristle defects including PS macrochaetae loss in Bar enhance deficiency are eliminated by a gain-of-function allele of ac-sc. Bar may thus be considered to be an activator of ac-sc essential for producing proneural clusters for microchaetae and possibly PS macrochaetae as well (M. Sato, 1999).

During late third instar, the expression domain of Bar in the prescutum is immediately adjacent to dpp and wg expression domains. dpp likely regulates Bar expression negatively. When dpp is expressed throughout the notum using UAS-dpp driven by ap-Gal4, Bar expression is totally abolished. Conversely, reduction in dpp activity ectopically induces Bar expression in the medial region of the future notum. Similar medial expansion of Bar expression is observed subsequent to reduction in the activity of hedgehog (hh), an inducer of dpp. Possible effects of wg on Bar expression were sought using wgts, in which Wg secretion but not production is temperature-sensitive. Bar expression in the lateral Bar prescutum is abolished after 48 hours (but not 24 hours) incubation at the restrictive temperature. armadillo (arm), a beta-catenin homolog, is a signal transducer of Wg signaling. Bar expression is lost in clones mutant for arm when generated in the lateral Bar prescutum, while Bar misexpression is present in lateral prescutum clones expressing a constitutively active form of arm. It is thus concluded that Bar expression in lateral prescutum is requires Wg signals, whose levels determine the ventral border of the Bar prescutum. During late third instar, the expression domain of Bar in the prescutum overlaps with those of pnr and iro. Since no appreciable change in Bar expression is detected in flies mutant for iro or pnr, Bar expression may be regulated independent of pnr and iro. Bar may be partially involved in wg repression (M. Sato, 1999).

It is concluded that a checker-board-like subdivision of future notum is regulated by putative prepattern gene expression. Future notum may be divided into square subdomains in a checker-board-like manner, each with its own unique combinations of prepattern gene expression. Putative prepattern genes, iro and pnr, form longitudinal domains. Bar homeobox genes form the anterior-most domain. This is the first demonstration of the presence of latitudinal, front to back, prepattern genes in the notum. Bristle formation in each subdomain may be positively regulated by a region-specific combination of prepattern genes. Consistent with this, microchaetae formation in the anterolateral prescutum (the lateral Bar prescutum), where Bar and iro are coexpressed, requires the concerted action of Bar and iro (M. Sato, 1999).

Bar homeodomain proteins are anti-proneural in the Drosophila eye: transcriptional repression of atonal by Bar prevents ectopic retinal neurogenesis

Atonal (Ato)/Math (Mammalian atonal homolog) family proneural proteins are key regulators of neurogenesis in both vertebrates and invertebrates. In the Drosophila eye, Ato is essential for the generation of photoreceptor neurons. Ato expression is initiated at the anterior ridge of the morphogenetic furrow but is repressed in the retinal precursor cells behind the furrow to prevent ectopic neurogenesis. Ato repression is mediated by the conserved homeobox proteins BarH1 and BarH2. Loss of Bar causes cell-autonomous ectopic Ato expression, resulting in excess photoreceptor clusters. The initial ommatidial spacing at the furrow occurs normally in the absence of Bar, suggesting that the ectopic neurogenesis within Bar mutant clones is not due to the lack of Notch (N)-dependent lateral inhibition. Targeted misexpression of Bar is sufficient to repress ato expression. Furthermore, evidence is provided that Bar represses ato expression at the level of transcription without affecting the expression of an ato activator, Cubitus interruptus (Ci). Thus, it is proposed that Bar is essential for transcriptional repression of ato and the prevention of ectopic neurogenesis behind the furrow (Lim, 2003).

Each ommatidium of the adult compound eye consists of eight photoreceptors that are generated by the proneural function of Ato expressed within and anterior to the furrow in the eye disc. The domain of Ato expression is juxtaposed to the Bar-expressing undifferentiated cells behind the furrow. Although Bar is also expressed in R1 and R6 photoreceptors, this study focuses specifically on the Bar expression in the undifferentiated cells and the Ato expression in adjacent anterior cells. These Bar-expressing undifferentiated cells will be referred as the 'basal cells' since their nuclei stay in the basal region while photoreceptor cell nuclei migrate apically, although cell bodies of both cell types are connected to the top and bottom of the eye disc epithelium. Nuclei of Ato-expressing cells are located basally during the stages 1 and 2, but migrate apically as they become R8 founder neurons posterior to the furrow (Lim, 2003).

Thus Ato expression is highly elevated in the absence of Bar behind the furrow, suggesting that Bar is necessary for downregulation of Ato expression. Furthermore, Bar represses ato expression at the transcriptional level through both 3'- and 5'-regulatory regions of ato. The 9.3 kb of ato 5' sequence (5'F:9.3) has been shown to be responsible for ato expression in the equivalence groups and the R8 founder cells in an Ato-dependent manner (stages 2-4). Sca and Egfr-mediated MAP kinase signaling may inhibit this enhancer function of 5'-regulatory element of ato within interommatidial regions to establish regularly spaced intermediate groups. By contrast, the 5.8 kb of ato 3' enhancer (3'F:5.8) is only activated anterior to the furrow to drive the initial stripe of ato expression (stage 1). How this enhancer activity is inhibited posterior to the stage 1 Ato domain of the eye disc is unknown. The results now indicate that the initial stripe (stage 1) of ato expression driven by 3'-regulatory element is strongly inhibited by Bar behind the furrow (Lim, 2003).

Ectopically elevated Ato expression within Bar loss-of-function clones is sufficient to induce the formation of mature ectopic photoreceptor clusters. This suggests that Bar mutations specifically eliminate the repression of initial ato expression with little effects on subsequent steps of photoreceptor recruitment. This is consistent with the observations that Sca, Egfr signaling and N-mediated lateral inhibitions function properly within Bar loss-of-function clones. Therefore, the major role of Bar during retinal neurogenesis appears to be the inhibition of initial stripe ato expression through 3'-regulatory elements of ato behind the furrow (Lim, 2003).

Bar is a DNA-binding homeodomain transcription factor. Mammalian homolog Barx2 was shown to bind directly to regulatory elements of several neural cell adhesion molecules, which contains target sites including the core sequence CCATTAGPyGA. Interestingly, the 5'F9.3 and 3'F5.8 regulatory regions of ato also have multiple potential Bar binding sites containing the same core sequence, suggesting that Bar may directly bind to these target sites of ato regulatory elements and repress ato transcription (Lim, 2003).

It is important to note that CiFL induced by Hh signaling can activate ato expression. Furthermore, Bar and CiFL are expressed complementarily to each other. These observations raise the possibility that ato repression by Bar may be mediated by Bar repression of CiFL. However, the results indicate that Bar function is independent of CiFL, supporting the idea that the primary cause of ato repression behind the furrow is a direct function of Bar as a repressor rather than indirect effects of the removal of the activator, CiFL. Furthermore, overexpression of CiFL by the lz-Gal4 driver in the presence of Bar does not activate ato expression, indicating that Bar-mediated ato repression is epistatic to an overexpression of CiFL activator (Lim, 2003).

Based on the findings, a model of Bar function in retinal neurogenesis is proposed. Ato is expressed within the furrow and is required for the generation of R8 founder neurons. Bar homeodomain proteins are expressed in the basal cells behind the furrow and represses ato expression, showing a complementary expression pattern to Ato. This function of Bar on ato-repression occurs independent of CiFL, a transcriptional activator of ato. Rather, Bar may directly repress ato transcription by binding to 3'- and 5'-regulatory regions of ato through its potential binding sites (Lim, 2003).

The finding that Bar inhibits the expression of the proneural gene ato as a transcriptional repressor in the eye disc raises the interesting issue of whether Bar can also repress the expression of other proneural genes in the eye or other tissues. Misexpression of BarH1 or BarH2 using a dpp-Gal4 driver shows increased expression of proneural gene scute in the eye and wing discs rather than repressing its expression, generating more sensory bristles. By contrast, the deficiency in Bar causes the loss of sensory interommatidial bristles in the eye. These results suggest that Bar can act as an activator for the expression of ASC proneural genes to generate bristle sensory neurons. Therefore, Bar can act as transcriptional activator as well as repressor for different proneural genes depending on developmental contexts in Drosophila. This dual function of Bar was also observed in mammalian Barx2. Barx2 has activator and repressor domains in the C- or N-terminal regions, respectively. Mbh1, another mammalian homolog of the Bar class genes, functions as either activator or repressor for the expression of neural bHLH genes in cell culture system. Therefore, the dual function of transcriptional activation and repression may be a general property of Bar family homeodomain proteins in the control of expression of neural target genes. These opposite actions of Bar may be dependent on the binding of their specific partners to the activator or repressor domain (Lim, 2003).

It has been shown that loss of groucho (gro) results in increased ato expression behind the furrow of the eye disc. Gro represses the expression of proneural genes during N-mediated lateral inhibition. It is interesting to note that Bar family homeodomain proteins have a conserved ~10 amino acid motif termed the FIL domain at the N-terminal region of the homeobox. This domain shows sequence similarity to the core region of the engrailed homology-1 (eh1) domain in Engrailed (En) repressor, which can directly interact with Gro co-repressor through its eh1 motif. Therefore, Bar may interact with Gro through its FIL domain for its repressor function (Lim, 2003).

Bar class homeodomain proteins are evolutionarily highly conserved from Drosophila to human. Vertebrate Bar homologs include Xenopus XBH1 and XBH2, mouse and human Barhl1 and Barhl2, rat Mbh1 [same gene as Barhl2], and murine and human Barx1 and Barx2 genes. Although in vivo function of Bar homologs has not been extensively analyzed, some members of the Bar class homeobox genes may be involved in the genesis and fate specification of neuronal cells. A mammalian homolog, Mbh1, is expressed in a complementary pattern to Mash1, a homolog of ASC, in the rat eye. Hence, Mbh1 may be involved in inhibition of Mash1 expression, similar to the ato repression by Drosophila Bar proteins (Lim, 2003).

In vertebrate eye development, a mammalian homolog of ato, Math5 (and/or Xath5), is crucial for the generation of retinal ganglion cells, which are the first neurons to arise and therefore may be analogous to the R8 founder cells in the Drosophila eye. The essential role of Math5 in the genesis of ganglion cells suggests that Math5 plays Ato-like proneural function in vertebrate eye development. It will be interesting to see whether a specific Bar homolog(s) may be involved in the repression of Math5, since the Drosophila Bar inhibits ato expression. In addition, Bar class genes are attractive candidates for many human genetic disorders, including Joubert syndrome and Rieger syndrome. The new function of Drosophila Bar in negative regulation of neurogenesis may provide insights into the function of Bar family genes in vertebrates and the molecular basis of human diseases associated with altered Bar function (Lim, 2003).


GENE STRUCTURE

BarH1

cDNA clone length - 3.1 kb

Bases in 5' UTR - 633

Exons - three

Bases in 3' UTR - 767


BarH2

cDNA clone length - 3.75 Kb

Bases in 5' UTR - 968

Exons - three

Bases in 3' UTR - 783


PROTEIN STRUCTURE

Amino Acids - 543 BarH1; 604 BarH2

Structural Domains

Except for one amino acid substitution, BarH1 and BarH2 homeodomains are identical in sequence. The phenylalanine residue in helix 3, conserved in all metazoan homeodomains examined thus far, is replaced by a tyrosine residue (Kojima, 1991).


BarH1 and BarH2 : Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 3 April 99

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