Ultrabithorax: Biological Overview | Evolutionary Homologs | Transcriptional Regulation | Targets of activity | Protein Interactions | Posttranscriptional regulation | Developmental Biology | Effects of Mutation | References

Gene name - Ultrabithorax

Synonyms - bithorax (bx)

Cytological map position - 89E1-3

Function - transcription factor

Keywords - bithorax complex

Symbol - Ubx

FlyBase ID:FBgn0003944

Genetic map position - 3-58.8

Classification - homeodomain - Antp class

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Beh, C. Y., El-Sharnouby, S., Chatzipli, A., Russell, S., Choo, S. W. and White, R. (2016). Roles of cofactors and chromatin accessibility in Hox protein target specificity. Epigenetics Chromatin 9: 1. PubMed ID: 26753000
Summary:
The regulation of specific target genes by transcription factors is central to understanding of gene network control in developmental and physiological processes, yet how target specificity is achieved is still poorly understood. This is well illustrated by the Hox family of transcription factors as their limited in vitro DNA-binding specificity contrasts with their clear in vivo functional specificity. This study generated genome-wide binding profiles for three Hox proteins, Ubx, Abd-A and Abd-B, following transient expression in Drosophila Kc167 cells, revealing clear target specificity and a striking influence of chromatin accessibility. In the absence of the TALE class homeodomain cofactors Exd and Hth, Ubx and Abd-A bind at a very similar set of target sites in accessible chromatin, whereas Abd-B binds at an additional specific set of targets. Provision of Hox cofactors Exd and Hth considerably modifies the Ubx genome-wide binding profile enabling Ubx to bind at an additional novel set of targets. Both the Abd-B specific targets and the cofactor-dependent Ubx targets are in chromatin that is relatively DNase1 inaccessible prior to the expression of Hox proteins/Hox cofactors. It is concluded that there is a strong role for chromatin accessibility in Hox protein binding, and the results suggest that Hox protein competition with nucleosomes has a major role in Hox protein target specificity in vivo.
Prasad, N., Tarikere, S., Khanale, D., Habib, F. and Shashidhara, L. S. (2016). A comparative genomic analysis of targets of Hox protein Ultrabithorax amongst distant insect species. Sci Rep 6: 27885. PubMed ID: 27296678
Summary:
In the fruitfly Drosophila melanogaster, the differential development of wing and haltere is dependent on the function of the Hox protein Ultrabithorax (Ubx). This study compare Ubx-mediated regulation of wing patterning genes between the honeybee, Apis mellifera, the silkmoth, Bombyx mori and Drosophila. Orthologues of Ubx are expressed in the third thoracic segment of Apis and Bombyx, although they make functional hindwings. When over-expressed in transgenic Drosophila, Ubx derived from Apis or Bombyx could suppress wing development, suggesting evolutionary changes at the level of co-factors and/or targets of Ubx. To gain further insights into such events, direct targets of Ubx from Apis and Bombyx were identified by ChIP-seq and compared with those of Drosophila. While majority of the putative targets of Ubx are species-specific, a considerable number of wing-patterning genes are retained, over the past 300 millions years, as targets in all the three species. Interestingly, many of these are differentially expressed only between wing and haltere in Drosophila but not between forewing and hindwing in Apis or Bombyx. Detailed bioinformatics and experimental validation of enhancer sequences suggest that, perhaps along with other factors, changes in the cis-regulatory sequences of earlier targets contribute to diversity in Ubx function.
Li, H., Qi, Y. and Jasper, H. (2016). Ubx dynamically regulates Dpp signaling by repressing Dad expression during copper cell regeneration in the adult Drosophila midgut. Dev Biol. PubMed ID: 27570230
Summary:
The gastrointestinal (GI) tract of metazoans is lined by a series of regionally distinct epithelia. To maintain structure and function of the GI tract, regionally diversified differentiation of somatic stem cell (SC) lineages is critical. The adult Drosophila midgut provides an accessible model to study SC regulation and specification in a regionally defined manner. SCs of the posterior midgut (PM) have been studied extensively, but the control of SCs in the middle midgut (MM) is less well understood. The MM contains a stomach-like copper cell region (CCR) that is regenerated by gastric stem cells (GSSCs) and contains acid-secreting copper cells (CCs). Bmp-like Decapentaplegic (Dpp) signaling determines the identity of GSSCs, and is required for CC regeneration, yet the precise control of Dpp signaling activity in this lineage remains to be fully established. This study shows that Dad, a negative feedback regulator of Dpp signaling, is dynamically regulated in the GSSC lineage to allow CC differentiation. Dad is highly expressed in GSSCs and their first daughter cells, the gastroblasts (GBs), but has to be repressed in differentiating CCs to allow Dpp-mediated differentiation into CCs. WThe Hox gene Ultrabithorax (Ubx) is required for this regulation. Loss of Ubx prevents Dad repression in the CCR, resulting in defective CC regeneration. This study highlights the need for dynamic control of Dpp signaling activity in the differentiation of the GSSC lineage and identifies Ubx as a critical regulator of this process.
Hessinger, C., Technau, G.M. and Rogulja-Ortmann, A. (2016). The Drosophila Hox gene Ultrabithorax acts both in muscles and motoneurons to orchestrate formation of specific neuromuscular connections. Development [Epub ahead of print]. PubMed ID: 27913640
Summary:
Hox genes are known to specify motoneuron pools in the developing vertebrate spinal cord and to control motoneuronal targeting in several species. However, the mechanisms controlling axial diversification of muscle innervation patterns are still largely unknown. This study presents data showing that the Drosophila Hox gene Ultrabithorax (Ubx) acts in the late embryo to establish target specificity of ventrally projecting RP motoneurons. In abdominal segments A2 to A7, RP motoneurons innervate the ventro-lateral muscles VL1-4, with VL1 and VL2 being innervated in a Wnt4-dependent manner. In Ubx mutants, these motoneurons fail to make correct contacts with muscle VL1, a phenotype partially resembling that of the Wnt4 mutant. Ubx regulates expression of Wnt4 in muscle VL2 and interacts with the Wnt4 response pathway in the respective motoneurons. Ubx thus orchestrates the interaction between two cell types, muscles and motoneurons, to regulate establishment of the ventro-lateral neuromuscular network.

Gupta, R. P., Bajpai, A. and Sinha, P. (2017). Selector genes display tumor cooperation and inhibition in Drosophila epithelium in a developmental context-dependent manner. Biol Open 6(11): 1581-1591. PubMed ID: 29141951
Summary:
During animal development, selector genes determine identities of body segments and those of individual organs. Selector genes are also misexpressed in cancers, although their contributions to tumor progression per se remain poorly understood. Using a model of cooperative tumorigenesis, this study shows that gain of selector genes results in tumor cooperation, but in only select developmental domains of the wing, haltere and eye-antennal imaginal discs of Drosophila larva. Thus, the field selector, Eyeless (Ey), and the segment selector, Ultrabithorax (Ubx), readily cooperate to bring about neoplastic transformation of cells displaying somatic loss of the tumor suppressor, Lgl, but in only those developmental domains that express the homeo-box protein, Homothorax (Hth), and/or the Zinc-finger protein, Teashirt (Tsh). In non-Hth/Tsh-expressing domains of these imaginal discs, however, gain of Ey in lgl- somatic clones induces neoplastic transformation in the distal wing disc and haltere, but not in the eye imaginal disc. Likewise, gain of Ubx in lgl- somatic clones induces transformation in the eye imaginal disc but not in its endogenous domain, namely, the haltere imaginal disc. These results reveal that selector genes could behave as tumor drivers or inhibitors depending on the tissue contexts of their gains.
Tsai, A., Muthusamy, A. K., Alves, M. R., Lavis, L. D., Singer, R. H., Stern, D. L. and Crocker, J. (2017). Nuclear microenvironments modulate transcription from low-affinity enhancers. Elife 6. PubMed ID: 29095143
Summary:
Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. This study reports that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation.
Gabilondo, H., Rubio-Ferrera, I., Losada-Perez, M., Del Saz, D., Leon, Y., Molina, I., Torroja, L., D, W. A. and Benito-Sipos, J. (2018). Segmentally homologous neurons acquire two different terminal neuropeptidergic fates in the Drosophila nervous system. PLoS One 13(4): e0194281. PubMed ID: 29634720
Summary:
This study identified the means by which segmentally homologous neurons acquire different neuropeptide fates in Drosophila. Ventral abdominal (Va)-neurons in the A1 segment of the ventral nerve cord express DH31 and AstA neuropeptides (neuropeptidergic fate I) by virtue of Ubx activity, whereas the A2-A4 Va-neurons express the Capa neuropeptide (neuropeptidergic fate II) under the influence of abdA. These different fates are attained through segment-specific programs of neural subtype specification undergone by segmentally homologous neurons. This is an attractive alternative by which Hox genes can shape Drosophila segmental neural architecture (more sophisticated than the previously identified binary "to live" or "not to live" mechanism). These data refine knowledge of the mechanisms involved in diversifying neuronal identity within the central nervous system.
ADe Las Heras, J. M., Garcia-Cortes, C., Foronda, D., Pastor-Pareja, J. C., Shashidhara, L. S. and Sanchez-Herrero, E. (2018)c. The Drosophila Hox gene Ultrabithorax controls appendage shape by regulating extracellular matrix dynamics. Development. PubMed ID: 29853618
Summary:
Although the specific form of an organ is frequently important for its function, the mechanisms underlying organ shape are largely unknown. In Drosophila, the wings and halteres, homologous appendages of the second and third thoracic segments, respectively, bear different forms: wings are flat whereas halteres are globular and yet both characteristic shapes are essential for a normal flight. The Hox gene Ultrabithorax governs the difference between wing and haltere development, but how Ultrabithorax function in the appendages prevents or allows flat or globular shapes is unknown. This study shows that Ultrabithorax down-regulates Matrix metalloproteinase1 expression in the haltere pouch at early pupal stage, which in turn prevents the rapid clearance of Collagen IV compared to the wing disc. This difference is instrumental in determining cell shape changes, expansion of the disc and apposition of dorsal and ventral layers, all of these phenotypic traits being characteristic of wing pouch development. These results suggest that Ultrabithorax regulates organ shape by controlling Matrix metalloproteinase1 expression and the extent and timing of extracellular matrix degradation.
Lu, D., Li, Z., Li, L., Yang, L., Chen, G., Yang, D., Zhang, Y., Singh, V., Smith, S., Xiao, Y., Wang, E., Ye, Y., Zhang, W., Zhou, L., Rong, Y. and Zhou, J. (2018). The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila. PLoS One 13(6): e0199353. PubMed ID: 29928011
Summary:
Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. The chromatin barrier activity of Fab-8 was studied and how it is affected by the zygosity of the transgene. Fab-8 was able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments were performed on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.
Kaschula, R., Pinho, S. and Alonso, C. R. (2018). microRNA-dependent regulation of Hox gene expression sculpts fine-grain morphological patterns in a Drosophila appendage. Development. PubMed ID: 30143542
Summary:
Disruptions of normal Hox gene expression can lead to severe morphological defects revealing a link between the regulation of Hox expression and pattern formation. This study explored these links focusing on the impact of microRNA regulation on the expression of the Drosophila Hox gene Ultrabithorax (Ubx) during haltere development. Through the combination of bioinformatic and transcriptomic analyses the miR-310/313 cluster (miR-310C) as a candidate regulator of Ubx. Several experiments confirm this. First, miR-310C and Ubx protein show complementary expression patterns in haltere imaginal discs; second, artificial activation of miR-310C expression in haltere discs leads to Ubx-like phenotypes. Third, expression of a fluorescent reporter bearing Ubx 3'UTR sequences is reduced when co-expressed with miR-310C. Fourth, deletion of miR-310C leads to Ubx upregulation and changes the array of mechanosensory sensilla at the base of the haltere. Fifth, artificial increase of Ubx levels within the miR-310C expression domain phenocopies the mechanosensory defects observed in miR-310C mutants. It is proposed that miR-310C-mediated repression delimits Ubx fine-grain expression contributing to the sculpting of complex morphologies in the Drosophila haltere. This work reveals a novel role of microRNA regulation in the control of Hox gene expression with impact on morphology.
BIOLOGICAL OVERVIEW

The homeotic genes encoded for by the Antennapedia and bithorax complexes control segment identity in the developing fly. This is developmental decision making for high stakes. Which segment should develop a wing? A leg? How many appendages should there be, and should appendages on different segments be the same or different from one another? Ultrabithorax regulates decisions regarding the number of wings and legs the adult will have.

Normal Drosophila have only one pair of wings, in contrast to the two pair found on butterflies. But Drosophila has the potential to develop a second set of wings. The haltere ( rhymes with "stare"), an external organ involved with balance, develops in place of a wing on the third thoracic segment. Given an appropriate mutation, the haltere could become a second set of wings.

A closer look at the work of Ubx in the determination of wing morphogenesis follows. Mutations of Ubx result in transformation of the dorsal and ventral appendages of the third thoracic segment (the haltere and third leg) into their counterparts on the second thoracic segment. More than a decade ago, it was concluded that normal Ubx expression in the third segment prevents second segment fate. In this manner Ubx mutations have the potential to produce a second set of wings (Struhl, 1982).

Can the repressive effects of Ubx be used to explain some of the difference between flies and butterflies? Three different studies both suggest the answer is no. Ubx is expressed in posterior thoracic segments in all insects, including butterflies and other modern four winged insects. There is, however, a lack of specificity homeotic transformation. Recently it has been shown that ectopic expression of Ultrabithorax, abdominal-A and Abdominal-B cause similar transformations of the fruitfly appendages: antennal tissue into leg tissue and wing tissue into haltere tissue. Thus the homeotic requirement to form appendages is, in some cases, non-specific (Casares, 1996). It is concluded that instead of there being a single evolutionary switch in regulation, there must have been multiple changes in many genes responsible for wing development regulation by Ubx, or for that matter, other homeotics (Warren, 1994 and Carroll, 1995).

However, regulation of larval abdominal prolegs does fit the expected result. Butterflies and Drosophila have diverged in the abdominal expression of Ubx (as opposed to thoracic expression). Early Ubx expression in the abdomen of butterflies is downregulated (shut off), allowing for the derepression of distal-less and the specification of prolegs; in Drosophila, Ubx expression remains high, inhibiting abdominal limb development (Warren, 1994). Thus, in the specification of prolegs, a single genetic change (Ubx expression) in the abdomen can explain the difference between flies and butterflies.

To complicate an already complicated picture, consider splice variants. UBX has a total of six different protein species (isozymes), generated from mRNA splice variants that are found in various Drosophila tissues. What possible function(s) could all these variants serve? For most multiply spliced RNAs the answer is not apparent, but for Ubx, a provocative answer is found.

One of the areas of developmental and molecular biology that has captured much attention and efforts to build a better understanding, is post-transcriptional processing of RNA. Genes are transcribed into high molecular weight nuclear RNA species that contain both the transcribed exons and the discarded introns. At a later time, this RNA is processed; the introns are removed and the exons are spliced together. It is this processed mRNA that serves to code for proteins.

Splicing factors are pivotal to the success of this process. These factors can recognize specific sequences in RNA, and based on sequence recognition, specifically remove unwanted introns and splice together the desired exons.

In RNAs subjected to alternative splicing (UBX provides a good example), different introns and exons are removed depending on particular tissue types. The result is different tissue specific splice variants, a total of six different protein isozymes coded for by the different splice variants of UBX. The UBX splice variants differ in the distance between the homeodomain and a domain responsible for interaction with Extradenticle, required as a coactivator on UBX target genes. It has been concluded that different UBX isozymes function effectively with EXD on different target genes dependent on the distance between the homeodomain and the interactive domain (Johnson, 1995).

This is a remarkable result. It means that the tissue specificity and developmental role of UBX is determined by splicing. What is true for UBX must be true for other transcription factors. The splicing factors available in a particular cell regulate the developmental fate of that cell by generating different splice variants of transcription factors. The arcane world of post-transcriptional regulation moves from a biochemical curiosity to the center ring of the developmental biology circus.

A study by S. D. Weatherbee (1998) is arguably the best study yet published about how gene regulation differs in homologous structures, and points to future studies for how differential gene regulation will be shown to account for the structural differences between species. The differentiation of the Drosophila haltere from the wing through the action of the Ultrabithorax (Ubx) gene is a classic example of Hox regulation of serial homology. This study reveals several features of the control of haltere development by Ubx which, in principle, are likely to apply to the Hox-regulated differential development of other serially homologous structures in other animals. Specifically, it has been shown that Ubx acts: (1) at many levels of regulatory hierarchies, on long-range signaling proteins and their target genes, as well as genes further downstream; (2) selectively on a subset of downstream target genes of signals common to both wing and haltere, and (3) independently on these diverse targets. This information is presented in terms of the effects of Ubx on gene expression in the three axes of appendage formation, since these axes are to a large extent independently regulated and independent gene regulation in the axes serves to structure the entire wing (Weatherbee, 1998).

In the anterior posterior axis, Ubx represses selected Dpp target genes. The expression pattern of en is essentially the same in the haltere disc as in the wing disc, indicating that Ubx is not regulating haltere identity by altering the expression of this compartmental selector gene. Similarly, the expression of dpp in the developing haltere on the anterior side of the AP compartment boundary resembles that in the wing disc. Because these discs give rise to very different appendages, there may be genes downstream of the Dpp signal that are regulated by Ubx. To identify these, an examination was carried out of how a number of genes involved in the development of specific wing characters are expressed and regulated in the developing haltere (Weatherbee, 1998).

From its cellular site of secretion, Dpp acts as a morphogen to organize wing growth, AP pattern, and to activate target gene expression over a distance. The optomotor blind (omb), spalt (sal), and spalt related (salr) genes are expressed in nested patterns centered on the Dpp stripe and are necessary for proper development of the central wing region, including veins II-IV. The expression of these Dpp target genes was examined in the haltere disc: although omb is expressed in the developing haltere pouch (straddling the Dpp stripe as it does in the wing disc), salr and sal are not expressed in the haltere pouch. These results show that the Dpp signal transduction machinery operates in the haltere disc but that selected wing target genes are not activated by the Dpp signal. To determine whether Ubx represses salr expression in the haltere disc, homozygous Ubx clones were generated. Indeed, salr is derepressed in Ubx clones in the anterior compartment of the haltere disc. As in the wing disc, salr expression in these clones depends on their distance from the Dpp source. To determine whether Ubx is sufficient to repress salr, salr expression was examined in CbxM1/+ wing discs in which Ubx is ectopically expressed along part of the DV boundary. In these wing discs salr expression is repressed in a cell autonomous fashion. Because sal/salr are required for the induction of vein development, the selective repression of salr by Ubx suppresses part of the Dpp-mediated AP wing patterning program in the haltere. As with the spatial patterning of wing veins, the pattern of intervein tissue is also determined by specific regulatory genes and critical for morphogenesis. The Drosophila Serum Response Factor (DSRF, or blistered) gene is expressed in future intervein tissue and required for the adhesion of the dorsal and ventral surfaces of the flat wing. The haltere, however, is more balloon-like; interestingly, DSRF expression is absent from the haltere pouch except for two crescents at the extreme dorsal and ventral edges of the anterior compartment. This difference is caused by Ubx regulation, because in Ubx clones in the haltere disc, repression of DSRF is relieved and a pattern of DSRF expression homologous to that in the wing forms within the boundaries of the clone. Conversely, ectopic expression of Ubx in wing discs extinguishes DSRF expression in a cell-autonomous manner (Weatherbee, 1998).

In the dorsoventral axis, Ultrabithorax represses Wingless in the posterior compartment and selectively represses genes along the dorsal-ventral boundary. It has long been assumed that the global coordinate systems in homologous appendages are the same and, indeed, the apterous selector gene is expressed in the dorsal compartment of the haltere disc as in the wing. However, it was found that Wg, which is expressed along both the anterior and posterior extent of the DV boundary in the wing disc, is not expressed in the posterior compartment of the haltere disc. Because Wg function along the DV boundary is required for growth and patterning of the wing disc, the absence of Wg in the posterior haltere disc probably contributes to its disproportionately smaller size in comparison to the anterior compartment. In posterior Ubx clones in the haltere disc, Wg is expressed along the DV boundary, suggesting that Ubx represses the posterior portion of the Wg expression pattern. The activation of Wg along the DV boundary occurs via the Notch receptor signaling pathway. This pathway also activates the "boundary" enhancer of the vestigial gene, which is activated along the entire anterior and posterior extent of the DV boundary in the haltere. These results demonstrate that the Notch pathway is active along the entire DV boundary but that Ubx selectively prevents Wg activation by this pathway in the posterior compartment. Wg is expressed in the anterior compartment of the haltere disc, yet its phenotypic effects are markedly different from those in the anterior of the wing disc (Weatherbee, 1998).

The most conspicuous difference is that in the wing, Wg activity along the DV boundary induces the formation of the prominent triple and double rows of bristles along the wing margin, whereas in the haltere, it does not. The formation of margin bristles is regulated by Wg via the induction of the proneural achaete (ac) and scute (sc) target genes and also requires the Cut transcription factor. In the haltere disc, Cut is expressed along the anterior DV boundary, whereas ac and sc are not induced. To determine if Ubx represses ac/sc activation by Wg, Ubx clones were examined. In the haltere disc, sc expression is derepressed in clones that touch or cross the anterior portion of the DV boundary. Conversely, sc expression is lost in anterior wing disc cells that ectopically express Ubx. This repression by Ubx is sensitive to the dosage of Ubx activity, since ectopic ac/sc expression is observed in Ubx/+ haltere discs. This ectopic expression corresponds with ectopic bristles found on the halteres of Ubx/+ adults. Further reductions of Ubx function in haltere discs cause greater derepression of sc on the DV boundary and a corresponding emergence of triple row bristles on the adult haltere. The haltere has several types of sense organs, including the proximally located pedicellular sensillae, which are not present on the wing. Correspondingly, sc is expressed in the presumptive pedicellar portion of the haltere disc but not in the equivalent part of the wing disc. In Ubx clones in this region of the haltere disc, sc expression is lost. Therefore, Ubx is required to positively regulate sc in this unique pattern in the haltere disc. Together with the repression of sc along the DV boundary of the haltere, these observations suggest that Ubx acts on two independent domains of the sc expression pattern, presumably via specific cis-regulatory elements controlling each aspect of sc gene expression (Weatherbee, 1998).

In the proximodistal axis, Ubx selectively represses one enhancer of the vestigial gene. vg is expressed and required in the cells that will give rise to the distal appendage fields of the wing and haltere imaginal discs. vg expression in the wing field is regulated by two distinct enhancers that are activated by different signaling pathways. vg expression is first activated along the DV boundary of the wing disc by the Notch pathway, through the boundary enhancer; later it is activated in the growing wing pouch by the Dpp and Wg signals, through the "quadrant" enhancer. Similarly, the boundary enhancer is activated in both the wing and haltere discs, however, the quadrant enhancer is silent in the haltere field. The repression of the quadrant enhancer in the haltere is sensitive to the dosage of Ubx and is partially derepressed in Ubx/+ haltere discs. More importantly, in Ubx clones in the haltere disc, the quadrant enhancer is fully activated. These results show that Ubx selectively represses a portion of the native vg wing expression pattern in the haltere disc through the quadrant enhancer (Weatherbee, 1998).

In these experiments five genes have been identified whose function is necessary for the formation or patterning of various wing characters but whose expression is negatively regulated by Ubx in the haltere disc. For each gene, their repression in the haltere disc correlates with the absence of, or difference between, haltere characters and those in the serially homologous wing. One means by which to test the significance of the repression of these genes in the haltere disc is to determine what effects their derepression might have upon haltere morphology. It is crucial to recognize that the effects of expressing target genes in the haltere does not depend only on the sufficiency of a given gene to induce a phenotype in the wing or at an ectopic site (legs, eyes, etc.). Another crucial factor is the architecture of the Ubx-regulated gene hierarchies in the haltere. There are three possible outcomes and interpretations for the ectopic expression of a differentially expressed gene:

The effects of ectopic expression of the vestigial gene was examined in the haltere and other tissues under the control of the GAL4/UAS system. Whereas vg expression in all other appendages and tissues causes wing-like outgrowths, in the haltere no significant change in adult appendage size or morphology was observed. However, striking differences between the morphology of the outgrowths formed on the second and third thoracic legs were observed. The former had clear wing-like morphology, whereas the latter had haltere-like morphology. The failure of ectopic vg expression to significantly alter haltere morphology and the distinct haltere-like character of the outgrowths formed in third thoracic legs suggests that Ubx acts on genes that are downstream of or parallel to vg in the genetic hierarchy. To test whether Ubx regulates genes downstream of vg, a search was carried out for candidate genes whose expression depends on Vg. The spalt and DSRF genes that are normally not expressed in leg imaginal discs are ectopically induced in first and second thoracic leg imaginal discs as a response to targeted expression of Vg, and may thus be activated in the developing wing through some mechanism that is dependent on Vg. The patterns of ectopic induction of sal and DSRF in T1 and T2 leg discs are reminiscent of their normal expression patterns in wing discs. In contrast to T3 leg discs, which also express Ubx, the central domains of ectopic induction of Sal and DSRF expression are suppressed. These results demonstrate that downstream targets of Vg are also regulated by Ubx, independent of the Ubx regulation of Vg itself. The repression of these and other targets by Ubx would then suggest why the deregulation of Vg expression in the developing haltere is insufficient to reprogram haltere development toward wing development and to alter the morphology of the adult haltere. Similarly, ectopic expression of the DSRF or Sal (M. Averof, personal communication to Weatherbee, 1998) transcription factors also does not alter haltere size, shape, or cell morphology. These results imply that there are genes downstream of DSRF and Sal whose expressions are necessary for the realization of a phenotype but which are repressed by Ubx in the haltere disc (Weatherbee, 1998)

Ectopic expression of the scute gene in the developing haltere is sufficient to induce ectopic sensory organs. Interestingly, near the DV boundary, large bristles resembling those of the wing margin are induced, whereas in more proximal regions, sense organs form that are characteristic of the haltere. This result suggests that the repression of sensory organ formation by Ubx at the DV boundary is largely at the level of the sc gene, whereas the character of the proximal sense organs is modified by Ubx action downstream of or parallel to scute. Thus, all three outcomes outlined above are obtained in these ectopic expression experiments, which reveal that Ubx acts independently on the five genes identified as well as on genes further downstream of or parallel to these regulators in the wing patterning hierarchy (Weatherbee, 1998).

Critical role for Fat/Hippo and IIS/Akt pathways downstream of Ultrabithorax during haltere specification in Drosophila

In Drosophila, differential development of wing and haltere, which differ in cell size, number and morphology, is dependent on the function of Hox gene Ultrabithorax (Ubx). This paper reports studies on Ubx-mediated regulation of the Fat/Hippo and IIS/dAkt pathways, which control cell number and cell size during development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded, caused considerable increase in haltere size, mainly due to increase in cell number. These phenotypes were also associated with the activation of Akt pathways in developing haltere. Although activation of Akt alone did not affect the cell size or the organ size, dramatic increase was observed in haltere size when Akt was activated in the background where expanded is down regulated. This was associated with the increase in both cell size and cell number. The organ appeared flatter than wildtype haltere and the trichome morphology and spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

Wing and haltere are the dorsal appendages of second and third thoracic segments, respectively, of adult Drosophila. They are homologous structures, although differ greatly in their morphology. The homeotic gene Ultrabithorax (Ubx), which is required and sufficient to confer haltere fate to epithelial cells, is known to regulate many wing patterning genes to specify haltere, but the mechanism is still poorly understood (Singh, 2015).

There are a number of differences between wing and haltere at the cellular and organ levels. Wing is a large, flat and thin structure, while haltere is a small globular structure, although both are made up of 2-layered sheet of epithelial cells. Space between the two layers of cells in haltere is filled with haemocytes. Cuticle area of each wing cell is 8 fold more than a haltere cell. Haltere has smaller and fewer cells than the wing. Trichomes of wing cells are long and thin, while haltere trichomes are short and stout in morphology. The ratio of anterior to posterior compartment size in the haltere (~2.5:1) is much different from that in the wing (~1.2:1). Haltere also lacks wing-type vein and sensory bristles. Haltere cells are more cuboidal compared to flatter wing cells (Roch, 2000). Thus, cell number, size and shape all add to the differences in the size and shape of the two organs (Singh, 2015).

However, cells of the third instar larval wing and haltere discs are similar in size and shape (Makhijani, 2007). The difference between cell size and shape becomes evident at late pupal stages (Roch, 2000). Wing cells become much larger, compared to haltere cells. At pupal stages, they also exhibit differences in the organization of actin cytoskeleton elements viz. F-actin levels are much higher in haltere cells compared to wing cells (Roch, 2000) (Singh, 2015).

In the context of final shape of wings and halteres, one needs to understand the mechanism by which Ubx influences cell size, shape and arrangement. It is possible that Ubx regulates overall shape of the haltere by regulating either cell size and/or shape. The current understanding of mechanisms by which wing and haltere differ at cellular, tissue and organ level is ambiguous (Sanchez-Herrero, 2013). For example, while removal of Ubx from the entire haltere, or at least from one entire compartment, leads to haltere to wing transformation with increased growth of Ubx minus tissues, mitotic clones of Ubx (using the null allele Ubx6.28) show similar sized twin spot in small clones (Crickmore, 2006, De Navas, 2006; Makhijani, 2007). Only when very large clones of Ubx6.28Ubx6.28 are generated, one can see increased growth compared to their twin spots (Crickmore, 2006). This suggests that unless a certain threshold level of growth factors is de-repressed, the haltere does not show any overgrowth phenotype (Singh, 2015).

There have been several efforts to identify functional and molecular mechanisms by which Ubx regulates genes/pathways to provide haltere its distinct morphology. Various approaches have been used to identify targets of Ubx that are expected to differentially express between wing and haltere, e.g., loss-of-function genetics, deficiency screens, enhancer-trap screening and genome wide approaches such as microarray analysis and chromatin immunoprecipitation (ChIP). Targets include genes involved in diverse cellular functions like components of the cuticle and extracellular matrix, genes involved in cell specification, cell proliferation, cell survival, cell adhesion, or cell differentiation, structural components of actin and microtubule filaments, and accessory proteins controlling filament dynamics (reviewed in Sanchez-Herrero, 2013; Singh, 2015).

Decapentaplegic (Dpp), Wingless (Wg), and Epidermal growth factor receptor (EGFR) are some of the major growth and pattern regulating pathways that are repressed by Ubx in the haltere (Weatherbee, 1998, Shashidhara, 1999; Prasad, 2003; Mohit, 2006; Crickmore, 2006, Pallavi, 2006; De Navas, 2006; Makhijani, 2007). However, over-expression of Dpp, Wg, Vestigial (Vg) or Vein (Vn) provides only marginal growth advantage to haltere compared to the wildtype. In this context, additional growth regulating pathways amongst the targets of Ubx were examined. Genome wide studies have identified many components of Fat/Hippo and Insulin-insulin like/dAkt signalling (IIS/dAkt) pathways as potential targets of Ubx. The Fat/Hippo pathway is a crucial determinant of organ size in both Drosophila and mammals. It regulates cell proliferation, cell death, and cell fate decisions and coordinates these events to specify organ size. In contrast, the IIS/dAkt pathway is known to regulate cell size (Singh, 2015).

Recent studies have revealed that the Fat/Hippo pathway networks with other signalling pathways. For example, during wing development, Fat/Hippo pathway activities are dependent on Four-jointed (Fj) and Dachous (Ds) gradients, which are influenced by Dpp, Notch, Wg and Vg. Glypicans, which play a prominent role in morphogen signalling, are regulated by Fat/Hippo signalling (Baena-Lopez, 2008). EGFR activates Yorkie (Yki; effector of Fat/Hippo pathway) through its EGFR-RAS-MAPK signalling by promoting the phosphorylation of Ajuba family protein WTIP (Reddy, 2013). However, EGFR negatively regulates events downstream of Yki (Herranz, 2012). The Fat/Hippo pathway is also known to inhibit EGFR signalling, which makes the interaction between the two pathways very complex and context-dependent. IIS/dAkt pathway is also known to activate Yki signalling and vice-versa. Thus, Fat/Hippo pathway may specify organ size by regulating both cell number (directly) and cell size (via regulating IIS/dAkt pathway) (Singh, 2015).

This study reports studies on the functional implication of regulation of Fat/Hippo and IIS/dAkt pathways by Ubx in specifying haltere development. Over-expression of Yki or down regulation of negative components of the Fat/Hippo pathway, such as expanded (ex), induced considerable increase in haltere size, mainly due to increase in cell number. Although activation of dAkt alone did not affect the organ size or the cell size, activation of Yki or down regulation of ex in the background of over-expressed dAkt caused dramatic increase in haltere size, much severe than Yki or ex alone. In this background, increase was observed in both cell size and cell number. The resulted haltere appeared flatter than wildtype haltere and the morphology of trichomes and their spacing resembled that of wing suggesting homeotic transformations. Thus, these results suggest a link between cellular growth and pattern formation and the final differentiated state of the organ (Singh, 2015).

The findings suggest that, downstream of Ubx, the Fat/Hippo pathway is critical for haltere specification. It is required for Ubx-mediated specification of organ size, sensory bristle repression, trichome morphology and arrangement. The Fat/Hippo pathway cooperates with the IIS/dAkt pathway, which is also a target of Ubx, in specifying cell size and compartment size in developing haltere. The fact that over-expression of Yki or downregulation of ex show haltere-to-wing transformations at the levels of organ size and shape, and trichome morphology and arrangement, suggest that regulation of the Fat/Hippo pathway by Ubx is central to the modification of wing identity to that of the haltere (Singh, 2015).

The observations made in this study pose new questions and suggest various interesting possibilities to study the Fat/Hippo pathway with a new perspective.

(1) It was observed that while Yki is nuclear in haltere discs, it appears to be non-functional. Yki is a transcriptional co-activator protein, which requires other DNA-binding partners for its activity. In this context, understanding the precise relationship between Yki and Ubx may provide an insight into mechanism of haltere specification (Singh, 2015).

(2) The Fat/Hippo pathway (along with the IIS/dAkt pathway) may be involved in the specification of cell size, trichome morphology and their arrangement, all of which are important parameters in determining organ morphology. Recent studies indicate that the Fat/Hippo pathway regulates cellular architecture and the mechanical properties of cells in response to the environment. It would be interesting to study the role of the Fat/Hippo pathway in regulating the cytoskeleton of epithelial cells during development. Haltere cells at pupal stages exhibit higher levels of F-actin than wing cells. One possible mechanism that is currently being investigated is lowering of F-actin levels in transformed haltere cells due to over-expression of Yki or down regulation of ex. This may cause flattening of cells during morphogenesis leading to larger organ size (Singh, 2015).

(3) Reversing cell size and number was sufficient to induce homeotic transformations at the level of haltere morphology. This suggests the importance of negative regulation of genetic mechanisms that determine cell size and number, in specifying an organ size and shape. As a corollary, Ubx-mediated regulation of Fat/Hippo and IIS/dAkt pathways provides an opportunity to study cooperative repression of cell number and cell size during organ specification (Singh, 2015).

(4) Certain genetic backgrounds investigated in this study showed severe effect on cell proliferation in haltere discs than in wing discs. This could be due to the fact that, the wing disc has already attained a specific size by the third instar larval stage (the developmental stage examined in this study), which is controlled by several pathways. Any change to this size may need more drastic alteration to the controlling mechanisms. As Ubx specifies haltere by modulating various wing-patterning events, there may still exist a certain degree of plasticity in mechanisms that determine the size of the haltere. However, in absolute terms, the haltere is also resistant to changes in growth control due to regulation by Ubx at multiple levels. Thus, differential development of wing and haltere provides a very good assay system to study not only growth control, but also to dissect out function of important growth regulators (tumour suppressor pathways) such as the Fat/Hippo pathway using various genome-wide approaches (Singh, 2015).


GENE STRUCTURE

There are two transcripts that differ in their 3'UTR length as determined by two termination signals. UBX is coded for by the following four or five elements: a 5' exon containing the 5'UTR and the N-terminal coding sequence, two micro-exons that are incorporated in certain splice variants, an optional B element that is made part of the C terminal exon in some splice variants, and a C terminal exon containing both the homeodomain and the 3'UTR. There are a total of six specific splice variants: two expressed in epidermis, mesoderm and the peripheral nervous system; two in the central nervous system, epidermis, mesoderm and peripheral nervous system, and two in the central nervous system only. The micro-exons each code for 17 amino acids; the B element codes for 9 amino acids. Therefore the smallest protein species contains 43 fewer amino acids than the largest (O'Connor, 1988, Kornfield, 1989 and Subramanian, 1994).

genomic DNA length - 76 kb

cDNA clone length - 3.2 and 4.6 kb

Bases in 5' UTR - 964

Bases in 3' UTR - 1580 and 2212


PROTEIN STRUCTURE

Alternative splicing of UBX transcript generates a family of five proteins (UBX isoforms) that function as transcription factors. All isoforms contain a homeodomain within a common 99 aa C-terminal region (C-constant region) joined to a common 247 aa N-terminal (N-constant) region by different combinations of three small optional elements. UBX isoforms expressed in D. melanogaster cells are phosphorylated on serine and threonine residues, located primarily within a 53 aa region near the middle of the N-constant region, to form at least five phosphorylated states per isoform. Temporal developmental profiles of UBX isoforms parallel those for the respective mRNAs; all isoforms are similarly phosphorylated throughout embryogenesis (Gavis, 1991).

Amino Acids - 389 for the longest protein, form Ib. The smallest splice variant is 346 amino acids long.

Structural Domains

The homeodomain is C-terminal. There is a 15 residue N-terminal extention to the homeodomain that includes a WPWM motif. This motif determines interaction with Extradenticle. The size and sequence of the region between the WPWM element and the homeodomain differ among the UBX isoforms. This variable region affects interaction with EXD (Johnson, 1995).

See four paralogous Hox clusters of mammals for homologies of Ultrabithorax with mammalian Hox proteins.


Ultrabithorax: Evolutionary Homologs | Transcriptional Regulation | Targets of activity | Protein Interactions | Posttranscriptional regulation | Developmental Biology | Effects of Mutation | References
date revised: 22 APR 97  

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