Gene name - Ultrabithorax
Synonyms - bithorax (bx)
Cytological map position - 89E1-3
Function - transcription factor
Keywords - bithorax complex
Symbol - Ubx
Genetic map position - 3-58.8
Classification - homeodomain - Antp class
Cellular location - nuclear
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:
(1) Ectopic expression of individual genes in the haltere could be sufficient to induce a wing character. This result would show that the regulation of this gene by Ubx is the key event to determine the difference of that character in the wing and haltere.
(2) There could be no effect on haltere morphology. Given that these genes are sufficient to induce ectopic phenotypes in the wing or elsewhere, this result could occur if downstream genes are independently regulated by Ubx and therefore prevented from being activated even when upstream activators are present.
(3) One could induce haltere characters or structures with intermediate identity. This would imply that Ubx modifies the morphology of characters through other genes in addition to the ectopically expressed gene (Weatherbee, 1998).
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).
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
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).
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.
date revised: 22 APR 97
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