Ultrabithorax


DEVELOPMENTAL BIOLOGY

Embryonic

UBXmRNA is first detected at about 2.5 hr after fertilization (stage 5) in a broad distribution that peaks anteriorly in parasegment 6(PS6) [Images] and rapidly decreases to low levels posteriorly. UBX is next detected in PSs 8, 10 and 12, where it generates a pair-rule pattern. Expression is rapidly detected in PSs 9 and 11, and then in PSs 5 and 7, destroying the alternating segment pair rule pattern. PS6 remains the band of strongest expression (McCall, 1994).

In visceral mesoderm, the musculature surrounding the gut, expression of Ubx is detected in PS7. In the central nervous system Ubx is expressed in a pattern similar to that of early epidermal expression: strong expression in PS6, with decreasing levels in PSs 7-12. Expression varies from cell to cell, suggesting specific roles in cell fate determination (Irvine, 1991).

Ubx specifies the development of two different metameres--parasegment 5, which is entirely thoracic, and parasegment 6, which includes most of the first abdominal segment. How does a single Hox gene specify two such different morphologies? In the early embryo, cells respond similarly to UBX protein in both parasegments. The differences between parasegments 5 and 6 can be explained by the different spatial and temporal pattern of UBX protein expression in these two metameres. UBX protein represses limb primordia before 7 hours, when Ubx is expressed in the abdomen, but not later, when UBX is first expressed in the T3 limb primordium. The regulation of one downstream target of UBX, the Distal-less gene, provides a model for this transition at the molecular level (Castelli-Gair, 1995).

To determine when the homeotic genes are required for specific developmental events Ultrabithorax, abdominal-A and Abdominal-B proteins have been expressed at different times during development using the GAL4 targeting technique. Early transient homeotic gene expression has no lasting effects on the differentiation of the larval epidermis, but it switches the fate of other cell types irreversibly (e.g. the spiracle primordia). One cell type in the peripheral nervous system makes sequential, independent responses to homeotic gene expression. There is also an in vivo competition between the bithorax complex proteins for the regulation of their down-stream targets (Castelli-Gair, 1994).

The developing central nervous system in many species expresses distinct segment-specific characteristics. These can be regulated by homeotic genes. The composition of CNS lineage differs between the thoracic and abdominal segments with respect to the presence or absence of specific glial and neuronal components. Segment specificity of neuroblast NB1-1 is determined in the neuroectoderm at the early gastrula stage (stage 7). Heterogenetic transplantation and mutant analysis show that the activity of either Ubx or abd-A is required for the expression of the abdominal variant of the lineage. Heat induction of Ubx or abd-A expression or their derepression in Polycomb mutant embryos can override thoracic determination several hours after gastrulation (stage 10/11). At that stage antibody stainings reveal both UBX and ABD-A to be present in NB1-1 during normal development (Prokop, 1994).

To gain further insights into homeotic gene action during CNS development, the role of the homeotic genes was characterized in embryonic brain development of Drosophila. Neuroanatomical techniques were used to map the entire anteroposterior order of homeotic gene expression in the Drosophila CNS. This order is virtually identical in the CNS of Drosophila and mammals. All five genes of the Antennapedia Complex are expressed in specific domains of the developing brain. The labial gene has the smallest spatial expression domain; it is only expressed in the posterior part of the tritocerebral anlage. This contrasts with previous reports that lab is expressed throughout the tritocerebral (intercalary) neuromere. The proboscipedia gene has the largest anteroposterior extent of expression, however, in contrast to other homeotic genes, pb is only found in small segmentally repeated groups of 15-20 cells per neuromere. These groups of pb-expressing cells range from the posterior deutocerebrum toward the end of the VNC. Since pb-expressing cells are found anterior to the lab-expressing cells in the brain, this is an exception to the spatial colinearity rule. (Spatial colinearity is conserved in the epidermis, where pb expression is posterior to lab expression). The Deformed gene is expressed in the mandibular neuromere and the anterior half of the maxillary neuromere and the Sex combs reduced gene is expressed in the posterior half of the maxillary neuromere and the anterior half of the labial neuromere. The Antennapedia gene is expressed in a broad domain from the posterior half of the labial neuromere toward the end of the VNC. The three genes of the Bithorax Complex are expressed in the VNC. Ultrabithorax gene expression extends in a broad domain from the posterior half of the T2 neuromere to the anterior half of the A7 neuromere, with highest expression levels in the posterior T3/anterior A1 neuromeres. The abdominal-A gene is expressed from the posterior half of the A1 neuromere to the posterior half of the A7 neuromere. For the above mentioned genes, the anterior border of CNS expression remains stable from stage 11/12 until the end of embryogenesis. In contrast, the anterior border of CNS expression for the Abdominal-B gene shifts at stage 14. Before this stage Abd-B expression extends from the posterior half of neuromere A7 to the end of the VNC; afterwards, it extends from the posterior half of neuromere A5 to the end of the VNC with the most intense expression localized to the terminal neuromeres. With the exception of the Dfd gene, the anterior limit of homeotic gene expression in the CNS is always parasegmental (Hirth, 1998).

For information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

Expression of UBX proteins via a heat-inducible promoter generate homeotic transformations of segmental identities in the embryonic cuticle and peripheral nervous system (PNS) and transform antennae into legs in the adult. The embryonic transformations are used to determine the identity functions of members of the UBX family and UBX mutant forms. Whereas UBX forms I and IV each induce the cuticle transformations, only form I induces the PNS transformations (Mann, 1990).

The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. Evidence is provided that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart (Lo, 2002).

Antp is strongly expressed in four consecutive pairs of cardioblasts in the anterior of the dorsal vessel. The three anterior cardioblast pairs of this domain of strong Antp expression are the posterior three tinman (tin) cardioblast pairs of segment A1, while the fourth pair corresponds to the anterior pair of the two seven up (svp) cardioblast pairs located between A1 and A2. There is also strong expression in at least six pericardial cells flanking the domain of strong cardioblast expression, all of which are non-Tin expressing pericardial cells. Weaker Antp expression is seen in a row of three or four consecutive cardioblast pairs in T3 immediately anterior to the domain of strong Antp, and also in the four tin cardioblast pairs of segment A2 (Lo, 2002).

Located posterior to the domain of Antp expression is a domain of Ubx expression in the midsection of the dorsal vessel. The highest levels of Ubx are observed in the tin cardioblasts of segments A3 and A4, while lower levels are seen in the svp cardioblasts at the A3/A4 border and in the cardioblasts of segments A2 and A5. In addition, the cardioblasts in the heart segments contain barely detectable levels of Ubx. There also appears to be Ubx expression in some of the pericardial cells within A2 to A5, but due to the low expression levels, it is difficult to determine their exact number and whether any of these are tin pericardial cells (Lo, 2002).

The spatially restricted expression of Antp and Ubx in portions of the aorta indicates that these two Hox genes function in the regulation of the A-P polarity of the dorsal vessel as well. Based upon loss- and gain-of-function experiments with Antp and Ubx, these two genes do not appear to be involved in the subdivision into aorta and heart. However, it is conceivable that Antp and Ubx are involved in the later subdivision of this anterior portion of the dorsal vessel into additional chambers that are seen in the adult stage after the remodeling of the dorsal vessel. In addition, Ubx has a role in the A-P patterning of the larval dorsal vessel that appears to be due to its expression in pericardial progenitors. It has been proposed that lymph glands and pericardial cells descend from a common type of progenitor cell, which form the lymph glands in T3/A1 and pericardial cells in more posterior segments. By contrast, in Ubx mutant embryos, the lymph gland is strongly expanded toward more posterior abdominal segments. Together, these observations suggest that during normal development the activity of Ubx within pericardial progenitors of the posterior portion of the aorta acts to suppress lymph gland formation from these cells (Lo, 2002).

Larval

Ubx is expressed strongly in thoracic segment 3 discs, haltere and third leg, and more weakly in posterior parts of the thoracic segment 2 discs, wing and second leg (Irvine, 1991).

An Ubx minigene constructed from three key Ubx upstream control regions is capable of supporting development of Ubx null mutants throughout larval life and beyond to pharate flies, thereby rescuing the larval lethality due to the homeotic mutation. The cuticle of these flies shows that the minigene provides at least partial Ubx function in each of the four compartments whose morphogenetic pathways are determined by Ubx. Long-range repressor elements in the chromosomal Ubx gene play an important role in the generation of Ubx expression patterns in imaginal discs (Castelli-Gair, 1992b).

Abdominal histoblast nests along the larval body wall give rise to the adult ectoderm and therefore the cuticle. The dorsal and ventral histoblast nests within the first abdominal (A1) segment are not segmentally homologous with the metathoracic (T3) haltere and leg discs, respectively, since they occur at distinct dorso-ventral locations during normal development and can be found together within the same segment in mutants of the bithorax complex (BX-C) where T3 is transformed towards A2-A4 or A1 towards T3. Histoblast patterning abnormalities are also observed in BX-C mutants. It appears that the Ubx locus is required to steer ectodermal cells toward an imaginal histoblast fate rather than a larval cell fate at specific regions within the first abdominal segment (Frayne, 1991).

The level of polyteny of the Drosophila salivary gland chromosomes was determined throughout the chromosome region 89E1-4, the locus of the Bithorax Complex. A zone of underreplication spans the 300 kb of DNA from the Ubx to Abd-B loci. From the centromere proximal end of the complex, a 70-kb-long gradual decrease of polytenization starts with the Ubx transcription unit and, after a floor corresponding to the abd-A locus, raises gradually back to the maximum over 70 kb in the region of the Abd-B transcription unit. The maximum relative level of underreplication is about 10-fold. The level of polyteny of chromosomes in a gland is estimated at about 1,000. Therefore, even at the lowest point of polyteny, the number of DNA duplexes assuring the continuity of the chromosomes can be estimated at 100 and certainly not limited to a unique double helix. In flies carrying the mutation Suppressor of DNA Underreplication [Su(UR)ES], the underreplication of the Bithorax Complex is fully suppressed. In the wild type, the Bithorax Complex forms a weak point featuring thinner bands separated by clefts or constrictions. In Su(UR)ES strain in contrast, the 89E1-4 band looks like a single solid band consisting of homogenous dense material. It is speculated that the wild-type Su(UR)ES protein hampers DNA replication of silenced domains and leads to their underreplication in salivary gland polytene chromosomes (Moshkin, 2001).

The Drosophila larval cardiac tube is composed of 104 cardiomyocytes that exhibit genetic and functional diversity. The tube is divided into the aorta and the heart proper that encompass the anterior and posterior parts of the tube, respectively. Differentiation into aorta and heart cardiomyocytes takes place during embryogenesis. Living embryos have been observed to correlate morphological changes occurring during the late phases of cardiogenesis with the acquisition of organ function, including functional inlets, or ostiae. Cardiac cell diversity originates in response to two types of spatial information such that cells differentiate according to their position, both within a segment and along the anteroposterior axis. Axial patterning is controlled by homeotic genes of the Bithorax Complex (BXC) that are regionally expressed within the cardiac tube in non-overlapping domains. Ultrabithorax (Ubx) is expressed in the aorta whereas abdominal A (abd-A) is expressed in the heart, with the exception of the four most posterior cardiac cells which express Abdominal B (Abd-B). Ubx and abd-A functions are required to confer an aorta or a heart identity on cardiomyocytes, respectively. The anterior limit of the expression domain of Ubx, abd-A and Abd-B is independent of the function of the other genes. In contrast, abd-A represses Ubx expression in the heart and ectopic overexpression of abd-A transforms aorta cells into heart cardiomyocytes. Taken together, these results support the idea that BXC homeotic genes in the cardiac tube conform to the posterior prevalence rule (Ponzielli, 2002).

The cardiac tube is also segmentally patterned and each metamere contains six pairs of cardioblasts that are genetically diverse. The transcription of seven up (svp), which is expressed in the two most posterior pairs of cardioblasts in each segment, is dependent on hedgehog (hh) signaling from the dorsal ectoderm. In combination with the axial information furnished by abd-A, the segmental hh-dependent information leads to the differentiation of the six pairs of svp-expressing cells into functional ostiae (Ponzielli, 2002).

The morphological and functional criteria defined in this study have allowed cardiomyocytes to be subdivided into two distinct populations that acquire different identities and differentiate according to their positions along the anteroposterior axis. Ubx is expressed in almost all cardiomyocytes of the aorta whereas abd-A is expressed in almost all cardiomyocytes of the heart. The lack (or a very low level) of Ubx expression in the T3 and A1 segments of the aorta suggests that cardiomyocytes in these segments may be exposed to a distinct mode of differentiation. In support of this hypothesis, morphological analysis has revealed distinct features in the most anterior region (segments T3,A1) of aorta. These particular traits were nonetheless difficult to unmask owing to a hindering of the aorta inside the embryo and to the presence of the ring and lymph glands surrounding the cardiac tube. Similarly, the lack of abd-A expression (and the strong Abd-B expression) in the four most posterior cardioblasts of the heart implies that these cells respond to specific genetic and differentiation programs that do not operate in more anterior heart cardiomyocytes. While no obvious morphological features permit these most posterior cardioblasts to be distinguished, their position in the caudal most region of the cardiac tube suggests that they are likely candidates to form the pacemaker center of the organ (Ponzielli, 2002).

In contrast to the situation in the ectoderm, the domains of expression of the BXC homeotic genes in the cardiac tube do not overlap, are contiguous and mutually exclusive. The same type of regionalized expression is also encountered in the visceral mesoderm in which, for example, Ubx is expressed in PS7 while abd-A expression encompasses the segments PS8 to PS12. Nevertheless, whatever the tissue, ectoderm or visceral mesoderm, the more posteriorly expressed gene represses (or dominates over) more anteriorly expressed genes, conforming to the phenotypic suppression or posterior prevalence rules. Accordingly, loss-of-function of abd-A leads to a posteriorization of Ubx expression and a concomitant transformation of the heart into aorta. Similarly, the anterior boundary of the expression domains of abd-A and Abd-B were not modified in Ubx and abd-A mutants, respectively. Reciprocally, overexpression of abd-A in the whole cardiac tube represses Ubx expression and transforms the most posterior aorta cardiomyocytes into heart cardiomyocytes. However, ectopic expression of Ubx also impairs the differentiation of cardioblasts, although to a lesser extent than when abd-A is overexpressed and it does not significantly repress Abd-A expression. This latter result suggests that Ubx and Abd-A may be in competition for common downstream targets (Ponzielli, 2002).

In Ubx embryos, or in Ubx, abd-A double mutants, differentiation of the most anterior region of the aorta is affected. This observation suggests that, as in the visceral mesoderm, Antennapedia (Antp) might be expressed in the anterior domain (segments T3, A1) of the aorta, in which the lymph glands and the ring gland are located and that Antp transcription is repressed by Ubx in segment A2 and more posterior segments. In the absence of Ubx function, the Antp expression domain could be extended posteriorly and lead to the formation of ectopic lymph and/or ring gland cells. Finally, the fact that an additional effect on cardioblast differentiation was observed in double mutant embryos when compared with each single mutation, suggests that Ubx and abd-A participate in cardiomyocyte differentiation independently of their role in axial patterning (Ponzielli, 2002).

The homeotic genes abd-A and Ubx are transcription factors which probably induce differential activation of particular gene networks which, in turn, could confer specific physiological function on distinct subsets of cardiomyocytes. For example, studies performed on the cardiac tube of another insect, Samia caecropia, provide good evidence that the electrophysiological properties of the cardiomyocytes are different in the aorta and in the heart. abd-A function may be necessary to activate genes responsible for heart activity or genes that participate in cardiomyocyte growth. Aorta and heart cardiomyocytes respond to a differential control of cell growth since, at the end of embryogenesis, the heart cardiomyocytes are at least two to three times larger than the aorta cardiomyocytes. Alternatively, Ubx could repress the growth of the aorta cardiomyocytes analogous to its role in haltere cells. Growth control of cardiomyocytes is probably not the unique function exerted by Ubx and abd-A in the cardiac tube, since in the absence of both gene activities the cells do not differentiate properly (Ponzielli, 2002).

The expression of homeotic Bithorax Complex proteins in the fat bodies of Drosophila larvae was analyzed by staining with specific antibodies. These proteins are differentially expressed along the anteroposterior (AP) axis of the fat body, with patterns parallel to those characterized for the larval and adult epidermis. Since fat body nuclei have polytene chromosomes, it was possible to identify the BX-C locus and show that it assumes a strongly puffed conformation in cells actively expressing the genes of the BX-C. Immunostaining of these polytene chromosomes provided the resolution to cytologically map binding sites of the three proteins: Ubx, Abd-A and Abd-B. The results of this work provide a system with which to study the positioning of chromatin regulatory proteins in either a repressed and/or active BXC at the cytological level. In addition, the results of this work provide a map of homeotic target loci and thus constitute the basis for a systematic identification of genes that are direct in vivo targets of the BX-C genes (Marchetti, 2003).

Ubx is intensely expressed in a contiguous region, with an anterior limit distal to, but near, the anterior crossbridge in the third thoracic segment (T3). The domain includes the gonad, and the posterior limit falls in a region corresponding approximately to segments A6/A7. The Abd-A protein is expressed anteriorly in a longitudinal line of cells in a region corresponding to the A2 segment. From that point posteriorly it is accumulated in almost all of the cells in a region that is co-extensive with abdominal segments A3-A7. Finally, the Abd-B protein is expressed to the posterior end of the fat body with an anterior limit in the middle of A4. It is interesting to note that although Ubx is detected in all the nuclei of its domain, Abd-A and Abd-B are only expressed in subsets of nuclei in their respective domains. However, in the region corresponding to segments A4-A6 all of the proteins are co-expressed in most nuclei. These observations demonstrate that the protein products of the BX-C are differentially expressed along the AP axis of the fat body in a manner reminiscent of their accumulation patterns in the epidermis. However, the similarity of expression patterns of the proteins between the two tissues is more evident at their anterior limits than in their posterior extent. Perhaps the most striking result is the overlap of the three proteins in the region around the gonads. It will be interesting to determine if this overlap of domains has some operational significance, or if it is functionally irrelevant (Marchetti, 2003).

Pupal

The Drosophila haltere is a much reduced and specialized hind wing that functions as a balance organ. Ultrabithorax (Ubx) is the sole Hox gene responsible for the differential development of the fore-wing and haltere in Drosophila. Previous work on the downstream effects of Ubx has focused on the control of pattern formation. This study provides the first detailed description of cell differentiation in the haltere epidermis, and of the developmental processes that distinguish wing and haltere cells. By the end of pupal development, haltere cells are 8-fold smaller in apical surface area than wing cells; they differ in cell outline, and in the size and number of cuticular hairs secreted by each cell. Wing cells secrete only a thin cuticle, and undergo apoptosis within 2 hours of eclosion. Haltere cells continue to secrete cuticle after eclosion. Differences in the shape of wing and haltere cells reflect differences in the architecture of the actin cytoskeleton that become apparent between 24 and 48 hours after puparium formation. Induction of Ubx mutant clones reveals that Ubx protein is not needed later than 6 hours after puparium formation to specify these differences, though it is required at later stages for the correct development of campaniform sensilla on the haltere. It is concluded that, during normal development, Ubx protein expressed before pupation controls a cascade of downstream effects that control changes in cell morphology 24-48 hours later. Ectopic expression of Ubx in the pupal wing, up to 30 hours after puparium formation, can still elicit many aspects of haltere cell morphology. The response of wing cells to Ubx at this time is sensitive to both the duration and level of Ubx exposure (Roch, 2000).

The cuticle of the adult abdomen of Drosophila is produced by nests of imaginal histoblasts, which proliferate and migrate during metamorphosis to replace the polyploid larval epidermal cells. In this report, a detailed description is presented of the expression of four key patterning genes, engrailed (en), hedgehog (hh), patched (ptc), and optomotor-blind (omb), in abdominal histoblasts during the first 42 h after pupariation, a period in which the adult pattern is established. In addition, the expression is described of the homeotic genes Ultrabithorax, abdominal-A, and Abdominal-B, which specify the fates of adult abdominal segments. The results indicate that abdominal segments develop in isolation from one another during early pupal stages, and that some patterning events are independent of hh, wg, and dpp signaling. Pattern and polarity in a large anterior portion of the segment are specified without input from Hh, and evidence is presented that abdominal tergites possess an underlying symmetric pattern upon which patterning by Hh is superimposed. The signals responsible for this underlying symmetry remain to be identified (Kopp, 2002).

The dorsal cuticle of a typical abdominal segment contains a stereotyped sequence of pattern elements. At the anterior edge of each segment is the acrotergite, a narrow strip of naked sclerotized cuticle (a1). The remainder of the tergite is covered by trichomes, and can be subdivided into four regions. From anterior to posterior these regions are: a lightly pigmented region with no bristles (a2 fate); a lightly pigmented region that contains two to three rows of microchaetes (a3); a darkly pigmented region with one to two rows of microchaetes (a4); and a darkly pigmented region with a single row of macrochaetes (a5). The tergite is followed by the unpigmented posterior hairy zone (PHZ), which is composed of both anterior (a6) and posterior (p3) compartment cells. All trichomes and bristles in the segment are oriented uniformly from anterior to posterior. Finally, at the posterior edge of the segment is a zone of thin, naked intersegmental membrane (ISM), which can be subdivided into anterior smooth (p2) and posterior crinkled (p1) regions (Kopp, 2002).

The adult abdominal pattern is established in the first 2 days of pupal development, concurrent with the proliferation and migration of histoblasts and the destruction of the larval epidermal cells (LECs.) The spatial and temporal evolution of en, hh, ptc, and omb expression is followed during this critical period. The cuticle of each abdominal hemisegment is formed by three major histoblast nests. The anterior dorsal nest (aDHN) is composed of anterior compartment histoblasts and produces the tergite and part of the PHZ (a1-a6), whereas the posterior dorsal nest (pDHN) is composed of posterior compartment cells and produces the intersegmental membrane and the remainder of the PHZ (p1-p3). The ventral histoblast nest, which produces the sternite and pleura, contains both anterior and posterior compartment cells. en, hh, ptc, and omb are expressed in similar patterns in dorsal and ventral histoblasts, and the description is limited to the dorsal abdomen (Kopp, 2002).

Segment identities in the abdomen are specified by the Ubx, abd-A, and Abd-B genes of the bithorax complex (BX-C). More precisely, BX-C genes control the development of parasegments (ps), which are composed of the posterior compartment of one segment and the anterior compartment of the following segment. Ubx controls the identity of ps6, which includes the anterior compartment of the first abdominal segment (A1); abd-A functions primarily in ps7-ps9 (A2-A4), although it also contributes to the identities of ps10-ps12; and Abd-B is the main determinant of the identities of ps10-ps12. In the pupal abdomen, Abd-B is expressed strongly in ps12 (A7) (in females; the last abdominal segment is rudimentary in males), weaker in ps11 (A6), and at very low levels in ps10 (A5). This pattern is consistent with the view that different levels of Abd-B expression promote distinct segment identities in the posterior abdomen. abd-A is expressed in ps7 (A2) through ps12 (A7), at levels gradually increasing from the anterior to the posterior parasegments. Ubx is expressed only in the anterior compartment of A1 (ps6) in the abdominal epidermis. Double staining for Ubx and hh-lacZ shows that the posterior boundary of Ubx expression coincides precisely with the ps6/ps7 boundary. Thus, Ubx and abd-A are expressed in adjacent nonoverlapping domains, contrasting sharply with their overlapping expression in the embryo. Ubx expression is eliminated from A1 in the abd-A gain-of-function mutant Uab5, suggesting that abd-A represses Ubx during the pupal stage (Kopp, 2002).

Modulation of AP and DV signaling pathways by the homeotic gene Ultrabithorax during haltere development

Suppression of wing fate and specification of haltere fate in Drosophila by the homeotic gene Ultrabithorax is a classical example of Hox regulation of serial homology (Lewis, E. B. 1978. Nature 276: 565–570) and has served as a paradigm for understanding homeotic gene function. DNA microarray analyses was used to identify potential targets of Ultrabithorax function during haltere specification. Expression patterns of 18 validated target genes and functional analyses of a subset of these genes suggest that down-regulation of both anterior–posterior and dorso-ventral signaling is critical for haltere fate specification. This is further confirmed by the observation that combined over-expression of Decapentaplegic and Vestigial is sufficient to override the effect of Ubx and cause dramatic haltere-to-wing transformations. These results also demonstrate that analysis of the differential development of wing and haltere is a good assay system to identify novel regulators of key signaling pathways (Mohit, 2005).

Suppression of wing fate and specification of haltere fate by Ubx is a classical example of Hox regulation, which has served as a paradigm for understanding the nature of homeotic gene function. Using microarray analyses and subsequent downstream validation by methods other than microarray, 18 potential targets have been identified of Ubx function during haltere specification. In addition, differential expression of Dpp at the transcriptional level has been observed between wing and haltere imaginal discs. Including previously known 13 targets, there are now as many as 32 well-established direct or indirect targets of Ubx function during haltere specification. Although Ubx may regulate additional downstream targets, the expression patterns of the genes identified suggest that negative regulation of D/V and A/P signaling is one of the important mechanisms by which Ubx specifies haltere development (Mohit, 2005).

The functional significance of down-regulation of these signaling pathways is confirmed by the dramatic homeotic transformations caused by ectopic activation of Dpp and/or Vg in developing haltere discs. These transformed halteres still lacked veins and wing margin bristles, indicating that Ubx specifies haltere development by additional mechanisms. Indeed, the EGFR pathway, which plays a significant role in specifying wing veins, is directly repressed by Ubx in haltere discs (S. K. Pallavi, unpublished observations reported in Mohit, 2005). Furthermore, over-expression of Dad in wing discs does not cause any obvious wing-to-haltere transformation nor do dppd6/dppd12 wings show such phenotypes. Thus, while over-expression of Dpp causes partial haltere-to-wing transformations, down-regulation of Dpp in wing discs has no such effect. Further investigation is needed to identify all the critical steps downstream of Ubx required to completely transform haltere to a wing or vice versa. Nevertheless, the dramatic homeotic transformations induced by the co-expression of just two genes (Dpp and Vg) suggest that down-regulation of these two steps by Ubx is critical to specify haltere fate (Mohit, 2005).

Although both Vg and Dpp are known to induce growth, it is believed that the observed homeotic transformation is due to re-patterning and trans-differentiation and not due to simple over-growth. Induction of over-growth in haltere leads to larger appendages, but not homeotic transformations. Furthermore, a recent report suggests that changes in cell division patterns alone do not lead to cell fate changes. Thus, Dpp/Vg-induced homeosis is a specific mechanism that overrides the effect of Ubx and suggests an important mechanism for Ubx function during haltere specification. Interestingly, in the mouse, signaling molecules such as Bmp2, Bmp7 and Fgf8 are downstream targets of Hoxa13 during the development of limbs and genitalia. Thus, down-regulation of Dpp and Wnt/Wg signaling pathways in Drosophila and Bmp and Fgf in mouse suggest a common theme underlying Hox gene function during appendage specification and development (Mohit, 2005).

The results presented in this report are significant in two ways. First, they suggest a mechanism by which halteres may have evolved from hind wings of lepidopteran insects. Ubx protein itself has not evolved among the diverse insect groups, although there are significant differences in Ubx sequences between Drosophila and crustacean Arthropods. Nevertheless, over-expression of Ubx derived from either a non-winged arthropod such as Onychophora or a four-winged insect such as Tribolium is sufficient to induce wing-to-haltere transformations in Drosophila. This suggests that, in the dipteran lineage, certain wing patterning genes have come under the regulation of Ubx. In such a scenario, it is likely that only a small number of genes will have their cis-regulatory sequences modified (converging mutations) to enable their regulation by Ubx. Considering the gross morphological differences between lepidopteran hind wings and halteres, any new target of Ubx will have greater influence on the entire hind wing morphology. Indeed, over-expression of Dpp and/or Vg caused dramatic haltere-to-wing homeotic transformations. Since such transformations were not observed by over-expressing their upstream regulators such as Hh, Ci, N or Wg, it is likely that direct targets of Ubx would be closer to Dpp and Vg in the hierarchy of gene regulation. Currently, chromatin immunoprecipitation experiments using haltere extracts are underway to identify those target genes (Mohit, 2005).

The second significant conclusion from the results described here is on the utility of differential development of wing and haltere as a good model system to identify additional components of both A/P and D/V signaling. Nine such genes have been identified, 8 of which show modulation of their expression patterns along the D/V axis. Based on restricted expression patterns and biochemical features of the encoded proteins, their possible involvement in maintaining the integrity of the D/V boundary as well as differences between dorsal and ventral compartments is predicted. Indeed, preliminary characterization of two genes suggests their probable roles to restrict Wg expression to the D/V boundary (Mohit, 2005).

A recent report has identified 16 potential genes downstream of mouse Hoxd cluster during the development of the most distal parts such as digits and genitals. Most of them have not been previously implicated in the early stages of either limb or genital bud development or as components of the known signal transduction pathways. Considering tissue- and developmental stage-specific expression of those genes, it is possible that those targets too could be novel modulators of known signal transduction pathways. Taken together, these results provide a framework for understanding the mechanisms by which Hox genes specify segment-specific developmental pathways (Mohit, 2005).

Distinct genetic requirements for BX-C mediated specification of abdominal denticles

Hox genes encode transcription factors playing important role in segment specific morphogenesis along the anterior posterior axis. Most work in the Hox field aimed at understanding the basis for specialised Hox functions, while little attention was given to Hox common function. In Drosophila, genes of the Bithorax complex [Ultrabithorax (Ubx), abdominalA (abdA) and AbdominalB (AbdB)] all promote abdominal identity. While Ubx and AbdA share extensive sequence conservation, AbdB is highly divergent, questioning how it can perform similar functions than Ubx and AbdA. This study investigated the genetic requirement for the specification of abdominal-type denticles by Ubx, AbdA and AbdB. The impact of ectopic expression of Hox proteins in embryos deprived for Exd as well as for Wingless or Hedgehog signaling involved in intrasegmental patterning was analyzed. Results indicated that Ubx and AbdA do not require Exd, Wg and Hh activity for specifying abdominal-type denticles, while AbdB does. These results support that distinct regulatory mechanisms underlie Ubx/AbdA and AbdB mediated specification of abdominal-type denticles, highlighting distinct strategies for achieving a similar biological output. This suggests that common function performed by distinct paralogue Hox proteins may also rely on newly acquired property, instead of conserved/ancestral properties (Sambrani, 2013b).

This study relies on a gain of function strategy, scoring phenotypes in the thorax, where none of these three BX-C proteins are expressed, but where Exd, Wg, and Hh are expressed. This allows circumventing the difficulty resulting from the incapacity to unambiguously identify posterior most abdominal segments, where AbdB acts, and avoid complications in interpreting results that would arise from cross regulation between BX-C genes in the abdomen. However the approach also questions whether the conclusion of this study applies for BX-C proteins activity in their endogenous expression domains. Regarding Exd requirements, maternal and zygotic loss results in abdominal segment fusion, where segments are fused by pairs: A1/A2-3/A4-5/A6-7/A8. Although denticle belts are highly disorganized, denticles are clearly of abdominal type in anterior abdominal segments. When present in A8, the belt of denticles is very much reduced, and in most embryos is absent. This indicates that Ubx and AbdA do not require Exd for the specification of abdominal-type denticles in their endogenous expression domain, while AbdB does. The complete segment fusion resulting from loss of Wg and Hh signaling make it impossible to unambiguously identify the A8 segments, and therefore does not allow addressing if AbdB also requires Wg and Hh signaling in its endogenous expression domain. Denticles are found in continuous lawn, that encompasses most abdominal segments. Therefore denticles in the Ubx and AbdA expression domains are clearly of abdominal types, indicating that Ubx and AbdA do not require Wg and Hh signaling for the specification of abdominal-type denticles. Thus, whenever possible, resident BX-C Hox protein activity in loss of exd, wg and hh function are consistent with the conclusion raised in the gain of function approach, further supporting that Ubx/AbdA and AbdB use distinct regulatory mechanisms for achieving a common function (Sambrani, 2013b).

Such a conclusion was recently reached by studying the molecular mechanisms underlying repression of the limb-promoting gene Dll by Ubx and AbdB. It was shown that the cofactor requirement and intrinsic protein domain requirement for Ubx versus AbdB repression of Dll was distinct. Ubx represses Dll by binding DNA cooperatively with the Exd and Hth cofactors, which relies on the UbdA domain, a domain specific to Ubx and AbdA and located C-terminal to the HD (Sambrani, 2013a). Surprisingly, Ubx DNA binding is dispensable, probably due to cooperative binding to DNA with Exd and Hth, DNA binding proteins that likely compensate for Ubx loss of DNA binding. By contrast, AbdB represses Dll without the help of the Exd and Hth, and DNA binding of AbdB is strictly required for repression. It was further established that in specifying posterior spiracles and regulating empty spiracles expression, Exd/Hth antagonize AbdB activity, showing that the AbdB/Exd partnership depends on the biological context. Mechanisms at the origin of cooperativity/antagonism are still to be discovered (Sambrani, 2013b).

The present study corroborates the conclusion reached by the analysis of Dll repression by Ubx and AbdB and extends it in several ways: first by using a distinct Hox biological activity as functional readout; second by including in the analysis the AbdA Hox protein; and third by examining additional genetic requirements (Wg and Hh signaling). The work therefore provides further support for the view that distinct molecular strategies underlie an apparent unicity in BXC protein controlled biological function (Sambrani, 2013b).

Given the observation that Ubx and AbdA are very similar, sharing a highly conserved HD as well as additional protein domains such as the HX and UbdA motifs, while AbdB lacks these domains and has a highly divergent HD, it is not surprising that the genetic requirements are similar for Ubx/AbdA and distinct for AbdB. More unexpected was the finding that Ubx and AbdA do not require Exd for specifying abdominal-type denticles, while AbdB does. This indeed contrasts with the known and previously described Exd requirement for Ubx in A1 segment identity specification and Dll repression, and also contrasts with the dispensability of Exd for A8 segment identity specification, posterior spiracle specification and Dll repression (Sambrani, 2013a). This highlights that requirement of Exd for Hox activity depends on the specific function examined, rather than being a general and universal requirement (Sambrani, 2013b).

A salient difference between the central Ubx/AbdA and posterior AbdB Hox proteins is the mode of Hox DNA binding. Posterior paralogue Hox proteins have usually a stronger affinity for DNA when binding as monomer than central class Hox proteins. This difference mainly stems from the ability of posterior but not central class Hox proteins to make extensive contacts with the DNA backbone. These differences provide a frame to understand the requirement of Exd/Pbx cofactor for central class Hox proteins, which upon interaction with Hox proteins raises their DNA binding affinity. In the case of specification of abdominal-type denticles, the contribution of Exd is likely different, as required for AbdB and not Ubx/AbdA activity. This suggests that Exd may be involved in regulating the activity, rather than DNA binding, a function previously suggested in the regulation of Deformed Hox protein function (Sambrani, 2013b).

In summary, this work together with the study of Dll repression by BX-C proteins highlights that distinct regulatory mechanisms and molecular strategies underlies common Hox protein functions. Thus while sequence divergence following gene duplication promotes functional divergence, it also generates novel gene regulatory mechanisms and molecular strategies that yet promotes a common biological output (Sambrani, 2013b).


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

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