Interactive Fly, Drosophila

Wingless in the leg disc

Homothorax is shown to limit Dpp and Wg expression. Expression of the Dpp and Wg targets omb and H15 is restricted to those cells that do not express Hth. To determine if hth inhibits target gene activation by Dpp and Wg, hth was either removed from its endogenous domain or either a GFP-Hth fusion protein or the murine hth homolog MEIS-1B was misexpressed in the distal portion of the leg disc. Removing hth function results in the expansion of wg and dpp target gene expression. Dorsally situated hth- clones result in the expansion of omb expression, as marked by the omb-lacZ reporter gene. Does hth repress Distal-less and dachshund? Similar to removing exd function, when hth loss-of-function clones were examined, dac was found to be only partially derepressed, and derepression was found to be more likely to occur in clones that arise near endogenous dac expression. hth- clones have no effect on Dll expression, regardless of where they are situated. However, when clones of GFP-Hth- or MYC-MEIS-expressing cells are generated, both Dll and dac can be repressed. These results suggest that the expression of Dll and dac requires two conditions: (1) the absence of Hth and (2) sufficient activity in the Dpp and Wg pathways. High levels of Wg and Dpp signaling are shown to repress the nuclear localization of Exd by repressing hth transcription. The direct action of both the Wg- and Dpp-signaling pathways is required to specify cell fates along the P/D axis. High levels of Wg and Dpp signaling are required to activate Dll, a determinant of distal cell fates, and to repress expression of dac, a determinant of intermediate fates along the P/D axis. At intermediate levels of Wg and Dpp signaling, dac, but not Dll, is activated. The distal edge of hth expression coincides with the proximal edge of dac expression, suggesting that the threshold of Dpp and Wg signaling required to activate dac is similar to that required to repress hth. To test this idea, either Wg or Dpp signaling was elevated in the hth expression domain by generating clones of cells that express either a membrane-tethered form of Wg or an activated Dpp receptor, Thickveins QD (TKV QD). When Wg-expressing clones were generated dorsally, where endogenous Wg levels are low but where Dpp is present at high concentrations, there was a loss of Hth protein and a shift of Exd protein to the cytoplasm. This suggests that sufficient levels of both Wg and Dpp signaling are required to repress Hth (Abu-Shaar, 1998).

High levels of Wg and Dpp signaling are shown to affect Hth and Exd, at least in part, by repressing hth transcription. The ability of Wg and Dpp to repress hth appears to be indirectly mediated by Dll and dac. Like Dll, Dac appears to have the capacity to repress hth. The expression patterns of Hth and Dll were examined in leg discs that were entirely devoid of dac function. In these discs, the hth domain appears expanded distally and the Dll domain appears to be expanded proximally, consistent with the idea that dac normally represses both hth and Dll (Abu-Shaar, 1998).

The domains of gene expression for Hth, Dac and Dll, as well the regulatory interactions between them, suggest that the leg is functionally divided into two major domains. The first is a proximal domain, which expresses hth, has nuclear Exd and does not express at least some of the potential target genes of the Wg- or Dpp-signaling pathways. The second is a distal domain, which does not express hth, has Exd localized to the cytoplasm, and expresses the targets of Wg, Dpp and Wg+Dpp signaling. These data suggest that the proximal domain is what has been referred to as the coxopodite, or an extension of the body wall, and is distinct from the distal domain, the telopodite. hth expression and nuclear Exd in the coxopodite would restrict the ability of the Wg and Dpp signals to activate their target genes. This idea is consistent with the observation that these two domains differ with respect to their requirement for Hh signaling: unlike the telopodite, which exhibits severe truncations upon the reduction of hh function, the coxopodite is less severely affected. These two domains also appear to have different cell surface properties; cells from one domain prefer not to mix with cells from the other domain. For example, Dll mutant clones almost always relocalize to the hth-expressing domain and hth mutant clones frequently sort into distal regions of the leg disc. This phenomenon is not observed in the wing disc, where hth and Dll are restricted to outside and within the wing pouch, respectively: hth or Dll mutant clones are positioned randomly in this tissue. The mutant phenotypes displayed by the loss of coxopodite gene function are qualitatively different from those displayed by the loss of telopodite gene function. Removal of coxopodite genes such as exd results in either ënonsenseí or proximal to distal cell fate transformations, whereas removal of telopodite gene functions such as Dll and dac results in deletions of the appendage. In summary, the data support the idea that the proximal and distal regions of the leg have independent origins and differ from each other primarily due to the expression of hth, which limits or alters the ability of proximal cells to respond to Wg and Dpp signaling (Abu-Shaar, 1998 and references).

dac and Dll are shown to mediate Wg and Dpp mediated repression of hth. The demonstration that Wg and Dpp signaling repressed hth transcription and Exdís nuclear localization was surprising, because these two signaling molecules induce Exdís nuclear localization in the endoderm of the embryonic midgut. An investigation was carried out into the possibility that the repression of hth by Wg and Dpp is indirect and perhaps mediated by dac and Dll, which are not expressed in the midgut. TKV QD-expressing clones were generated and Hth, Dll and Dac were examined. Loss of function clones of Dll and dac were generated. When Dll- clones were generated before ~72 hours of development, hth was found to be derepressed and Exd was nuclear. However, clones generated after ~72 hours have no effect on hth or Exd, suggesting that there is an alternative mechanism for maintaining hth repression. Like Dll, Dac appears to have the capacity to repress hth. The ability of Dac to repress hth expression was confirmed by generating dac- clones. These dac- clones suggest that there might be other regulators of hth in addition to dac and Dll. Completely removing dac function results in viable animals that have deletions along the P/D axes of their legs. The expression patterns of Hth and Dll were examined in leg discs that were entirely devoid of dac function. In these discs, the hth domain appears expanded distally and the Dll domain appears to be expanded proximally, consistent with the idea that dac normally represses both hth and Dll. It is an apparent paradox that Wg and Dpp repress hth in the leg disc while these same signals activate hth expression and nuclear Exd in the midgut endoderm. This may be explained because in the leg, Wg and Dpp repress hth indirectly, by activating the hth repressors Dll and dac. In the absence of Dll or dac, hth is derepressed in the leg disc, even in cells that receive high levels of the Wg and Dpp signals. In contrast, in the embryonic endoderm, dac and Dll are not activated by Wg and Dpp, nor are any other known hth repressors, allowing hth to be activated in these cells (Abu-Shaar, 1998).

Many of the genes that pattern Drosophila are expressed throughout development and specify diverse cell types by creating unique local environments that establish the expression of locally acting genes. This process is exemplified by the patterning of leg microchaete rows. hairy (h) is expressed in a spatially restricted manner in the leg imaginal disc and functions to position adult leg bristle rows by negatively regulating the proneural gene achaete, which specifies sensory cell fates. While much is known about the events that partition the leg imaginal disc and about sensory cell differentiation, the mechanisms that refine early patterning events to the level of individual cell fate specification are not well understood. In the third instar leg imaginal disc, h is expressed along both the D/V and A/P axes. D/V axis expression appears as a single stripe in the anterior compartment of the disc immediately adjacent to the A/P compartment boundary. A/P axis expression appears as two wedge-shaped blocks in the distal leg segments on either side of the A/P compartment boundary. After disc eversion, the D/V axis stripe forms two of the four longitudinal leg stripes. The A/P axis expression forms five circumferential stripes at the first through fifth tarsal segments. The remaining two longitudinal stripes will be positioned along the A/P axis, intersecting the D/V axis stripes at the distal tip of the leg. These stripes do not appear until 2-3 hours after puparium formation (APF). This paper concerns itself with Hairy expression in the D/V axis (Hays, 1999).

To assess the roles of Dpp and Wg in the regulation of the D-h and V-h enhancer elements, the expression of D-h-lacZ and V-h-lacZ was assayed in legs that were mutant for either dpp or wg. Expression from D-h-lacZ is reduced in leg discs that are mutant for dpp, and is expanded to produce a full D/V axis stripe in discs that are mutant for wg. Conversely, V-h-lacZ expression is severely reduced in wg mutant discs, and duplicated in dpp mutant discs. These findings are in keeping with what has been demonstrated for other Dpp and Wg target genes and suggest that the D-h and V-h enhancers are targets of Dpp and Wg signaling, respectively. The roles of Dpp and Wg in D/V-h regulation were further examined by making somatic clones lacking components of the Dpp and Wg signaling pathways. Given the antagonism between Dpp and Wg in the leg, it was necessary to analyze clones mutant for a component of each pathway. D-h expression was examined in clones mutant for wg and Mothers against dpp (Mad). Mad is a downstream effector in the Dpp signaling pathway that has been shown to bind DNA and transcriptionally regulate some Dpp target genes directly. Dorsal Mad;wg clones that intersect the D-h stripe show loss of h expression, except for variable low level expression in a single row of cells immediately adjacent to the A/P boundary. It can reasonably be concluded that loss of D-h results from the loss of Mad, since there is no duplicated Wg in these clones. These results not only support the finding that D-h is a target of Dpp signaling, it identifies Mad as a potential transcriptional regulator of D-h. Thus it is proposed that D/V-h expression is regulated in a non-linear pathway in which Ci plays a dual role. In addition to serving as an upstream activator of Dpp and Wg, Ci acts combinatorially with them to activate D/V-h expression (Hays, 1999).

Several questions remain open regarding the placement of leg sensory organs. There is much to be learned about the regulation of the eight Ac leg stripes. In the absence of H function, ac is expressed in four broad stripes, which may represent four zones that are competent to express ac. The expression of ac in these zones could be under the control of independent cis-regulatory elements as are D-h and V-h. The D/V axis stripes would likely be regulated by high level Dpp and Wg signaling. The A/P axis stripes could be sensitive to low levels of Dpp and Wg signaling. This may also be the case for the A/P-h stripes, whose regulation is also not yet understood. Alternatively, the whole leg may be competent to express ac with the interstripes established by H and an unknown factor that represses the expression of ac in the four non-H-expressing interstripe regions (Hays, 1999).

The combination of the two secreted signaling molecules Wg and Dpp induces the formation of the proximodistal (P/D) axis in the leg of Drosophila. It was originally suggested that the Wg/Dpp combination may establish an organizer at the distal tip that controlled patterning along the P/D axis and that this organizer is characterized by expression of the homeobox gene aristaless. Even if such an organizer does exist then it is shown that al is not absolutely required for its activity because removing al at the tip using a null allele does not prevent formation of the P/D axis, although it does prevent the formation of the structures normally found at the tip of the leg. However, there is an absolute requirement for Distal-less activity in the formation of the P/D axis in the part of the leg that is more distal than the most proximal segment (the coxa). Yet, Dll protein does not show a graded distribution -- this presents a paradox between where Dll is expressed and where its activity is required -- late in development Dll protein can be detected only in the tarsus and distal tibia, but the genetic data reveal that Dll function is also required cell autonomously in more proximal regions, the femur and all of the tibia (Campbell, 1998).

If the model suggesting cell fate along the P/D axis as specified by Wg and Dpp directly proves to be correct, then one static, simplified version of this model could hold that different cell fates are established above strict concentration thresholds of Wg/Dpp, which in turn would correspond to precise distances from the sources of these molecules. However, one possible problem with this simplified model is growth: as the imaginal disc grows in size, the distance of any one cell from the source will vary so that for such a strict model to produce precise patterning, all cell fates may have to be established simultaneously. Relevant data suggest this is not the case. To account for growth, it has been proposed that different target genes may require Wg and Dpp for different periods of time before expression becomes independent of these signals. Following growth, this could result in overlapping domains of target genes, and these domains could be established at different times in development. However, an alternative way of viewing this model is to propose that different cell fates are established not simply on the basis of how much Wg and Dpp they receive but by how long they receive it. Early in development most of the presumptive leg cells will receive a specific level of Wg and Dpp, but as the disc increases in size, the presumptive proximal cells at the edge of the disc will begin to receive less Wg and Dpp, as they become situated further and further from the sources: they will also experience this specific level of Wg and Dpp for a shorter period of time than more centrally located, presumptive distal cells. Consequently, the length of time a cell receives this specific level of Wg and Dpp may provide positional information along the P/D axis: the longer it receives it the more distal it becomes. It is proposed that the present results with Dll may provide some evidence for such a dynamic version of this model. These results suggest presumptive intermediate level cells express Dll early in development but it is lost later, whilst presumptive distal cells show continuous expression. If it is assumed that a cell expresses Dll above a certain threshold of Wg/Dpp, then its expression may be lost during development at the edge of its expression domain when these cells become situated further from the sources of Wg and Dpp, as the disc grows in size (Campbell, 1998 and references).

One problem with this model is that maintenance of Dll expression does not appear to require continuous Wg and Dpp signaling. Dll expression is not lost in clones of a Dpp receptor, thick veins, or a Wg signal transducer, dishevelled, even when these are made during the second instar, i.e. at a time when the present results suggest Dll is still transiently expressed in some cells. There are at least two possible explanations for this: (1) the above model is correct but it is impossible to determine timing of gene function by making clones because this ignores the possibility of perdurance of gene products, or (2) the model is incorrect, but this may be because it assumes that Wg and Dpp are the only limiting factors controlling Dll expression (and patterning along the P/D axis): there may be an additional signal, possibly derived from the presumptive tip, which is also required for Dll expression. A temporal mechanism for axis formation is more evident in vertebrate appendages where positional identity along the P/D axis appears to be determined by such a mechanism: the longer a cell spends in the progress zone, the region behind the tip of the developing limb, the more distal it becomes. Consequently, the P/D axis is determined in a proximal to distal sequence. In Drosophila, there is contradictory evidence as to the order in which segments are specified along the P/D axis and further studies are required to resolve this question (Campbell, 1998 and references).

To determine whether Wg signaling is required for Frizzled-3 expression, examination was made of the effects of reduction and misexpression of Wg signals on Dfz3-lacZ expression. In a wg hypomorphic mutant (wg^CX3) background, the area of Dfz3-lacZ expression in leg discs decreases with a reduction of Wg expression. All embryonic Dfz3 expression other than that occurring along the dorsal edge and weak expression in brain disappears in a wg null mutant, (wg^CX3). When UAS-wg^ts is driven by ptc-Gal4, Dfz3-lacZ misexpression occurs in anterior cells along the anteroposterior compartment border in a cell-non-autonomous fashion. Similar but cell-autonomous misexpression of Dfz3-lacZhas been noted in flip-out clones expressing DArm, a constitutively active form of Arm. From these findings and expression of Dfz3-lacZ, it is concluded that Dfz3 expression is positively regulated by Wg signaling, which gives the opposite effect on Dfz2 expression (Cadigan, 1998). Consistent with this conclusion, at least in leg and wing discs, Dfz2 and Dfz3 show virtually complementary expression. That Dfz3 expression along the dorsal edge of an embryo where DWnt4 is expressed is insensitive to the absence of wg activity suggests that dorsal-edge Dfz3 expression may be due to DWnt4 signaling (A. Sato, 1999).

Wingless targets in the abdomen

To understand better the regulation of decapentaplegic in the abdomen, genomic fragments from the 3' region of dpp were tested for the ability to drive lacZ expression in the pupal epidermis. dpp express in the histoblasts and in the LEC is controlled by separate enhancer elements located between 100 to 105 kb on the standard dpp genomic map (10 kb 3' of the transcriptional termination site). Histoblast expression is regulated by two distinct regions. Fragments from between 109.5 kb and 113.5 kb on the dpp genomic map drive lacZ expression in the developing pleura, but not in the sternite or most of the tergite. Accordingly, this region is referred to as the pleural enhancer. Unlike the endogenous dpp pattern, some of the fragments from the 109.5-113.5 kb region drive persistent, rather than transient, expression in the lateral tergite. The tergite expression is controlled in part by a distinct element, located between 112.3 kb and 113.5 kb. A second enhancer region active in histoblasts (the ëcircumferential enhancerí) is located between 117.2 kb and 118.9 kb. This fragment drives expression in a stripe that extends around almost the entire segment, interrupted only at the ventral midline and near the spiracle. Presumably the activity of this enhancer is normally repressed in the tergite and sternite territories by other regulatory regions. Sequences responsible for dpp expression along the dorsal midline have not been identifed (Kopp, 1999).

Both the pleural and circumferential histoblast enhancers are responsive to hh. Expression of the BS 3.21 reporter construct, which is representative of the pleural enhancer, is strongly expanded to the anterior in the hh^Mir gain-of-function mutant, whereas expression of the BS 4 construct, which contains the circumferential enhancer, is duplicated. Both enhancers are repressed by wg, although to differing extents. BS 4 expression in the tergite (but not in the pleura) is completely eliminated in hs-wg pupae grown at high temperature overnight, whereas BS 3.21 expression is only weakly affected. dpp expression in the LEC is controlled by an entirely separate region. Fragments located between 98.5 kb and 106.9 kb drive expression in a correct dpp pattern in the LEC, but not in the histoblasts. Interestingly, this region is devoid of imaginal disc enhancers. The fragments BS 1.1 (98.5-100.3 kb), BS 2 (100.2-104.5 kb) and BS 2.1 (104.7-106.9 kb) produce very similar expression patterns, suggesting that dpp expression in the LEC is controlled by several redundant enhancers. Unlike the endogenous dpp gene, the BS 2 and BS 2.1 reporters are also expressed in the third instar larval epidermis (Kopp, 1999).

In Drosophila, the Hox gene Abdominal-B is required to specify the posterior abdomen and the genitalia. Homologs of Abdominal-B in other species are also needed to determine the posterior part of the body. The function of Abdominal-B in the formation of Drosophila genitalia has been studied, and the absence of Abdominal-B in the genital disc of Drosophila is shown to transform male and female genitalia into leg or, less frequently, into antenna. These transformations are accompanied by the ectopic expression of genes such as Distal-less or dachshund, which are normally required in these appendages. The extent of wild-type and ectopic Distal-less expression depends on the antagonistic activities of the Abdominal-B gene (as a repressor), and of the decapentaplegic and wingless genes (as activators). Absence of Abdominal-B also changes the expression of Homothorax, a Hox gene co-factor. These results suggest that Abdominal-B forms genitalia by modifying an underlying positional information and repressing appendage development. It is proposed that the genital primordia should be subdivided into two regions, one of them competent to be transformed into an appendage in the absence of Abdominal-B (Estrada, 2001).

In the genital disc, the transcription of Dll depends, as in the leg disc, on dpp and wg signals. Abd-B represses Dll expression. Moreover, increasing Abd-B levels in the Dll domain suppresses Dll transcription. The antagonistic activities of dpp/wg and Abd-B in determining the Dll distribution was analyzed. Mutations in PKA ectopically activate wg and dpp expression. PKA minus clones in the genital primordia activate Dll, although only in some places. This activation is not mediated by changes in Abd-B levels. Similarly, although Dll is derepressed in many late Abd-B minus clones, derepression of either dpp or wg was not observed. It is concluded that there is an antagonism between the activation of Dll by dpp/wg signaling and its repression by Abd-B. This is not mediated by changes in the expression of either dpp, wg or Abd-B (Estrada, 2001).

To characterize this antagonism further, Abd-B minus clones that were made were also unable to transduce the dpp signal. This signal requires the presence of the type II receptor encoded by the gene punt. In put;Abd-B double mutant clones, Dll is not activated, indicating that, in the absence of Abd-B, Dpp (and possibly Wg) are still required to activate Dll. Abd-B minus clones far from the wild-type Dll domain fail to activate Dll ectopically, suggesting that activation of Dll in the absence of Abd-B depends on the range of diffusion of Dpp and Wg, as in the leg disc and in the anal primordium (Estrada, 2001).

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