Table of contents

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 ‘nonsense’ 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 Exd’s nuclear localization was surprising, because these two signaling molecules induce Exd’s 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 (wgCX3) 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, (wgCX3). When UAS-wgts 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).

The origins of the Drosophila leg revealed by the cis-regulatory architecture of the Distalless gene

Limb development requires the elaboration of a proximodistal (PD) axis, which forms orthogonally to previously defined dorsoventral (DV) and anteroposterior (AP) axes. In arthropods, the PD axis of the adult leg is subdivided into two broad domains, a proximal coxopodite and a distal telopodite. This study shows that the progressive subdivision of the PD axis into these two domains occurs during embryogenesis and is reflected in the cis-regulatory architecture of the Distalless (Dll) gene. Dll protein in the thorax was first detected during embryonic stage 11, and continues to be visualized in this region until the end of embryogenesis. Early Dll expression, governed by the Dll304 enhancer, is in cells that can give rise to both domains of the leg as well as to the entire dorsal (wing) appendage. A few hours after Dll304 is activated, the activity of this enhancer fades, and two later-acting enhancers assume control over Dll expression. The LT enhancer is expressed in cells that will give rise to the entire telopodite, and only the telopodite. By contrast, cells that activate the DKO ("Distalless Keilin Organ") enhancer will give rise to a leg-associated larval sensory structure known as the Keilin's organ (KO). Cells that activate neither LT nor DKO, but had activated Dll304, will give rise to the coxopodite. In addition, the trans-acting signals controlling the LT and DKO enhancers are described; surprisingly, the coxopodite progenitors begin to proliferate ~24 hours earlier than the telopodite progenitors. Together, these findings provide a complete and high-resolution fate map of the Drosophila appendage primordia, linking the primary domains to specific cis-regulatory elements in Dll (McKay, 2009).

To determine how each of the cell fates in the limb primordia is specified, genetic experiments were carried out to identify the regulators of the LT and DKO enhancers. Consistent with LT's dependency on wg and dpp for leg disc expression, LT is activated in the embryo in cells that receive both inputs, as monitored by anti-Wg and anti-PMad staining. To determine whether wg is required for LT activity, a temperature-sensitive allele of wg was used to allow earlier Dll activation. Switching the embryos to the restrictive temperature at stage 11 resulted in the absence of LT activity, despite the presence of Dll protein (probably derived from Dll304 activity. In addition, ectopic activation of the wg pathway [using an activated form of armadillo (arm*)] resulted in more LT-lacZ-expressing cells (McKay, 2009).

Like wg, the dpp pathway is necessary for LT-lacZ expression in leg discs. Paradoxically, dpp signaling represses Dll in the embryo because dpp mutants show an expansion in Dll304-lacZ expression. By contrast, LT-lacZ is not expressed in dpp null embryos. LT-lacZ, but not Dll protein, was also repressed by two dpp pathway repressors, Dad and brk. Conversely, stimulation of the dpp pathway [using an activated form of the Dpp receptor (TkvQD)] resulted in ectopic activation of LT ventrally (McKay, 2009).

Taken together, these data demonstrate that LT is activated by Wg and Dpp in the embryonic limb primordia, just as it (and Dll) is in the leg disc. Similarly, DKO activity also requires Wg and Dpp input (McKay, 2009).

Although LT is activated by wg and dpp in the leg primordia, these signals are also present in each abdominal segment. Consequently, there must be additional factors that restrict LT activity to the thorax. One possibility is that LT is repressed by the abdominal Hox factors, such as Dll304. Alternatively, LT might be regulated by Dll, itself. In Dll null embryos LT-lacZ was initially expressed in a stripe of cells instead of a ring, but then expression decayed. Ectopic expression of Dll resulted in weak ectopic expression of LT-lacZ in the thorax and abdomen. These data suggest that LT activity is restricted to the thorax in part because of the earlier restriction of Dll304 activity to the thorax (McKay, 2009).

The related zinc-finger transcription factors encoded by buttonhead (btd) and Sp1 are also expressed in the limb primordia and are also required for ventral appendage specification. In strong btd hypomorphs, the activity of LT was still detected but the number of cells expressing LT-lacZ was decreased and its pattern was disrupted. LT-lacZ expression was completely eliminated in animals bearing a large deficiency that removes both btd and Sp1. By contrast, Dll304 was activated normally in these animals (data not shown). Importantly, LT-lacZ expression was rescued by expressing btd in these deficiency embryos. By contrast, expressing Dll, tkvQD, or arm* did not rescue LT expression in these deficiency embryos. Ectopic expression of btd resulted in weak ectopic activation of LT-lacZ in cells of the thorax and abdomen. Strikingly, the simultaneous expression of Dll and btd resulted in robust ectopic expression of LT-lacZ in abdominal segments in the equivalent ventrolateral position as the thoracic limb primordia. btd and Dll were not sufficient to activate LT in wg null embryos (data not shown). These data indicate that the thoracic-specific expression of the LT enhancer is controlled by the combined activities of btd and/or Sp1, Dll and the wg and dpp pathways (McKay, 2009).

Although the data suggest that LT is activated by a combination of Wg, Dpp, Btd and Dll, these activators are also present in the precursors of the KO, which activate DKO instead of LT. Because the KO is a sensory structure, the role of members of the achaete-scute complex (ASC) that are expressed in these cells was tested. In embryos hemizygous for a deficiency that removes the achaete-scute complex, LT-lacZ expression was expanded at the expense of the Ct-expressing cells. Consistently, ectopic expression of the ASC gene asense (ase) repressed LT and increased the number of Ct-expressing cells. These data suggest that there is a mutual antagonism between the progenitors of the telopodite and those of the KO. It was also found that DKO-lacZ expression in the leg primordia was lost in Dll or btd null embryos, consistent with the loss of KOs in these mutants. DKO activity was also lost from the limb primordia in embryos deficient for the ASC. These results indicate that DKO is activated by the same genes that promote LT expression but, in addition, requires proneural input from the ASC (McKay, 2009).

One of the most interesting findings from this work is that the temporal control of Dll expression in the limb primordia by three cis-regulatory elements is linked to cell-type specification. The earliest acting element, Dll304, is active throughout the appendage primordia. At the time Dll304 is active, the cells are multipotent and can give rise to any part of the dorsal or ventral appendages, or KO. A few hours later, Dll304 activity fades, and two alternative cis-regulatory elements become active. Together, these two elements allow for the uninterrupted and uniform expression of Dll within the appendage primordia. However, their activation correlates with a higher degree of refinement in cell fate potential: LT, active in only the outer ring of the appendage primordia, is only expressed in the progenitors of the telopodite. By contrast, DKO, active in the cells within the LT ring, is only expressed in the progenitors of the KO. Thus, although the pattern of Dll protein appears unchanged, the control over Dll expression has shifted from singular control by Dll304 to dual control by LT and DKO. Moreover, not only is there a molecular handoff from Dll304 to LT and DKO, the two later enhancers both require the earlier expression of Dll. Thus, the logic of ventral primordia refinement depends on a cascade of Dll regulatory elements in which the later ones depend on the activity of an earlier one (McKay, 2009).

The high-resolution view of the embryonic limb primordia provided in this study allows clarification of some contradictions that currently exist in the literature. Initial expression of Dll in the thorax overlaps entirely with Hth-nExd (referring to nuclear Extradenticle). Subsequently, hth expression is lost from most, but not all, of the Dll-expressing cells of the leg primordia. The first reports describing these changes failed to recognize the persistent overlap between Dll and Hth-nExd in some cells. As a result, and partly because of the analogy with the third instar leg disc, the predominant view of this fate map became that the Dll-positive, Hth-nExd-negative cells of the embryonic primordia gave rise to the telopodite, while the surrounding Hth-positive cells gave rise to the coxopodite. The expression pattern of esg, a gene required for the maintenance of diploidy, was also misinterpreted as being a marker exclusively of proximal leg fates. Counter to these earlier studies, the current experiments unambiguously show that the Dll-positive, Hth-nExd-negative cells in the center of the primordia give rise to the KO, the ring of Dll-positive, Esg-positive, Hth-nExd-positive cells gives rise to the telopodite, and the remaining Esg-positive, Dll-negative cells give rise to the coxopodite (McKay, 2009).

The spurious expression of DKO-lacZ in Dll-non-expressing cells outside the leg primorida complicates the interpretation of several experiments. Attempts to refine DKO activity by changing the size of the cloned fragment proved unsuccessful. Nevertheless, the evidence supports the idea that DKO-positive, Dll-positive cells of the leg primordia give rise to the Keilin's organ, and not the adult appendage (McKay, 2009).

The progenitors of the coxopodite begin to proliferate at approximately 48 hours of development, consistent with previous measurements of leg imaginal disc growth, whereas the progenitors of the telopodite do not resume proliferating for an additional 12 to 24 hours. According to estimates of the cell cycle time in leg discs, this difference in the onset of proliferation results in one to two additional cell divisions in the coxopodite, consistent with images of late second instar leg discs presented in this study. Why might the telopodite and coxopodite begin proliferation at different times? One possibility is that the cells of the coxopodite give rise to the peripodial epithelium that covers the leg imaginal disc, and therefore require additional cell divisions relative to the telopodite. It is also possible that the telopodite is delayed because the neurons of the Keilin's organ serve a pathfinding role for larval-born neurons that innervate the adult limb. Perhaps this pathfinding function requires that the KO and telopodite remain associated with each other through the second instar. Consistently, the leg is the only imaginal disc that has not invaginated as a sac-like structure in newly hatched first instar larvae (McKay, 2009).

A possible explanation for the delay in the onset of telopodite proliferation is the persistent co-expression of hth and Dll in these cells; hth (and tsh) expression is turned off in these cells at about the same time they begin to proliferate. Consistent with this idea, maintaining the expression of hth throughout the primordia blocks the proliferation of the telopodite. Also noteworthy is the finding that the genes no ocelli and elbow have been shown to mediate the ability of Wg and Dpp to repress coxopodite fates. Together with the current findings, it is possible that the activation of these two genes in the LT-expressing progenitors is the trigger that turns off hth and tsh in these cells (McKay, 2009).

The experiments suggest that once LT is activated, and under normal growth conditions, there is a lineage restriction between the telopodite and coxopodite. By contrast, previous lineage-tracing experiments using tsh-Gal4 concluded that the progeny of proximal cells could adopt more distal leg fates. However, tsh is still expressed in the telopodite progenitors far into the second instar, providing an explanation for these results. In contrast to this early restriction, there is no evidence for a later lineage restriction within the telopodite. For example, the progeny of a Dll-positive cell can lose Dll expression and contribute to the dac-only domain (McKay, 2009).

Interestingly, the lineage restriction between coxopodite and telopodite is not defined by the presence or absence of Hth-nExd or Tsh because both progenitor populations express hth and tsh after their fates have been specified. By contrast, when these two domains are specified, the telopodite expresses Dll, while the coxopodite does not, suggesting that Dll may be important for the lineage restriction. However, later in development, some cells in the telopodite lose Dll expression and express dac, but continue to respect the coxopodite-telopodite boundary. Thus, either Dll expression in the telopodite is somehow remembered or the telopodite-coxopodite boundary can be maintained by dac, which is expressed in place of Dll immediately adjacent to the telopodite-coxopodite boundary. Also noteworthy is the finding that clones originating in the coxopodite can contribute to the trochanter, the segment inbetween the proximal and distal components of the adult leg that expresses both Dll and hth in third instar imaginal discs. However, the progeny of such clones do not contribute to fates more distal than the trochanter. Likewise, a clone originating in the telopodite can also contribute to the trochanter, but will not grow more proximally into the coxa. Thus, the lineage restriction uncovered here seems to be determined by distinct combinations of transcription factors expressed in the coxopodite and telopodite progenitors at stage 14. The progeny of cells that express Dll, tsh and hth can populate the telopodite or trochanter, whereas the progeny of cells that express tsh and hth, but not Dll, can populate the coxopodite or trochanter. In light of Minute-positive results, however, the lineage restriction between coxopodite and telopodite does not satisfy the classical definition of a compartment boundary. A similar non-compartment lineage restriction has also been documented along the PD axis of the developing Drosophila wing (McKay, 2009).

Wingless control Distal-less expression in the Drosophila appendages through functional 3' enhancers

The expression of the Hox gene Distal-less (Dll) directs the development of appendages in a wide variety of animals. In Drosophila, its expression is subjected to a complex developmental control. This study examined a 17kb genomic region in the Dll locus that lies downstream of the coding sequence; control elements of primary functional importance were found for the expression of Dll in the leg and in other tissues. Of particular interest is a control element, which has been called LP, which drives expression of Dll in the leg primordium from early embryonic development, and whose deletion causes severe truncation and malformation of the adult leg. This is the first Dll enhancer for which, in addition to the ability to drive expression of a reporter, a role can be demonstrated in the expression of the endogenous Dll gene and in the development of the leg. In addition, the results suggest that some enhancers, contrary to the widely accepted notion, may require a specific 5' or 3' position with respect to the transcribed region (Galindo, 2011).

The genomic region immediately downstream of the Dll transcription unit has remained virtually unexplored since the gene was first characterised at the molecular level. The only enhancer described in this region was a maxillary enhancer which requires Dfd to drive Dll expression. This enhancer was contained within the ETD6 fragment, affected in the DllB allele, but not in DllJ. Beyond the ETD6 fragment lie several kilobases of genomic DNA without any major transcripts. In contrast, the genomic region upstream of Dll had been extensively investigated in search of control regions involved in the leg expression of Dll. This has identified at least four new enhancers in the Dll downstream region than can drive expression in the embryonic and early larval leg primordia (LP), late larval leg disc primordia (LL), leg bracts (BR) and wing margin (WM). In addition, the results have helped refine the location of the maxillary enhancer (MX) (Galindo, 2011).

The known control elements for leg expression included an early embryo enhancer (304), a late embryo and early larval enhancer (215/LT), a Keilin organ enhancer (DKO) and a self-maintenance element (M) . These spread over 20 kb upstream of the Dll transcription unit, and together they seemed to account for the whole pattern of expression of Dll. This conclusion was based mostly on the pattern of expression they impose on reporter genes. The only functional information available on regulatory regions was the rescue experiments with the 312 and 313 minigenes, and these suggested that the upstream region was able to rescue the lack of Keilin's organs in a Dll null mutant. This study has revisited and extended these rescues, and it was observe that a few of the rescued individuals can develop into pharate adults displaying a severe leg phenotype, which indicates that this 5′ region is not enough to support a complete Dll gene expression pattern in leg development. This conclusion is supported by the phenotypes of the DllJ and Dll1092 regulatory mutants and, most strikingly, by the newly induced DllR28 mutant. This mutant is a relatively small deletion and its phenotype is remarkably similar to the minigene rescues, which suggests that the enhancer that it affects accounts for the most crucial part of the leg function of the 3′ region (Galindo, 2011).

The crucial regulatory element disrupted by DllR28 is the LP enhancer. The deleted region is well covered by reporter fragments 5 and 1, with extensive overlap among them, and the only leg enhancer is LP, present only in fragment 1. Therefore, it is unlikely that any other leg enhancer which is covered by the DllR28 mutation has passed unnoticed, and the LP enhancer can be mapped to a 0.8 kb interval up to some 3 kb downstream of the Dll1092 insertion site. Impairing the function of this enhancer has dramatic consequences resulting in deformities in medial leg and truncation of the distal segments. These regions derive from cells that fell within the LP-expressing territory up to first instar. It was observed that in late third instar the morphology of the DllR28 imaginal discs is abnormal, expression of Dac is extended distally and the expression of Dll is weakened both in the central domain and in the peripheral ring. Therefore, the activity of the LP enhancer is required for the early determination of leg PD fates and the subsequent efficient distal expression of Dll. The LP enhancer is not only functionally different from 304 and 215/LT, but also its timing and the regulation of its expression are also different. LP starts to work in stage 11, soon after 304 and earlier than 215/LT. It integrates positive effects from three main signalling pathways, Wg, Dpp and EGFR, and it does not absolutely require Dll for its own expression (Galindo, 2011).

LP is the only Dll enhancer described to date with any functional significance in leg development. The combination of 215/LT + M can drive expression of lacZ in a central domain in the leg disc which is coincident with the endogenous Dll, but to date no leg-specific regulatory mutation has been mapped to this region. Another major caveat against the central role attributed to the combination of the 215/LT and M enhancers comes from the fact that both this combination and 215/LT itself are dependent on Dll expression and therefore they may represent an autoactivatory input to reinforce the expression of Dll, rather than the actual trigger of Dll expression in the leg. It is possible that 215/LT contains a 'shadow' leg enhancer whose functionality would be required to reinforce and maintain the activity of the downstream leg enhancers in extreme physiological conditions or during certain developmental periods (Galindo, 2011).

An additional leg enhancer, expressed from early-mid third instar in leg imaginal discs, was found that was called LL. LL is an autoregulatory enhancer, which autonomously requires Dll. Its pattern of expression coincides with the endogenous Dll domain, and in this respect it is similar to the other autoregulatory enhancer described to date, the M enhancer. Thus, it would seem that Dll expression may require a variety of enhancers with an autoactivatory component: 215/LT, M and LL (Galindo, 2011).

An integrated model of the regulation of Dll in the legs would be as follows: At embryonic stage 10, Dll expression is activated in the single primordium for the Keilin's organ (the vestigial larval leg), the leg and the wing imaginal disc, and is required for the formation of these three structures. This activation of 304 is achieved by Wg, while Dpp, EGFR and the Hox proteins Ubx and AbdA act as repressors; hence this mixed appendage primordium is located in the thoracic segments only and at the dorsal edge of the ventral stripe of wg expression. Slightly later, at stage 11, 304 ceases to act, the wing primordium loses Dll expression, and separates and moves away dorsally. Dll expression remains in the leg and Keilin primordia but is now driven by LP, which interprets inputs differently than 304: thus, while LP is similarly activated by Wg, it is also activated by Dpp and EGFR, which were repressors of 304. A requirement has been described for EGFR signalling in leg development between 6 and 7 h of development (stage 11) with concomitant transient activation of MAPK activation. This precise timing indicates that EGFR activates Dll through the LP enhancer. Later on, during stages 12 and 13, 215/LT becomes active and collects activatory inputs from Wg, Dpp and Dll itself to reinforce the action of LP. This mode of regulation remains during first instar, and is responsible for the specification of most of the imaginal leg (the telopodite), giving raise to trochanter, femur, tibia, and tarsus. At the first to second instar transition, the activity of LP ceases, the leg imaginal disc separates from the Keilin organ and the expression of Dll disappears from the presumptive femur and distal tibia, which acquire the expression of dac. Expression of Dll remains in the distal part of the leg (tibia and tarsus), driven by continuing Wg and Dpp signalling through 215/LT. At early third instar, the expression of Dll becomes independent of Wg and Dpp and seems to rely exclusively on autoactivatory maintenance driven by 215/LT + M and the new 3′ autoactivatory enhancer described here, LL. This self-maintained expression remains until the late pupa, when sensory-organ specific expression driven by the BR enhancer appears in the bracts of the leg bristles (Galindo, 2011).

While this model accounts for Dll regulation in Drosophila, and presumably other holometabolous insects with separate larval and imaginal leg primordia, it is likely that a very similar mechanism operates in less derived hemimetabolous insects and other arthropods, which develop their legs directly at embryogenesis. These less derived arthropods also display dynamic Dll expression showing the disappearance of Dll expression from the presumptive medial leg (femur and tibia in insects), which in Drosophila is correlated with inactivation of the activatory 3′ enhancer LP. This reduction in Dll expression does not occur in the antenna of any of these species, and this differential regulation contributes to the different pattern and morphology of these appendages (Galindo, 2011).

It was already suspected that the Dll wing margin enhancer had to lie in the downstream region, since the upstream region could not drive any expression in the wing imaginal disc. This work has shown that the minigene rescues with the 312 and 313 fragments produce pharate adults in which the wing margin has a typical Dll phenotype of lack of bristles. In consequence, there must be a wing enhancer in this downstream region. Two regions were found that can drive GFP expression in the wing margin. The first one, shared by fragments 3 and 7 may be the same as the LL enhancer, and it is active in the wing margin late in pupal development. It could be a manifestation of the self-regulatory enhancer LL in the wing margin, but in any case it is probably irrelevant since the expression of Dll is required for the determination of the wing margin bristles earlier, in late third instar. The second one, the enhancer that has been called WM is most likely the missing wing margin enhancer. WM is contained in Fr1, like LP, but probably 3′ of it since the DllR28 deletion does not affect the wing margin. WM can drive GFP expression only in a narrow line of cells at the presumptive wing margin itself, while Dll protein expression is stronger in the wing margin, but then decays gradually in the wing pouch, in what has been interpreted to be a graded response to wg. The most likely explanation is that this enhancer may need to act in conjunction with another element elsewhere in the Dll locus, most likely an autoactivatory enhancer. Thus, cells in the early wing disc close to the margin would switch Dll expression on, but as the disc grows some of these cells will find themselves further away from the margin, and outside of the functional Wg gradient. In these cells, some weaker Dll expression would still remain thanks to the self-maintenance activity of Dll. In this scenario, the gradient of Dll protein observed in the wing disc (strong levels near the margin, weaker in the blade), would be the result of the life history of the disc cells, while the pattern of Fr1-GFP would just represent a snapshot of the cells currently exposed to Wg. It would be interesting to test this possibility in the context of previous and recent re-assessments of the long-range Wg gradient hypothesis (Galindo, 2011).

Finally, a BR enhancer was found that is co-expressed with Dll-Gal4 and probably represents the driver required for the function of Dll in the leg bracts. Bracts are determined by directional EGFR signalling from the bristle. It was known that a typical phenotype in different combinations of Dll mutant alleles and in Dll somatic clones was the lack of the bracts, which are characteristic of medial and distal leg segments. This study has shown through the small deletion in DllR28 that this phenotype maps to the downstream region of Dll, and the corresponding control region was identified in the overlap of Fr5 and Fr6 since both fragments can drive expression of reporter genes in the bracts (Galindo, 2011).

Two cautionary lessons could be obtained from these results. First, some enhancers may have specific positional requirements with respect to the coding region in order to function efficiently. In this respect, the LP, LL and WM enhancers did not work or worked much less efficiently when placed 5′ of the Gal4 transcription unit, but did drive expression of GFP when placed downstream of the transcription unit. Since the objective of the present work was not to study the positional specificity of enhancers, the results do not permit a completely watertight interpretation, but some of the alternatives can be discarded on close inspection (Galindo, 2011).

The nature of the vector backbone is unlikely to be the cause of the difference, since both use the same hsp70 minimal promoter, which is standard for many Drosophila vectors. In addition, PTGal has been used in the characterization of several regulatory regions, with at least 14 publications listed by Pubmed. Finally, this positional effect was not present in the MX or BR enhancers, both of which could drive correct expression either upstream or downstream of both reporters. In the case of MX, this fragment works in three different constructs (lacZ, Gal4 and GFP). The difference between LP, LL and WM 5′ and 3′ reporters could also be due to a specific requirement for these enhancers to be situated at a minimal distance from the promoter; this minimal spacing could be achieved more easily when situated 3′ of the transcription unit. However, examination of the distances between the LP and LL enhancers and the hsp70 promoter in fragments 1 and 7 constructs does not support this explanation either, due to the 5′ position of the enhancers within the 5 kb inserts and to the small size of the GFP coding sequence (1.2 kb). In any case, any argument based on construct distances fades if it is considered that the endogenous distance of LP to the Dll promoter is much larger at nearly 40 kb. Still, other possibilities cannot yet be discarded, such as the presence in fragments 7 and 1 of uncharacterized insulators, located 3′ to the actual LP and LL enhancers. To definitely prove this 3′ position effect, cloning of the LP and LL enhancers 5′ of the hsp70 promoter and the GFP reporter in the pH-Stinger vector would be required (Galindo, 2011).

Regardless of the precise basis of this position effect, its functional significance may reflect some constraint in the control of the transcription of Dll, or it may help to prevent the ectopic activation of genes further downstream, and therefore represent a more general safety mechanism in the control of gene expression. In a similar study, the downstream region of the wingless gene was investigated and regulatory regions for the eye, wing and ventral (leg and antenna) imaginal discs identified. Although the patterns of expression of the reporter genes closely resembled endogenous Wg, some details in their pattern and activation timing differed with respect to the endogenous protein. Small differences like these have been usually disregarded, but may stem from the fact that regulatory regions have been largely characterised in reporter constructs in which the genomic region was cloned upstream of the lacZ reporter gene, even if their native position is downstream of the coding region. These results beg further research that might challenge the prevalent view that the 5′ or 3′ positioning of enhancers is not as important as distance to the promoter, and may illuminate new models of enhancer–promoter communication (Galindo, 2011).

From these results, a second cautionary principle arises. Even if the pattern of expression of a reporter construct is similar to the endogenous gene product, one cannot necessarily conclude that the DNA region cloned in such a construct is either absolutely required or fully sufficient to control the expression of the gene. Similarly, in vitro binding assays inform as to the potential ability of DNA fragments to bind certain proteins, not of the functional outcome in vivo. Multiple enhancers, either similar or unrelated, can contribute towards the final output in both normal and extreme conditions. Therefore, expression data of reporter constructs should be complemented with functional information in order to obtain meaningful insights into the regulation of the genes under study (Galindo, 2011).

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 ‘circumferential enhancer’) 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 hhMir 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).

Wingless and the PNS

Cell proliferation and cell type specification are coordinately regulated during normal development. Cyclin E, a key G1/S cell cycle regulator, is regulated by multiple tissue-specific enhancers resulting in dynamic expression during Drosophila development. This study further characterized the enhancer that regulates cyclin E expression in the developing peripheral nervous system (PNS) and shows that multiple sequence elements are required for the full cyclin E PNS enhancer activity. Wg signaling is important for the expression of cyclin E in the sensory organ precursor (SOP) cells through two conserved TCF binding sites. Blocking Wg signaling does not completely block SOP cell formation but does completely block SOP cell proliferation as well as the subsequent differentiation (Deb, 2008).

The results reveal that cyclin E expression in developing PNS precursor cells is regulated by a large enhancer containing multiple sequence elements, including two TCF-binding sites that mediate the regulation by Wg signaling. While these TCF-binding elements are essential for the activity of the PNS enhancer, proximal and distal elements in the 4.6-PNS sequence appear to be important for full activity. The importance of Wg in the regulation of the PNS expression of cyclin E is supported by the fact that wg mutant embryos displayed decreased cyclin E expression in the developing PNS cells. This reduction in cyclin E expression in wg mutant embryos was accompanied by an inhibition of BrdU incorporation in the developing PNS, and an inhibition of the determination of the Pros and Elav expression cells in the developing PNS. It is possible that the block in differentiation into the Pros and Elav positive cells is a consequence of the inhibition of cyclin E expression or perturbations to the cell proliferation. However it is also possible that the observed differentiation block in PNS is due to a function of Wg that is independent of PNS cell proliferation. Further studies will be needed to resolve this issue (Deb, 2008).

In addition to wg, a number of other mutations such as achaete/scute (ac/sc) complex and da have also been reported to block PNS precursor proliferation and affect the expression of several cell cycle genes. Ac/Sc complex proteins and Da are bHLH proteins that are important in all aspects of es-PNS precursor differentiation while bHLH protein Atonal (ato) and Da are required for all aspects of ch-PNS precursor development. Recent studies of the expression of the Cdk inhibitor Dap during cell type specification revealed that Dap expression is directly regulated by the same developmental mechanisms that control the differentiation of these cell types. Therefore it will be interesting to test if bHLH proteins such as Da also directly regulate cyclin E expression in the developing PNS cells (Deb, 2008).

Table of contents

wingless continued: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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