Promoter Structure

spineless alleles broken in the upstream region of the gene have no effect on spineless expression in the leg, but alter expression in the antenna to resemble that normally seen in the leg. The effects of a translocation broken at 4 kb to 5 kb (ssD114.3) are described. spineless is expressed in a transient ring in the antennal and leg discs in this mutant. This indicates that the antennal expression pattern of spineless is controlled by the region upstream of 4 kb to 5 kb, whereas the tarsal pattern of expression is likely controlled by a downstream, perhaps intronic, region. Consistent with their effects on spineless expression, mutants located in the upstream region cause transformation of the distal antenna to tarsus. However, only the distal part of the third antennal segment and the arista are affected. Detailed examination of one upstream mutant (ssD114.7) indicates that the antennal tarsus produced has second leg identity (Duncan, 1998).

The transformation of antenna to leg is a classical model for understanding segmental fate decisions in Drosophila. The spineless (ss) gene encodes a bHLH-PAS transcription factor that plays a key role in specifying the identity of distal antennal segments. This report identifies the antennal disc enhancer of ss and then uses enhancer-lacZ reporters to work out how ss antennal expression is regulated. The antennal determinants Distal-less (Dll) and homothorax (hth) are key activators of the antennal enhancer. Dll is required continuously and, when present at elevated levels, can activate the enhancer in regions devoid of hth expression. In contrast, homothorax (hth) is required only transiently both for activation of the enhancer and for specification of the aristal portion of the antenna. The antennal enhancer is repressed by cut, which determines its proximal limit of expression, and by ectopic Antennapedia (Antp). Repression by Antp is not mediated by hth, suggesting that ss may be a direct target of Antp. ss+ is not a purely passive target of its regulators: ss+ partially represses hth in the third antennal segment and lies upstream of Dll in the development of the maxillary palp primordia (Emmons, 2007; full text of article).

This study used lacZ reporters to identify the enhancers responsible for most aspects of ss expression during embryonic and imaginal development. Antennal expression is driven by two large fragments from the ss 5' region, B6.9 and EX8.2. Both of these fragments drive expression in the antennal segment of the embryo and in the distal portion of the pupal antenna. B6.9 is also expressed in the antennal disc through most or all larval development. Dissection of B6.9 allowed localization of the larval antennal enhancer to a fragment of 522 bp. The B6.9 and 522 reporters were used as a proxy for ss expression in experiments to determine the effects of potential upstream regulators of ss. This strategy has its strengths and weaknesses, but has been made necessary by an inability to generate antisera against Ss. A major strength of the approach is that it was possible to assess the effects of regulators on individual enhancers. It is likely that monitoring endogenous ss expression would give results that are less clear cut since both the antennal and tarsal enhancers of ss are active within the antenna. A potential weakness is that the reporters may not faithfully reproduce the normal expression of ss. However, as far as is possible to tell, the antennal reporters reproduce ss expression very well. The expression of B6.9 and EX8.2 in the embryonic antennal segment and the pupal antenna corresponds very closely to that of endogenous ss. Expression of B6.9 and 522 in the larval antennal disc appears very similar or identical to that of ss+, and the transient requirement for hth+ in the activation of these reporters corresponds well to the transient requirement for hth+ in aristal specification. The tarsal enhancer P732 likely also reproduces the spatial pattern of ss+ expression as its tarsal expression domain corresponds well to the region deleted in ss mutants (Emmons, 2007).

The results of this dissection of the B6.9 fragment were surprising. Removal of the left-hand 2 kb of B6.9 to produce S4.9 resulted in the loss of antennal specificity; S4.9 reporters are expressed in both antennal and leg discs. The E2.0 subfragment of S4.9 shows a similar expression pattern, and expression of this fragment in both leg and antennal discs is independent of Hth, but requires Dll continuously. On further subdivision of the E2.0 fragment, it was found that antennal and leg expression are separable; the 522 fragment is largely specific for the antenna, whereas the 531 fragment drives expression primarily in leg discs. To summarize, antennal specificity is present in B6.9, lost in S4.9 and E2.0 and regained in 522. How can sense be made of this? The region deleted from B6.9 to produce S4.9 clearly plays an important role in enforcing antennal specificity. Since this region contains a PRE, one might suspect that it functions in larval stages to maintain repression of the enhancer outside of the antennal segment. However, that the E2.0 fragment has lost the requirement for Hth in both the antenna and leg (S4.9 has not been tested) suggests that the PRE-containing region might function in both locations. One possibility is that this region represses the enhancer in both antennal and leg discs. In the antenna, this repression can be overcome by the combined action of Hth and Dll, while in the leg Dll alone is not sufficient for activation. When the PRE-containing region is deleted, repression is absent or reduced, so that Dll can activate the enhancer without assistance from Hth, and expression is seen in both antennal and leg discs. Why then is antennal specificity restored in the 522 subfragment? Perhaps this fragment is lacking a subset of Dll interaction sites so that it can no longer be activated by Dll alone, but requires combined activation by Hth and Dll. Although this model is consistent with many of the results, it does not provide a ready explanation for the leg specificity of the 531 fragment (Emmons, 2007).

In addition to activation by combined Hth and Dll, the ss antennal disc enhancer is repressed by Cut and by ectopic Antp. Each of these regulators will be discussed separately. It was found that hth+ is required only transiently for activation of the B6.9 reporter. hth clones induced in the embryo or first instar lose expression of B6.9 autonomously in both A3 and the aristal primordia. However, some time in the second of early third instar. Regulatory instar expression of B6.9 becomes independent of hth. Consistent with this transient requirement, it is shown that hth+ is required only early in larval development for specification of the arista. hth clones induced in the first and second instars show a transformation of the entire antenna to a leg-like appendage. However, clones induced after this time show normal aristal development. These temporal requirements are reflected in the expression pattern of hth: hth is expressed throughout the antennal primordium early in development, but in the second or early third instar is repressed in the central domain, which will produce the arista (Emmons, 2007).

The stable activation of B6.9 by Hth suggests that this fragment contains a 'cellular memory module'. The presence of a PRE within B6.9 is consistent with this idea. The ss locus binds Polycomb protein in salivary gland chromosomes and was recently shown to contain PREs by chromatin immunoprecipitation. In the latter work, ss PREs were localized to within the E1.6 subfragment of B6.9 as well as the EX8.2 fragment, both of which showed pairing dependent suppression in this work. PREs are generally thought of as functioning to stably repress genes. However, PREs can also be associated with activating elements to form memory modules that mediate stable activation. It seems likely that B6.9 contains such a module that responds to Hth. Like a memory module from the hedgehog gene, activity of the ss module is set sometime around the second instar. Surprisingly, it was found that activation of the 522 reporter by Hth can also be persistent, although not as stable as for B6.9. The 522 fragment does not appear to contain a PRE, suggesting that Hth may directly recruit factors to the 522 element that cause semi-stable transcriptional activation (Emmons, 2007).

ss is not a completely passive target of hth; ss partially represses hth in antennal discs, which causes hth to be expressed at a lower level in A3 than in A2. This repression appears to be important for normal development as ectopic expression of Hth can delete A3. Moreover, clones ectopically expressing Hth are largely blocked from entering A3 from the proximal (A2) side, suggesting that the different levels of Hth present in A2 and A3 cause a difference in cell affinities between these segments. Hth-expressing clones are similarly restricted to the two most proximal segments in leg discs, although here there is no endogenous expression of hth more distally (Emmons, 2007).

In contrast to hth, Dll is required continuously for expression of both B6.9 and 522 as Dll clones induced even very late in development lose expression of these reporters. This continuous requirement for Dll indicates that stable activation of the B6.9 memory module by Hth does not by itself commit the reporter to expression; rather, activation by Hth appears to render B6.9 open to interaction with Dll and perhaps other positive factors (Emmons, 2007).

Three lines of evidence suggest that Dll is the primary activator of the ss antennal enhancer. (1) It was found that expression of B6.9 and 522 is sensitive to the dosage of Dll+. Expression of both reporters is reduced in animals carrying only one dose of Dll+, and for 522, expression is enhanced in clones having extra doses of Dll+. This dose sensitivity suggests that ss is a direct target of Dll. (2) It was found that expression of both reporters is often induced within clones expressing ectopic Dll, even in the apparent absence of Hth expression. Such activation is seen in clones in the distal leg, wing and elsewhere. (3) It was found that the embryonic antennal enhancer carried by B6.9 is absolutely dependent upon Dll+, but independent of hth. Taken together, these observations suggest that Dll is a primary activator of the ss antennal enhancers. Hth may provide antennal specificity by boosting the level of activation by Dll in the antennal disc (Emmons, 2007).

Surprisingly, it was found that the regulatory relationship between ss and Dll is reversed in the maxillary palp. Here, ss is expressed prior to Dll and is required for the normal initiation of Dll expression. Although some Dll expression ultimately takes place in the palp primordium in ss animals, this expression is weak and occurs in only a few cells. It has not been worked out how ss is activated in the palp. However, it seems likely that dpp plays a role as the 531 subfragment of B6.9 drives expression in a stripe in the region of the palp that roughly coincides with a stripe of dpp expression. The positioning of ss upstream of Dll in the palp may explain why the region ventral to the antenna is so sensitive to ectopic expression of Ss. Strong activation of Dll here by ectopic Ss combined with endogenous expression of hth might be expected to cause frequent induction of ectopic antennae, as is observed. Since ss is normally expressed in the palp, why should earlier ectopic Ss cause the palp primordium to develop as antenna? It seems likely that timing is key, but level of Ss expression could also be important (Emmons, 2007).

The reciprocal regulatory roles of ss and Dll in the antenna and palp suggest a particularly close relationship between these genes. This relationship is reinforced by the finding that ss is required for the development of bracts in the femur, as is Dll (Emmons, 2007).

The finding that Dll and Hth are both activators of the ss antennal reporters is consistent with the proposal that antennal identity is defined by the combined activity of these regulators. However, the results indicate that this model is an oversimplification. Examination of clones expressing Dll, Hth, or both proteins together revealed little correlation between activation of the B6.9 and 522 antennal reporters and combined expression of Dll and Hth. Strikingly, Dll-expressing clones often activate the reporters ectopically without any apparent concomitant expression of Hth, and clones expressing both proteins usually do not activate the reporters. These experiments also reveal strong context dependence. Examples include the leg, where Dll-expressing clones can activate the reporters distally, but not proximally (where endogenous hth expression occurs) and the wing disc, where clones expressing Dll or both Dll and Hth activate the reporters in the wing pouch, but not at all in the notum. The level of expression of both proteins also appears to be key as high levels of Dll can activate the reporters in the leg in the absence of Hth and elevated levels of Hth can repress expression in the normal antennal domain. Previous results have shown that antennal structures can be induced by ectopic expression of Dll in the wing hinge region or proximal leg (which express hth endogenously) or by combined expression of Dll and Hth elsewhere. While this is true, the results indicate highly variable effects in such ectopic expression experiments and fail to detect the strongly synergistic activation of antennal identity by combined Hth and Dll implied by the model. The results indicate that Dll is the primary activator of the ss antennal reporters, that Hth serves to promote this activity and that activation by Dll and Hth is highly context-dependent (Emmons, 2007).

Consistent with direct control of the antennal reporters by Dll and Hth, two highly conserved regions within the 522 fragment contain apparent binding sites for Dll, Hth, and the Hth dimerization partner Extradenticle. The functional importance of these binding sites is currently being tested (Emmons, 2007).

This study has show that the proximal boundary of B6.9 and 522 expression is defined by repression by cut. This repression likely explains why ectopic Cut causes a transformation of arista to tarsus. cut has been shown to define the proximal expression limit of distal antenna (dan) and distal antenna related (danr); since ss lies upstream of these genes , it seems very likely that their regulation by cut is indirect. The mechanism of action of Cut is not well understood, since only one direct target has been characterized in Drosophila (Emmons, 2007).

Ectopic expression of Antp in the antenna represses the B6.9 and 522 reporters. This finding was expected, since it is well known that expression of Antp or other Hox genes in the antenna causes a transformation to leg. The conventional view is that this transformation results from the repression of hth by ectopic Hox proteins. Repression of hth early in development would be expected to lead secondarily to loss of ss expression and loss of distal antennal identity. However, it was found that clones expressing Antp repress the B6.9 and 522 reporters even when these clones are induced very late in development, long after the requirement for activation by hth has passed. Late repression of the antennal reporters by Antp must therefore occur independently of hth and could be direct. One possibility, currently being tested, is that Antp might compete with Dll for binding to the 522 enhancer. Late repression of the ss antennal enhancer by Antp is consistent with the effects of Antp-expressing clones on antennal identity: such clones induced in the mid to late third instar cause transformations of distal antenna to leg (Emmons, 2007).

Clones induced late that ectopically express Antp in a sustained fashion were examined. In contrast, previous work studied the effects of pulses of Antp expression induced by one-hour heat shocks in a heat shock/Antp line. It had been found that transformations of arista to tarsus were induced by such pulses only when they were administered at the end of the second instar. Why do pulses of Antp at this time cause a stable, heritable transformation of the distal antenna? The current results suggest an explanation. The period sensitive to Antp pulses coincides roughly with when the ss antennal enhancer becomes independent of hth. This correlation suggests that pulses of Antp in the second instar cause heritable transformations by interfering with the stable activation of ss by Hth. Recently, it has been reported that ectopic Antp does not repress hth in the antenna early in larval development. This observation suggests that Antp might act directly on the ss antennal enhancer to prevent its stable activation by Hth (Emmons, 2007).

The regulation of ss by ectopic Antp suggests that Antp may normally play a significant role in repressing ss antennal enhancer activity in the legs. Although this idea has not been tested directly, it seems unlikely that Antp is primarily responsible for keeping the ss antennal enhancers inactive in the leg. Antp null clones do cause activation of the ss target gene dan in leg discs, implying ectopic activation of ss. However, this activation occurs only proximally, with the distal leg appearing to develop independently of Antp. Expression of Antp in the proximal leg may account for why Dll-expressing clones fail to activate B6.9 or 522 in this location. Ectopic activation of the ss antennal enhancers in the leg primordia of the embryo is not seen in an Antp null mutant (Emmons, 2007).

These studies suggest that antennal structures are specified in a combinatorial fashion by Hth, Dll, Ss and probably other factors. In A3, all three proteins are required for normal antennal identity. In ss antennae, hth continues to be expressed in A3 (although at elevated levels), as does Dll. Despite this continued expression of hth and Dll, A3 develops without antennal characteristics and produces only naked cuticle. Thus, Hth and Dll are unable to specify A3 characters in the absence of Ss. Conversely, assuming that ss is stably activated in the antenna by Hth, as is B6.9, then hth clones induced late would show persistent expression of both ss and Dll in A3. Such clones are transformed to leg, implying that Ss and Dll have no ability to direct A3 identity in the absence of Hth. Taken together, these observations suggest that Hth, Dll and Ss must act together to specify A3 identity. This requirement for combined action accounts for why ectopic expression of Ss does not induce A3 tissue in the medial leg, since hth is not normally expressed here. The view of combinatorial control suggests that many A3-specific target enhancers might be identifiable in genome searches as regions that contain clustered binding sites for Hth, Dll and Ss; tests of this prediction will be presented elsewhere (Emmons, 2007).

In contrast to A3, the aristal primordium appears to be specified by ss and Dll acting together in the absence of hth expression. hth is expressed in the aristal region early in development, where it functions to establish ss expression, but it is soon repressed here. Therefore, for most of development, the arista is specified by Ss and Dll acting without input from Hth. Consistent with this picture, the arista adopts leg identity in ss null mutants, and ectopic expression of ss causes the distal tip of the leg to develop as arista (Emmons, 2007).

In ss mutants, the distal antenna is terminated by a single tarsal segment (the fifth). In contrast, in ss mutants that lack only antennal enhancer activity (e.g. the breakpoint mutations ssD114.3 and ssD114.7, the distal antenna develops with a near complete set of tarsal segments. This difference likely reflects the activity of the tarsal enhancer in the antenna. In support of this view, the ss tarsal enhancer drives expression in the segmented base of the arista, a region known as the basal cylinder. This region transforms to tarsal segments 2-4 in Antp-induced transformations of antenna to leg. However, the question arises as to why normal antennal expression of ss causes the proximal arista to develop as basal cylinder, whereas ss expression driven by the tarsal enhancer alone causes this same region to develop as tarsal segments. Likely, the key difference is that expression driven by the tarsal enhancer is transient, whereas expression driven by the antennal enhancer is sustained. Perhaps transient expression of ss allows growth and subsegmentation to produce a full set of tarsal segments, whereas sustained expression inhibits growth, producing the basal cylinder. Consistent with this idea, sustained expression of ss driven by the GAL4 method can cause deletion of tarsi in the legs. The levels of expression driven by the tarsal and antennal enhancers may also be important as flies having only one dose of ss show a partial transformation of the basal cylinder to tarsus. The ss tarsal enhancer drives weak expression in A3 as well as in the basal cylinder, likely accounting for the presence of some specialization of A3 in ss mutants lacking the antennal enhancers (Emmons, 2007).

The view that antennal identity is specified by the combined action of Hth, Dll and Ss contradicts the now prevalent view that antennal identity is determined solely by hth. The major evidence supporting the latter view is that early hth clones transform the entire antenna to leg, and ectopic expression of Hth can induce ectopic antennal structures in the anal plates. Moreover, Dll shows little antennal specificity, being expressed in the distal portions of all of the ventral appendages, and ss expression in the antenna is dependent upon hth+. Should hth be viewed as the antennal 'selector' gene? hth does not seem to be a selector in the same sense as the Hox genes; it is expressed very broadly in the embryo and in other imaginal discs and plays no role in activating ss in the antennal segment of the embryo. Moreover, the ability of ectopic Hth to induce antennal structures is very limited: transformations of anal plate to distal antenna have been reported following ectopic expression of Hth or Meis1, a mammalian homolog. However, others have been unable to reproduce this effect by ectopic expression of Hth, matching the results of this study. That anal plates are susceptible to transformation at all is likely due to the fact that Dll and ss are coexpressed here in normal development. A further dissimilarity is that hth acts only as an establishment regulator of ss in the antennal disc, unlike the continuous requirements usually seen for the Hox genes. Ultimately, assessment of the importance of hth will depend on whether its function in the antenna is conserved. The expression pattern of hth in the antenna does appear to be conserved in the milkweed bug Oncopeltus. However, localization of nuclear Exd (a proxy for Hth expression) indicates that Hth is not differentially expressed in the antenna and leg of the cricket. Expression of hth in the crustacean Porcellio also appears to be identical in the second antenna and the legs. Characterization of hth, Dll and ss expression and function in additional arthropods will be required to assess properly the importance of these genes in antennal specification (Emmons, 2007).

Control of the spineless antennal enhancer: direct repression of antennal target genes by Antennapedia

It is currently thought that antennal target genes are activated in Drosophila by the combined action of Distal-less, homothorax, and extradenticle, and that the Hox gene Antennapedia prevents activation of antennal genes in the leg by repressing homothorax. To test these ideas, a 62bp enhancer was isolated from the antennal gene spineless that is specific for the third antennal segment. This enhancer is activated by a tripartite complex of Distal-less, Homothorax, and Extradenticle. Surprisingly, Antennapedia represses the enhancer directly, at least in part by competing with Distal-less for binding. Antennapedia is required in the leg only within a proximal ring that coexpresses Distal-less, Homothorax and Extradenticle. It is concluded that the function of Antennapedia in the leg is not to repress homothorax, as has been suggested, but to directly repress spineless and other antennal genes that would otherwise be activated within this ring (Duncan, 2010).

This report examines the regulation of an enhancer from the antennal gene ss that drives expression specifically in the third antennal segment (A3). The work provides the first look at how the homeodomain proteins Dll, Hth, and Exd function in the antenna to activate antennal target genes. These proteins form a trimeric Dll/Hth/Exd complex on the enhancer, suggesting that Dll acts much like a Hox protein in antennal specification. This work also reveals how the Hox protein Antp functions in the leg to repress antennal development. The conventional view has been that the primary function of Antp is to repress hth in the distal leg, which then prevents the activation of all downstream antennal genes. However, this study found that Antp represses the ss A3 enhancer directly. This repression is essential within a proximal ring in the leg that coexpresses the antennal gene activators Dll, Hth, and Exd. Antp competes with Dll for binding to the enhancer, and this competition is part of a molecular switch that allows the ss A3 element to be activated in the antenna, but represses its activation in the leg. The results suggest that repression of antenna-specific genes in the proximal ring is the sole function of Antp in the leg imaginal disc (Duncan, 2010).

At 62 bp, the ss A3 enhancer (called D4) is one of the smallest enhancers to be identified in Drosophila, and yet it is quite strong; only a single copy is required to drive robust expression of lacZ reporters. The enhancer is also very specific, driving expression in A3 and nowhere else in imaginal discs. It has been proposed that antennal identity in Drosophila is determined by the combined action of Dll, Hth, and Exd. Consistent with this proposal, all three of these factors were found to be required for D4 expression. Although these activators are coexpressed in both A2 and A3, D4/lacZ expression is restricted to A3 by Cut, which represses the enhancer in A2. Like ss itself, D4/lacZ is also repressed by ectopically expressed Antp (Duncan, 2010).

A previous report (Emmons, 2007) showed that the antennal expression pattern of ss is reproduced by lacZ reporters containing a 522 bp fragment from the ss 5' region. This fragment contains five conserved (41%-90% identity) domains, each of which was deleted and tested for effect on expression in vivo. Expression in the arista and the third antennal segment (A3) prove to be under separate control; expression in the arista requires domains 1, 3 and 5, whereas expression in A3 is lost only when domain 4 is deleted. Moreover, reporters containing domain 4 alone show expression in A3 and nowhere else in imaginal discs. Thus, domain 4 is both necessary and sufficient for A3-specific expression. Domain 4 (D4) is 62 bp in length and is highly conserved, being invariant at 50/62 base pairs in the 12 Drosophila species sequenced (Duncan, 2010).

Surprisingly, Dll, Hth, Exd, Cut, and Antp all act directly upon D4. The activators Hth and Exd bind with strong cooperativity to directly adjacent sites. Their joint binding site matches the optimum site for in vitro binding of the mammalian homologs of Hth and Exd (Meis and Prep), consistent with the robust activity of the enhancer in vivo. Mutation of either of these sites abolishes activity of the enhancer. The coactivator Dll binds three sites in D4; one of these sites (Dlla) is required for almost all activity of the enhancer. Dll shows strong cooperativity with Hth and Exd for binding to D4, indicating that Dll interacts physically with these proteins. This interaction requires DNA binding, as Dll protein containing a missense change that blocks DNA binding (a change of asn51 to ala in the homeodomain) shows no ability to associate with D4-bound Hth and Exd. A curious feature of the cooperativity seen in the binding studies is that although Hth and Exd increase the affinity of Dll for D4, Dll appears to have little effect on the affinity of Hth and Exd for the enhancer. Since Hth and Exd already bind cooperatively with one another, it may be that additional cooperative interactions with Dll have little effect. Alternatively, it may be that Hth and Exd interact with Dll only after binding DNA. If so, Hth and Exd would be expected to increase Dll binding to D4, but Dll would have little effect on the binding of Hth and Exd, as observed. Interactions between Dll and Hth in the absence of DNA have been reported in immunoprecipitation experiments. However, this study was unable to repeat these observations. Moreover, the finding that the asn51 mutant of Dll fails to associate with D4-bound Hth and Exd argues strongly against such interactions (Duncan, 2010).

The repressor Cut also acts directly upon D4. Binding of Cut requires two sites, one overlapping Dlla and the other overlapping the joint Hth/Exd site. These binding sites suggest that D4 is controlled by Cut in much the same way that a structurally similar Abdominal-A (Abd-A) regulated enhancer from the rhomboid gene is controlled by the repressor Senseless (Sens). In the rhomboid enhancer, adjacent Hth and Exd sites are also present, and these create a binding site for Sens. Activity of the rhomboid enhancer is controlled by a competition between binding of the Sens repressor and binding of the activators Abd-A, Hth, and Exd. It seems likely that D4 is controlled similarly, with the repressor Cut competing for binding with the activators Dll, Hth, and Exd. It will be of interest to determine whether enhancers similar to D4 are used more widely to control Cut targets involved in its role as an external sense organ determinant (Duncan, 2010).

A key finding in this work is that Antp represses D4 by direct interaction. Antp binds a single site in D4, which overlaps or is identical to the Dlla binding site. Like Dll, Antp binds cooperatively with Hth and Exd. Using purified proteins, it was showm that binding of Dll and Antp to the Dlla site is mutually exclusive. This indicates that Antp represses the enhancer at least in part by competing with Dll for binding. Similar competition may occur at other enhancers; when Antp expression is driven artificially in the distal leg, variable deletions of the tarsal segments occur. These defects might arise because Antp competes with Dll for binding to its target genes in the distal leg. In most other contexts examined, Antp is an activator of transcription; why it fails to activate D4 is not clear. The similar behavior of Dll and Antp in binding to D4 supports the idea that Dll behaves like a Hox protein in activating D4 (Duncan, 2010).

Although the initial focus of this study was on the antenna, the finding that Antp interacts directly with D4 led to an examination of D4 regulation in the leg, where Antp is normally expressed. In second leg imaginal discs, Antp is required only in a proximal ring of cells that coexpresses Dll and Hth. This ring appears in the early third instar, and is of uncertain function. Large Antp clones in T2 leg discs that do not enter this ring appear to develop completely normally, regardless of whether they are located distal or proximal to the ring. However, clones that overlap the ring show activation of D4/lacZ within the ring cells. Importantly, such clones have no effect on the expression of Dll or Hth within the ring. By examining Antp clones of increasing age the following sequence of events is inferred. First, D4/lacZ is activated in cells of the ring that are included within Antp clones. Second, many such clones begin expressing the antennal markers Ss and Cut, indicating a transformation to antenna, and round up as if they have lost affinity for neighboring cells. Third, such clones appear to extend and move distally in the disc (Duncan, 2010).

The events described for Antp clones in the leg make sense of several previously enigmatic observations. It has been noted that many Antp clones in the leg do not transform to antenna and appear to develop normally. The finding that only clones that overlap the proximal ring undergo transformation accounts for this observation. Antp clones that do contain transformations usually show apparent nonautonomy in that not all cells in the clone are transformed to antenna. The current results account for this observation as well, since within an Antp leg clone only those cells located in the proximal ring undergo transformation to antenna; cells located elsewhere in the clone retain normal leg identity. Most importantly, these observations provide an explanation for why ss is controlled directly by Antp. Antp clones have no effect on hth or Dll expression in the proximal ring. Therefore, Antp must function in the ring at the target gene level to repress antennal genes that would otherwise be activated by combined Hth and Dll (and Exd). Since several such targets are known, it seems likely that several, perhaps many, antennal genes in addition to ss are repressed directly by Antp (Duncan, 2010).

Transformed Antp clones in the leg often show ectopic hth expression in distal locations. If hth is not directly controlled by Antp in the leg, as this study suggests, then why is hth ectopically expressed within such clones? A likely explanation is that downstream antennal genes that have become activated in such clones feed back to activate hth. This interpretation is strongly supported by the finding that ectopic expression of the antennal genes ss, dan, or danr in the distal leg causes ectopic activation of hth. Thus, the distal expression of hth seen in Antp leg clones is likely a consequence rather than a cause of the transformation to antenna. Whether repression of hth in the antenna by ectopic Antp is also indirect is not clear. Dll is also expressed ectopically in transformed Antp leg clones, suggesting that it is also subject to feedback activation by downstream antennal genes (Duncan, 2010).

The function of the proximal Dll- and Hth-expressing ring in the proximal leg is not well understood. The ring is highly conserved among the insects, and may serve as a boundary between the proximal and distal portions of the legs. In the context of this work, a striking feature of the ring is that it contains a microcosm of gene expression domains corresponding to the three major antennal segments. Thus, proceeding from proximal to distal through the ring, cells express hth alone, hth + Dll, and hth + Dll + strong dachshund. These expression combinations are characteristic of the A1, A2, and A3 antennal segments, respectively. Looked at in this way, the ring would appear to resemble a repressed antennal primordium within the leg (Duncan, 2010).

It has been known for almost thirty years that Antp is required in the leg to repress antennal identity. However, an understanding of how this repression occurs has been lacking. The current results indicate that Antp functions within the proximal ring to directly repress antennal genes that would otherwise be activated by combined expression of Dll, Hth, and Exd. This appears to be the only function of Antp in the leg, at least during the third instar larval stage. The results are entirely consistent with the idea that second leg is the 'ground state' ventral appendage (the limb type that develops in the absence of identity specification) and that the role of Antp in the leg is to preserve this ground state by repressing the activation of 'head-determining' genes (Duncan, 2010).

Transcriptional Regulation

In both the antenna and leg, spineless expression is shown to depend on Distal-less (Dll), a master regulator of ventral appendage formation The Dll gene is required for the development of all leg segments distal to the coxa. To test whether spineless lies downstream of Dll in limb development, spineless expression was examined in a weak Dll loss-of-function mutant, DllPK. This allele survives to the pharate adult stage when heterozygous with Dll null alleles such as DllB, and causes the deletion of distal limb structures. spineless expression is almost completely eliminated in the tarsus, antenna, and maxillary palp of DllPK/DllB heterozygotes. Thus, spineless lies downstream of Dll in all three of these appendages. In the antenna, spineless expression is reduced in animals that carry only one dose of Dll+. This presumably accounts for the weak transformation of distal antenna to leg seen in most Dll mutant heterozygotes (Duncan, 1998).

To monitor Dll expression in relation to spineless, a monoclonal antibody was isolated against Dll protein. Dll is expressed uniformly in the central portions of the leg and antennal imaginal discs. In the early third instar, when spineless is first expressed in the leg, the outer edge of the spineless tarsal ring coincides precisely with the proximal limit of Dll expression. As the leg disc grows, the boundary of Dll expression expands beyond the spineless tarsal ring, so that a proximal zone of cells that express Dll, but not spineless, is created. In the antenna, Dll expression extends more proximally than spineless at all stages examined (Duncan, 1998).

Hox genes regulate the same character by different strategies in each segment

Hox genes control regional identity along the anterior-posterior axis in various animals. Each region contains morphological characteristics specific to that region as well as some that are shared by several different regions. The mechanism by which one Hox gene regulates region-specific characteristics has been extensively analyzed. However, little attention has been paid to the mechanism by which different Hox genes regulate the same characteristics in different regions. This study shows that two Hox genes in Drosophila, Sex combs reduced and Ultrabithorax, employ different mechanisms to achieve the same out-put, the absence of sternopleural bristles, in the prothorax and metathorax, respectively. Sternopleural bristles are characteristics of the mesothorax, and it was found that spineless is involved in their development. Analysis of the regulatory relationship between Hox genes and spineless indicated that ss expression is repressed by Sex combs reduced in the prothorax. Since sole misexpression of ss could induce ectopic sternopleural bristle formation in the prothorax irrespective of the expression of Sex combs reduced, spineless repression appears to be critical for inhibition of sternopleural bristles by Sex combs reduced. In contrast, spineless is expressed in the metathorax independently of Ultrabithorax activity, indicating that Ultrabithorax blocks sternopleural bristle formation through mechanisms other than spineless repression. This finding indicates that the same characteristics can be achieved in different segments by different Hox genes acting in different ways (Tsubota, 2008).

This study found that three genes, Antp, ss and al, are involved in sternopleural bristle formation. In the al mutant, no appreciable Ac expression in the T2 leg disc is detected and sternopleural bristles are not formed, indicating that the requirement of al is absolute. In contrast, Ac expression is detectable in the ss mutant T2 leg disc and in the Antp mutant clones, indicating that the requirement of both ss and Antp for ac expression is not absolute. However, sternopleural bristles were never found in the ss mutant, despite the fact that Antp expression was unaffected in the ss mutant clone in the T2 leg disc. In contrast, Antp mutant cells, in which ss is expressed normally, formed sternopleural bristles. In addition, sole misexpression of ss in the T1 segment produces sternopleural bristles ectopically, while that of Antp did not. Therefore, ss appears to be necessary and sufficient for sternopleural bristle formation, while Antp appears to be insufficient and not necessarily required. Moreover, Ac expression is ectopically induced in the T1 leg disc by misexpression of ss but not of Antp and in the ss mutant T2 leg disc is very weak, highly restricted, and only transient. This indicates that ss but not Antp appears to be one of the major activators of ac expression. Taken together, ss appears to be much more fundamental for sternopleural bristle formation than Antp (Tsubota, 2008).

The initiation of ac expression coincides with the initiation of ss expression. Since al and Antp are already expressed before ac induction in the early third instar stage, the timing of ac induction may be determined by the regulation of ss expression. Interestingly, the residual Ac expression seen in the ss mutant leg disc is first observed in the mid third instar as in the wild-type leg disc. This implies that at least one additional gene (referred to as X hereafter), whose expression or function is activated at the same stage as the initiation of ss expression, may be involved in ac induction. One possibility may be a gene functioning in hormonal regulation. Nonetheless, the ability of the sole misexpression of ss to induce ectopic ac expression and sternopleural bristle formation strongly indicates that ss is much more fundamental than X (Tsubota, 2008).

The restriction of ac expression to the overlap between the ss and al expression domains indicates the importance of determining the distal limit of ss expression and the proximal limit of al expression. Analysis of clones lacking ss activity or misexpressing ss indicates that ss has a repressive activity on al expression. How can al be expressed in the overlap domain? In the overlap domain, ss represses al expression when misexpressed at high levels but does not when misexpressed at approximately endogenous levels. The level of ectopic Al expression in the ss mutant clone located in a region proximal to the normal al expression domain is lower than that of endogenous Al expression. Moreover, Al expression in the wild-type leg disc gradually decays at its proximal edges. Considering all of these observations, the following hypothesis is suggested: al expression is activated according to the proximodistal information and the proximal limit of the al expression domain may be determined by a balance between activation according to the proximodistal information and repression by ss. The activation force may dominate the repressive activity of ss in the overlapping region but may gradually decay towards the proximal edges of the al expression domain. In contrast, ss expression does not appear to be regulated by al. As with the case of al activation, it may be possible that ss is repressed according to the proximodistal information (Tsubota, 2008).

The morphological identities of the T1 and T3 segments, including the absence of sternopleural bristles, are determined by Scr and Ubx, respectively. Analyses of the T1 leg disc with Scr mutant clones and the T2 leg disc with ectopic Scr activity indicate that both ss and Antp are repressed by Scr in the T1 leg disc. In addition, there is a possibility that the expression or function of gene X is repressed by Scr. Weak Ac expression is transiently observed in the ss mutant T2 leg disc, indicating that ac expression can be weakly activated without ss activity in the presence of gene X and Antp activity. In addition, Scr does not appear to repress ac expression directly, since ectopic induction of ac by ss misexpression in the T1 leg disc was not associated with an alteration in Scr expression. If gene X is active in the T1 leg disc, sole misexpression of Antp is expected to activate ac expression at least weakly and transiently. However, no ectopic Ac expression was found upon sole misexpression of Antp. Therefore, the activity of gene X is likely to be repressed in the T1 leg disc. For evaluating the significance of these three genes on Scr-dependent inhibition of sternopleural bristle formation, the ability of ss misexpression to induce ectopic ac expression and sternopleural bristle formation without affecting Scr expression is of crucial importance. At present, whether ac expression and sternopleural bristle formation can be induced solely by ss or only in a combination of ss and Antp and/or gene X is unclear. However, ss misexpression induced Antp expression and, thus, at least ss and Antp were coexpressed upon sole misexpression of ss. As for gene X, if it is not activated by ss misexpression, the results indicate that ac expression and sternopleural bristle formation can be induced without gene X activity at least in the presence of both ss and Antp expression. In contrast, if ac expression and sternopleural bristle formation require gene X activity, ss misexpression must activate gene X. After all, the results indicate that sole misexpression of ss can fulfill at least a minimum requirement for ac expression and sternopleural bristle formation. In other words, if Scr could not repress ss expression, ac expression would be activated and sternopleural bristles would be formed irrespective of the expression and function of Antp and gene X. Therefore, Scr must repress ss expression and this appears to be a key step to block sternopleural bristle formation in the T1 segment (Tsubota, 2008).

In contrast to the T1 leg disc, strong Ss expression was observed in the wild-type T3 leg disc and it is unaltered in Ubx mutant clones. Therefore, Ubx appears to act through a mechanism unrelated to ss expression. How does Ubx function? Simultaneous expression of both ss and Antp seemed insufficient for ac expression and sternopleural bristle formation in the T3 segment, since Antp misexpression failed to induce Ac expression in the T3 leg disc, in which ss is prominently expressed. It may be possible that Ubx represses ac expression directly. Alternatively, Ubx may compromise the function of the Ss protein directly or indirectly through regulation of its downstream gene products. Another possibility is that Ubx acts through repression of gene X activity. These possibilities are not mutually exclusive with each other (Tsubota, 2008).

The occurrence of ac expression and sternopleural bristle formation in the absence of Antp activity indicates that the absence of sternopleural bristles is not the ground state. However, the number of sternopleural bristles is variable in that condition, indicating that the complete formation of sternopleural bristles is not also the ground state. Since ss misexpression experiment suggests that sternopleural bristles can be formed as long as ss is expressed, one possible aspect of the ground state may be the expression of ss and the production of at least some kind of bristles. Antp may have acquired the ability to modify this state to produce the current-type of sternopleural bristles. On the other hand, Scr may have evolved the ability to block sternopleural bristle formation by acquiring the activity to repress ss expression and Ubx by acquiring another, yet unknown function. Taken together, the current state of sternopleural bristles in all three thoracic segments appears to be the derived state (Tsubota, 2008).

Targets of Activity

The transient early expression of spineless in the leg suggests that spineless plays a role in the establishment of the tarsal region. Support for such a role is provided by the finding that bric à brac (bab) lies downstream of spineless. In wild type, bab expression is initiated in the tarsal region in the mid-third instar; at disc eversion, bab expression can be seen to extend from the middle of the first tarsal segment through the fifth segment. In spineless null mutants, bab expression is abolished in the leg (Duncan, 1998).

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc (see Dan and Danr). Hernandez and Fernandez are the names of twin brothers in Tintin comic-books. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).

To test whether hern and fer are sufficient to induce eye or antennal development, they were expressed ectopically using the GAL4/UAS system. When either the hern or the fer genes are misexpressed in the leg discs with dpp-GAL4 or Dll-GAL4 (EM212) drivers, distal legs are transformed to aristae. These transformations are accompanied by the ectopic expression of hth, sal, and ss, three genes expressed in the antennal primordium but not in the distal region of mature wild-type leg disc. Clones expressing either the hern or the fer genes in the leg or wing disc have smooth borders and frequently activate the sal and hth genes cell-autonomously. In dpp-GAL4/UAS-fer or ptc-GAL4/UAS-hern leg (or wing) discs, the expression of ss is also activated. Curiously, although ss is downstream of hth in the antenna and leg, ectopic ss in the leg disc can also activate hth in a few cells (Suzanne, 2003).

The hth or ss genes, together with Dll, are sufficient to develop ectopic distal antennae when expressed in different regions of the adult. The hern or fer genes are also able to elicit this transformation in the leg and they activate hth and ss. Conversely, when high levels of the Hth or Ss products are induced in the leg discs, ectopic expression of the hern and fer genes is found. To study the interactions between these genes in normal development, the relationship between Dll, hth, ss, and hern/fer in the antennal primordium was examined. A reduction of Hth activity using a dominant negative form of hth (UAS-EN-HTH1-430) results in a decreased activity of the MD634 and AC116 GAL4 lines, which reveal hern and fer expression, respectively. Similarly, in antennal discs of a Dll strong hypomorph or a ss null mutation, the expression of hern and fer disappears. These results suggests that hth, Dll, and ss are required to maintain hern and fer expression in the antenna. By contrast, high levels of hern or fer may reduce hth expression. In dpp-GAL4/UAS-fer or dpp-GAL4/UAS-hern larvae, the expression of hth (and sal) in the third antennal segment is eliminated or strongly reduced dorsally (where levels of hern and fer are high) and does not change or is ectopically activated ventrally (where levels of hern and fer are low). Similarly, fer-expressing clones are able to downregulate hth expression in the antennal primordium. These results suggest that levels of hern and fer expression may be important for a normal antennal development (Suzanne, 2003).

The differentiation of legs or antennae depends on the activity of the hth and Antp genes. The ss gene, however, is also able to transform distal leg (and also maxillary palp and rostral membrane) into distal antenna, and the absence of ss, like that of hth, transforms antenna into leg. Although ss seems to be downstream of Dll and hth in antenna specification, ectopic ss can activate hth in some cells of the leg disc. Similarly, misexpression of ss in the rostral membrane induces Dll expression. It seems, therefore, that ss can trigger an antennal genetic program when misexpressed in certain places (Suzanne, 2003).

The fer and hern genes are both required and sufficient to make part of the distal antenna. Four different genes, hth, ss, hern, and fer, are able to form distal antenna, together with Dll, when ectopically expressed. Their mutual regulation seems to differ when misexpressed in the leg disc or when normally expressed in the antennal primordium. In the leg disc, hern or fer activates hth and ss and, reciprocally, hth and ss induce hern and fer expression. Moreover, even ss can promote hth transcription, although just in a few cells. Taken together, these results suggest that the four genes can form distal antenna by activating each other's transcription when ectopically expressed (Suzanne, 2003).

In the third antennal segment, Dll, hth, and ss are required to activate hern/fer expression. Since ss is downstream of Dll and hth in the antenna, the activation of hern/fer by Dll and hth could be mediated by ss. It is noted, however, that the levels of hern and fer may modulate hth expression. Moderately increased levels of fer can activate hth in dpp-GAL4/UAS-fer discs but, when the levels of hern or fer in the antenna are highly increased, the transcription of hth is prevented. These results suggest that the total amount of hern and fer expression may be regulated in the antennal primordium. Accordingly, in clones mutant for danr (hern), the expression of dan (fer) is upregulated. Also supporting the conclusion that levels of hern and fer have to be regulated, it was found that, in ey-GAL4/UAS-hern or ey-GAL4/UAS-fer flies, where levels of either hern or fer are highly increased in the eye–antennal disc, both the eye and the antenna disappear (Suzanne, 2003).

The hern and fer genes can form ectopic aristae and eye tissue, but only in a limited number of regions of the adult cuticle. This is similar to what happens with other genes making ectopic antennae (hth, ss) or eye (eye-specification genes). This is due to the particular developmental context of the region where the genes are ectopically activated (Suzanne, 2003).

Many organisms respond to toxic compounds in their environment by inducing regulatory networks controlling the expression and activity of cytochrome P450 monooxygenase (P450s) detoxificative enzymes. In particular, black swallowtail (Papilio polyxenes) caterpillars respond to xanthotoxin, a toxic phytochemical in their hostplants, by activating transcription of the CYP6B1 promoter via several regions located within 150 nt of the transcription initiation site. One such element is the xenobiotic response element to xanthotoxin (XRE-Xan) that lies upstream of consensus XRE-AhR (xenobiotic response element to the aryl hydrocarbon receptor) and OCT-1 (octamer-1 binding site) element known to be utilized in mammalian aryl hydrocarbon response cascades. Two-plasmid transfections conducted in Sf9 cells have indicated that XRE-Xan, XRE-AhR and a number of other proximal elements, but not OCT-1, are critical for basal as well as xanthotoxin- and benzo[alpha]pyrene-induced transcription of the CYP6B1 promoter. Four-plasmid transfections with vectors co-expressing the Spineless (Ss) and Tango (Tgo) proteins, the Drosophila melanogaster homologues of mammalian AhR and ARNT, have indicated that these proteins enhance basal expression of the CYP6B1 promoter but not the magnitude of its xanthotoxin and benzo[alpha]pyrene induction. Based on these results, it is proposed that these Drosophila transcription factors modulate basal expression of this promoter in a ligand-independent manner and attenuate its subsequent responses to planar aryl hydrocarbons (benzo[alpha]pyrene) and allelochemicals (xanthotoxin) (Brown, 2005).

Protein Interactions

The Drosophila spineless gene encodes a basic-helix-loop-helix-PAS transcription factor that is required for proper specification of distal antennal identity, establishment of the tarsal regions of the legs, and normal bristle growth. ss is the closest known homolog of the mammalian aryl hydrocarbon receptor (Ahr), also known as the dioxin receptor. Dioxin and other aryl hydrocarbons bind to the PAS domain of Ahr, causing Ahr to translocate to the nucleus, where it dimerizes with another bHLH-PAS protein, the aryl hydrocarbon receptor nuclear translocator (Arnt). Ahr:Arnt heterodimers then activate transcription of target genes that encode enzymes involved in metabolizing aryl hydrocarbons. Ss functions as a heterodimer with the Drosophila ortholog of Arnt, Tango (Tgo). The ss and tgo genes have a close functional relationship: loss-of-function alleles of tgo were recovered as dominant enhancers of a ss mutation, and tgo-mutant somatic clones show antennal, leg, and bristle defects almost identical to those caused by ss minus mutations. The results of yeast two-hybrid assays indicate that the Ss and Tgo proteins interact directly, presumably by forming heterodimers. Coexpression of Ss and Tgo in Drosophila SL2 cells causes transcriptional activation of reporters containing mammalian Ahr:Arnt response elements, indicating that Ss:Tgo heterodimers are very similar to Ahr:Arnt heterodimers in DNA-binding specificity and transcriptional activation ability. During embryogenesis, Tgo is localized to the nucleus at sites of ss expression. This localization is lost in a ss null mutant, suggesting that Tgo requires heterodimerization for translocation to the nucleus (Emmons, 1999).

Ectopic expression of ss causes coincident ectopic nuclear localization of Tgo, independent of cell type or developmental stage. In the embryo, ss is expressed in the antennal segment, the gnathal segments, the leg anlage, and the peripheral nervous system. Strong nuclear accumulation of Tgo is seen in the antennal segment, which expresses the highest level of ss. Nuclear accumulation of Tgo is also observed in the gnathal segments (mandibular, maxillary, and labial), but the intensity of staining is relatively weak compared to the antennal segment. This correlates with the relatively weak expression of ss in the gnathal segments, when compared to the antennal segment. Nuclear localization of Tgo in the antennal and gnathal segments is dependent on ss, since it is not seen in a ss null mutant. The expression of ss in the appendage primordia and the peripheral nervous system also correlates with Tgo nuclear accumulation. Sensory cells that express ss are in close proximity to the tracheal cells that express trh. To distinguish these, embryos were labeled with anti-Trh and anti-Tgo. Non-tracheal cells that show nuclear Tgo are observed in the location of ss-expressing sensory cells. This non-tracheal Tgo nuclear accumulation is absent in ss mutant embryos. These results indicate that Tgo accumulates in the nuclei of ss-expressing antennal, gnathal and sensory cells, consistent with the formation and nuclear accumulation of Ss:Tgo heterodimers in vivo. Surprisingly, no significant Tgo nuclear accumulation is seen in the limb primordia, even though ss is expressed in these cells. This may reflect regulatory events idiosyncratic to the limb primordia, or a lack of sensitivity of the immunostaining, since the limb primordia express ss at considerably lower levels than the antennal segment. Tgo nuclear accumulation is also observed in the cells of the dorsal vessel. Since, sim, ss and trh are not expressed in the dorsal vessel, an additional bHLH-PAS protein may function in combination with Tgo in controlling the development or physiology of these cells, which comprise the Drosophila circulatory system. When ectopic expression of a UAS-ss transgene is driven by en-Gal4, Tgo is found to accumulate in nuclei in circumferential ectodermal en stripes. Similarly, expression of ss in mesodermal cells (driven by twi-GAL4) causes nuclear accumulation of Tgo in the mesoderm. These experiments support the conclusion that Ss and Tgo interact in vivo, and suggest that their interaction and nuclear accumulation does not depend on additional, spatially-restricted, factors. Despite the very different biological roles of Ahr and Arnt in insects and mammals, the molecular mechanisms by which these proteins function appear to be largely conserved (Emmons, 1999).

How did Ss and Ahr come to have such different functions in vertebrates and arthropods? One possibility is that Ahr functioned as some type of chemosensory protein in an ancestral organism. In vertebrates, this function became utilized by all cells to sense aryl hydrocarbon toxins, whereas in arthropods it became intimately associated with the specification of a major chemosensory organ, the antenna. It is hoped that studies of organisms from other lineages will shed light on how Ss and Ahr came to adopt such different roles (Emmons, 1999).

Legs and antennae are considered to be homologous appendages. The fundamental patterning mechanisms that organize spatial pattern are conserved, yet appendages with very different morphology develop. The distal antenna (dan) and distal antenna-related (danr) genes encode novel 'pipsqueak' motif nuclear proteins that probably function as DNA binding proteins serving as sequence-specific transcription factors but may serve instead as more general chromatin modification factors. dan and danr are expressed in the presumptive distal antenna, but not in the leg imaginal disc. Ectopic expression of dan or danr causes partial transformation of distal leg structure toward antennal identity. Mutants that remove dan and danr activity cause partial transformation of antenna toward leg identity. Therefore it is suggested that dan and danr contribute to differentiation of antenna-specific characteristics. Antenna-specific expression of dan and danr depends on a regulatory hierarchy involving homothorax and Distal-less, as well as cut and spineless. It is proposed that dan and danr are effector genes that act downstream of these genes to control differentiation of distal antennal structures (Emerald, 2003).

Dan plays an important role in specification of antennal identity downstream of spineless (ss), but rescue of the spineless mutation by Dan suggests that there may be additional genes acting downstream from ss to specify antennal identity. spineless mutants lead to ectopic expression of Antennapedia and concomitant loss of Dan/Danr expression and cause a strong phenotypic transformation of distal A3 and arista to tarsus. To determine whether morphological transformation depends on loss of Dan/Danr, use was made of Gal4 to direct Dan expression in the ss mutant discs. ptcGal4 directed expression of Dan causes strong suppression of the arista-to-tarsus transformation in the ss mutant antenna. ptcGal4 is expressed in a stripe of cells adjacent to the AP boundary in the antenna region of the disc. Dan expression does not repress ectopic expression of Antp in the ptcGal4 stripe of the mutant discs. This suggests that Dan can direct antennal differentiation in the presence of Antp, and overcome the ability of Antp to cause transformation to tarsus. Remarkably, this transformation can affect the entire distal arista, even though ectopic Dan is expressed in only a subset of Antp-expressing cells. These observations suggest that Dan plays an important role in specification of antennal identity (Emerald, 2003).

An additional line of evidence to indicate that both genes contribute to distal antenna identity comes from examining genetic interactions with spinelessaristapedia. ssa mutants lose Dan/Danr expression and express Antennapedia ectopically in the antenna disc. Restoring Dan expression is able to partially suppress the transformation to antenna, implicating Dan as an effector of ssa function. The consequences of removing one copy each of Dan and Danr was examined in a ss mutant background. The spineless114.4 allele shows a mild transformation of the basal capsule of the arista when heterozygous, suggesting that the reduced level of ss activity in this allele is not sufficient to support normal development. Removing one copy of danr using the danrex35 deletion in this background causes a modest increase in the size of the basal capsule and in the number of ectopic bristles. The dan danrex56 deletion causes a stronger phenotype, with the basal capsule adopting a two-segment structure with multiple bracted bristles and obvious tarsal morphology. Flies heterozygous for the dan danrex56 deletion are morphologically normal. Thus, reduction of both Dan and Danr gene dose leads to a more severe phenotype under conditions where ss activity is limiting. Even more extreme arista transformation phenotypes are observed when one copy of ss is removed in animals homozygous for the dan danrex56 deletion (Emerald, 2003).

Loss of Hth activity has been shown to cause transformation of arista to tarsus, presumably because of loss of ss. It has been suggested that uniform expression of Hth in second and early third instar antennae might be responsible for its role in specification of distal antenna identity. However, the results of this study indicate that Hth can have a non-autonomous effect on the expression of Dan in the antenna. Hth-expressing cells sort out from the distal part of the leg. Nonetheless they are able to induce Dan expression in cells that remain integrated in the distal leg. This observation is best explained by a non-autonomous induction of Dan in response to a signal from Hth-expressing cells. Responsiveness to this signal apparently requires Dll, which limits it to the distal region. These effects are presumably mediated by regulation of ss, which is required for Dan and Danr expression. These observations provide an explanation for the apparently non-autonomous role of Hth together with Dll in the distal antenna (Emerald, 2003).

ss is also required to induce Dan and Danr and to repress Antp expression. Repression of Antennapedia may be mediated in part by repression of Cut. The findings described above implicate Dan and Danr as downstream effectors of ss that promote development of distal antennal structures. Remarkably, expression of Dan or Danr under Gal4 control can restore antenna development and prevent transformation of antenna to leg in the ss mutant, even when Antp is present. A striking feature of these results is that there appears to be non-autonomous activity. Transformation is blocked in cells expressing Dan and Danr, as well as in nearby cells that do not express these proteins. The identity of the genes responsible for these non-autonomous effects in antenna specification remains to be determined. In view of recent reports of non-autonomous effects of vein/EGFR signaling in development of distal leg pattern, it will be of interest to learn if there is a link to this pathway in the non-autonomous effects of Dan and Danr (Emerald, 2003).

spineless: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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