Transcriptional Regulation

aristaless expression appears to be under the control of wingless and decapentaplegic signaling pathways. Ectopic expression of wg can induce both ectopic al expression, but only in regions expressing high levels of dpp (Campbell, 1993).

BarH1 and BarH2 play essential roles in the formation and specification of the distal leg segments of Drosophila. In early third instar, juxtaposition of Bar-positive and Bar-negative tissues causes central folding that may separate future tarsal segments 2 from 3, while juxtaposition of tissues differentially expressing Bar homeobox genes at later stages gives rise to segmental boundaries of distal tarsi including the tarsus/pretarsus boundary. Tarsus/pretarsus boundary formation requires at least two different Bar functions: early antagonistic interactions with a pretarsus-specific homeobox gene, aristaless, and the subsequent induction of Fas II expression in pretarsus cells abutting tarsal segment 5. Bar homeobox genes are also required for specification of distal tarsi. Bar expression requires Distal-less but not dachshund, while early circular dachshund expression is delimited interiorly by BarH1 and BarH2 (Kojima, 2000).

aristaless (al) is a homeobox gene expressed at the center of leg and antennal discs from early third instar onwards. Initially, the Al expression domain and early Bar ring overlap slightly in leg discs. Al/Bar overlapping can be more clearly seen in early antennal discs. Up to 90 hours AEL, the central part of the leg disc is strictly divided into two regions, Bar-positive/Al-negative and Bar-negative/Al-positive circular domains. In antennal discs, such discrimination in Bar/Al expression may be incomplete. Regionally exclusive expression of Bar and Al may be due to mutually antagonistic interactions between Bar and Al. When Al expression is examined in mid to late third instar larval leg discs having Bar minus clones, Al expression is seen to invade the Bar minus presumptive tarsus region, while Al expression is considerably attenuated by Bar misexpression along the anteroposterior compartment border in mid third instar discs. Ectopic patches of Bar expression are frequently observed in the presumptive pretarsus of hypomorphic al leg discs in late third instar. Bar derepression due to reduction in Al activity is more clearly observed in antennal discs; on a hypomorphic al mutant background, Bar is expressed in the centralmost region even at late third instar larval stages. That no appreciable repression of Bar is detected when al is misexpressed may indicate the involvement of factors other than Al in Bar repression in presumptive pretarsus. In addition, the lack of Al invasion into Bar minus clones in leg discs at early third instar larval stages may indicate that Bar is dispensable for repressing al when their expression is initiated (Kojima, 2000).

During Drosophila leg development, the distal-most compartment (pretarsus) and its immediate neighbor (tarsal segment 5) are specified by a pretarsus-specific homeobox gene, aristaless, and tarsal-segment-specific Bar homeobox genes, respectively; the pretarsus/tarsal-segment boundary is formed by antagonistic interactions between Bar and pretarsus-specific genes that include aristaless. Drosophila Lim1 is involved in pretarsus specification and boundary formation through its activation of aristaless. Ectopic expression of Lim1 causes aristaless misexpression, while aristaless expression is significantly reduced in Lim1-null mutant clones. Pretarsus Lim1 expression is negatively regulated by Bar and is abolished in leg discs lacking aristaless activity, which is associated with strong Bar misexpression in the presumptive pretarsus. No Lim1 misexpression occurred upon aristaless misexpression. The concerted function of both Lim1 and aristaless is required to maintain Fasciclin 2 expression in border cells and form a smooth pretarsus/tarsal-segment boundary. Lim1 is also required for femur, coxa and antennal development (Tsuji, 2000).

To isolate genes that possibly act with al in pretarsus specification, a search was made for genes expressed in the pretarsus but not the segment immediately adjacent to it at late third instar stages. P0092 is an enhancer trap line, in which lacZ expression in leg and antennal discs was found to be similar to al expression in these tissues. lacZ is coexpressed in virtually all Al-positive cells in the pretarsus, tibia, femur and possibly coxa in leg discs, and the arista and first antennal segment in antennal discs. Although Al expression is restricted to ventral cells in the tibia and dorsal cells in the femur, coxa and first antennal segment, lacZ expression has been noted in both ventral and dorsal cells uniformly, which gives rise to complete circular expression. In wing and haltere discs, in which al is also expressed, no appreciable expression of P0092-lacZ is observed. Nucleotide sequence analysis indicates that the putative P0092 gene encodes a LIM-homeodomain protein identical in amino acid sequence to Lim1 (Tsuji, 2000).

Flies neither homozygous nor hemizygous for the P0092 P insertion show any obvious morphological defects. Thus, Lim1 loss-of-function mutants generated by imprecise P-element excision and six independent larval or pupal lethal mutant lines were obtained. These frequently produce pharate adults with apparent defects in mouth parts, leg and antennal morphology, making it possible to examine the roles of Lim1 in leg and antennal development (Tsuji, 2000).

In legs and antenna completely lacking al activity, all pretarsus structures and arista are lost, respectively. In moderate hypomorphic al mutants, claws are frequently lost without loss of other pretarsus structures such as pulvilli and empodia, while in weak hypomorphic mutants, claws and aristae are not lost but only reduced in size. Lim1 minus mutants are very similar in leg phenotype to moderate al hypomorphic mutants. In about half of all cases, the antenna are absent from the Lim1 minus half head. When antennae is present, arista is deformed and reduced in size. That is, Lim1 minus arista are morphologically similar to those of weak hypomorphic al mutants. These findings indicate that Lim1 is essential for proper development of pretarsus and arista as well as al, although Lim1 mutant phenotypes are much less severe than a1 minus mutant phenotypes. In Lim1 minus legs that are simultaneously homozygous for al, not only claws but also empodia and pulvilli are frequently lost. The concerted function of Lim1 and al would thus appear to be required for normal pretarsus/aristal development (Tsuji, 2000).

Apart from the future pretarsus, Lim1 is expressed circularly in proximal segments such as the coxa, femur and tibia. In Lim1 minus flies, the femur is extensively reduced in size and the coxa is missing for the most part or present only as a small bulb-like structure, suggesting the requirement of Lim1 for proper development of the femur and coxa. Although the tibia is bent and fused with the femur, morphological analysis indicates the presence of essentially normal characteristic structures of the tibia, such as transverse rows of bristles, preapical bristles, tibial sense organs and tibial sensilla trichodea; tibial sense organs and tibial sensilla trichodea are structures situated near the proximal tibial end. The tibial phenotype may thus possibly derive from secondary effects of the femoral deformation. In late third instar, Dll expression is evident in the central region spanning from the most distal tip to distal half of the tibia and in the future trochanter. Consistent with shortening of the femur, appreciable reduction in mass has already taken place in the region flanked by the central Dll domain and the proximal Dll ring at late third instar. In Lim1 minus leg and antennal discs, Al expression in the proximal region, such as in the femur, coxa and first antennal segment, is virtually absent. In Lim1 mosaic clones in the femur or coxa, Al expression is abolished cell autonomously. Tibial Al expression remains in Lim1 discs but mosaic analysis clearly indicates substantial reduction in Al expression in Lim1 clones. al is expressed considerably prior to Lim1 and thus Lim1 may thus be involved in maintenance of pretarsus al expression. But loss of Al expression would not completely explain the femoral and coxal defects, since al is dispensable for normal development of the femur and coxa (Tsuji, 2000).

Mutually antagonistic interactions between al and Barare essential for the strict separation of Al and Bar domains, leading to localized Fas2 induction by Bar in border cells. Although the absence of Lim1 shows little Bar misexpression in the pretarsus, increased Bar misexpression in Lim1;al leg discs could indicate the involvement of Lim1 in the repression of Bar expression in the pretarsus. Remarkable decrease in Fas2 expression in putative Lim1;al mutant border cells indicates that Fas2 expression requires al and Lim1 functions, in addition to cell non-autonomous functions of Bar. Lim1 may be involved in pretarsus specification and boundary formation only through its activation of al. Low al expression in Lim1 single mutants may still be sufficient for maintaining the normal expression of Bar and Fas2, but with further reduction in al expression in Lim1;al double mutants, Bar misexpression and loss of Fas2 expression may result. Alternatively, Lim1 may act independently of al, and simultaneous reduction in al and Lim1 expression may cause Bar misexpression and reduction of Fas2 expression in the double mutants. These considerations are not mutually exclusive (Tsuji, 2000).

The morphogenesis of specialized structures within the CNS relies on the nonautonomous activity of cell populations that play the role of organizers. In the Drosophila visual system, cells on the dorsal and ventral margins of the developing visual cortex express the Wnt family member Wingless (Wg) and the TGF-beta Decapentaplegic (Dpp). The activity of these morphogens in establishing cortical cell fates sets the stage for the guidance of photoreceptor axons to their retinotopic destinations in the Drosophila brain. One role for Wg in cortical development is to induce and maintain the expression of Dpp, a key step in the assignment of dorsoventral cell identities. Dpp is induced early in cortical development, shortly after the onset of Wg expression in a few dorsal and ventral margin cells, and is maintained by Wg activity until at least the time of retinal axon pathfinding. Wg is a developmental signal in many different tissues, and acts by regulating different target gene sets to elicit a constellation of different cell fates. Wingless-controlled targets include distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, labial in the gut, and sloppy-paired in the embryonic CNS. Conversely, Dpp belongs to a Hedgehog-controlled circuit in the wing (Song, 2000 and references therein).

A regulatory mechanism is described that relays Wg signal reception to the tissue-specific expression of target genes in the visual cortex. In a screen for mutants in which photoreceptor axons project aberrantly to their destinations in the brain, a mutation in combgap was discovered. Retinal axon navigation defects in combgap animals are due to the role of cg in the establishment of cortical cell identity. cg represses the expression of Wg target genes in a positionally restricted manner in the visual cortex. wg+ induction of its cortical cell targets occurs via the downregulation of cg. Combgap is thus a tissue-specific relay between Wingless and its target genes for the determination of cell fate in the visual cortex (Song, 2000).

A combgap mutation was recovered in a screen for mutants with aberrations in retinal axon projections. On the basis of its effects on target region gene expression and the outcome of mosaic analysis, it is evident that a role for combgap in the specification of cortical cell identity underlies its requirement for the establishment of retinotopic connectivity in the visual system. In cg loss of function animals, three markers under wg+ control are expressed in expanded dorsal and ventral portions of the retinal axon target field. The requirement for cg to repress the markers within these domains is autonomous. The lamina midline region, however, appears phenotypically normal in homozygous or mosaic cg animals. This positionally restricted requirement for cg+ activity is correlated with the pattern of cg expression, since cg is not expressed in the midline region where it is not required. Since wg+ misexpression is sufficient to induce wg+-dependent markers in the midline region, another regulatory system must control these markers there. Hence, the consequences of wg signal reception at different dorsoventral positions within the cortical precursor field would appear to involve a set of regulatory molecules that divide the cortex into specific domains for pattern formation (Song, 2000).

At hatching, approximately 40 cortical cell precursors form a disc-shaped epithelium on the ventrolateral surface of each brain hemisphere. The epithelium is divided into lamina and medulla precursor zones, which can be distinguished by the expression of Cubitus interruptus (Ci) in the prospective lamina cortex. Cells in two domains at the prospective dorsal and ventral margins of the adult optic lobe begin to express wingless in the mid-first instar stage. dpp expression begins after the onset of wg expression and continues in domains immediately adjacent to the Wg-positive cells. Two additional dorsoventral-specific markers are optomotor blind (omb) and aristaless. Omb is expressed in dorsal and ventral domains that include both the Wg- and Dpp-positive cell populations. Omb-positive glia migrate from these domains toward the lamina midline. aristaless, as assayed by the al04352 enhancer trap insertion (al-lacZ), is expressed in a graded pattern with respect to distance from the Wg-positive cells. The expression of omb, dpp, and al-lacZ is induced by ectopic wg+ expression and absent under conditions of wg loss of function. These observations indicate that Wg is responsible for the expression of these three markers (Song, 2000).

The cell autonomy of combgap function was determined by generating somatic cgk11504 clones using the FLP, FRT method. Within cg clones outside of the midline region, Omb, Dpp, and al-lacZ are all expressed ectopically. Clones or portions of clones that fall within the midline region (30% of those examined) appeared phenotypically normal, consistent with the lack of a cg requirement for the midline region in homozygous animals. There are also position-specific effects observed within cg clones. For example, not all cells within a cg clone expressed the marker Dpp. The position-specific ectopic gene activation in cg clones might reflect the activity of other signals involved in cortical cell fate determination. cg thus behaves as an autonomous repressor of omb, dpp, and al-lacZ expression, except in the midline region where it is not required (Song, 2000).

The constellation of genes under Wingless control displays considerable tissue specificity. Wingless-controlled targets include Distal-less and vestigial in the wing, engrailed in the embryonic ectoderm, and sloppy-paired in the embryonic CNS. Though Dpp and Omb belong to a Hedgehog-controlled circuit in the wing, they are under Wg control in the visual cortices of the brain. With respect to the control of cell fate, Wg signal transduction apparently follows a canonical pathway from a pair of redundant receptors at the cell surface to the cytoplasmic control of Armadillo stability and nuclear translocation. This raises the question of how the tissue specificity of wg target gene expression is achieved (Song, 2000).

The observations that cg regulates dpp, optimotor blind and aristaless in the visual cortex place cg in a second tier of regulation, as a component of a tissue-specific relay mechanism between the Wg signal transduction pathway and the target genes that are wg dependent in visual system cortical cells. The evidence in support of this hypothesis is as follows: (1) epistasis analysis with the wg pathway negative regulator Axn places the requirement for cg downstream of the cytoplasmic complex that includes APC, GSK-beta, and Armadillo; (2) the induction of at least three downstream effectors of wg+ activity is mediated by negative regulation of cg expression -- cg expression is reduced in the dorsal and ventral domains of the cortical lamina where these wg target genes are expressed and ectopic cg expression blocks wg target gene expression within these domains; (3) ectopic wg+ clones repress cg expression, yielding Cg-negative domains in which wg target genes are ectopically expressed. The presence of consensus Pangolin binding sites in the first intron of cg suggests cg may be a direct target of Wg signal transduction. How the Armadillo/Pangolin complex might participate in the negative regulation of cg is unclear. Cg might act by binding directly to wg target gene regulatory elements as a transcriptional repressor (Song, 2000).

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

Initially, tests were performed to see whether Wg and Dpp directly pattern the proximodistal axis of the tarsus by determining their role in activation of the aristaless (al) gene in the center of the disc. al encodes for a homoeodomain protein required for development of structures found at the tip of the leg, including the claws. Previous studies indicated that al expression is activated by Wg and Dpp and this was confirmed with loss of function studies: al expression is absent from the center of wg and dpp mutant discs. However, this does not rule out al being activated by a secondary signal, which in turn is activated by Wg and Dpp. To test this, al expression was monitored in discs containing clones of cells mutant for genes required for transduction of Wg or Dpp signals, including arrow (arr), which encodes for a Wg co-receptor, and thickveins (tkv), which encodes a Dpp receptor (clone founder cells were generated before the onset of al expression). Central al expression is absent in discs consisting largely of arr mutant clones, but, as in wgts discs, such large clones would remove any putative secondary signal, and, in fact, further analysis revealed that al is still expressed in arr mutant cells located outside of the very center. Similarly, al can still be detected in tkv mutant cells. Thus, Wg and Dpp signalling are required, but not directly, to induce al, suggesting that it is activated by a secondary signal, which in turn is activated by Wg and Dpp (Campbell, 2002).

The EGF-receptor (EGFR) signalling pathway was tested as a potential activator of al because the claws are lost in Egfr hypomorphic mutants (the claws are also lost in al mutants. Use of a temperature-sensitive (ts) allele showed that Egfr is required for development of almost all of the tarsus. After a 24-h shift to the restrictive temperature during the first half of the third instar, Egfrts adults have leg truncations with the size of the truncation increasing with temperature. At lower temperatures only the most distal structures, the claws, are lost, but at high temperatures tarsal segments II–V are absent. Intermediate truncations result at temperatures between these extremes. These shifts affect only patterning of the tarsus, apart from at the highest temperature, 33°C, when development of more proximal regions is occasionally disrupted; this may reflect a low level EGFR signalling requirement for proliferation as has been shown in the eye disc. The temperature-dependent truncations of the tarsus suggest that development of the most distal region requires the highest level of EGFR activity, whereas more proximal regions require progressively less; that is, the tarsus may be patterned by a distal-to-proximal gradient of EGFR activity (Campbell, 2002).

Similar results were obtained analyzing marker gene expression in Egfrts discs, including al, Bar (B, expressed in segments IV and V) and rotund (rn, expressed in segments II–IV). Loss of EGFR activity results in loss of al, B and rn expression, but al is lost at a lower temperature than B, which in turn is lost at a lower temperature than rn, indicating that the more distal the marker, the higher the EGFR activity level required for expression. Clonal analysis with Egfrts showed that this response to EGFR is cell autonomous, and that again, al requires higher EGFR activity than B. It was not possible to do similar tests for rn because at temperatures above 31°C Egfrts clones do not survive in distal regions, raising the possibility that rn expression may be lost in Egfrts discs simply because of reduced growth or cell survival in this region. However, expression of other genes, including wg and dpp, appears normal in Egfrts discs. This is also true for the Wg and Dpp target Dll, clearly demonstrating that Wg, Dpp and Dll are not sufficient to distalize the leg (Campbell, 2002).

Ectopic activation of EGFR signalling results in autonomous, ectopic expression of al and B in mid-third-instar discs. Curiously, not all regions of the disc respond identically, with ventral regions being the most responsive and lateral regions the least. The reason for this is unclear but it indicates that factors in addition to EGFR may be regulating expression of tarsal genes such as al, at least outside of their normal domains. Only the presumptive tarsus is responsive to ectopic EGFR activity; this is most evident in adult appendages where no defects in patterning can be observed outside of here. Other regions may be refractive to ectopic EGFR activity because expression of tarsal genes requires Dll, which is expressed only in distal regions under Wg and Dpp control (Campbell, 2002).

In addition to activating genes, EGFR signalling is required to repress genes in distal regions, and again different genes appear to be differentially sensitive, with some, such as B and rn, possibly being both activated and repressed above different thresholds. B, rn and dac are repressed in the center of wild-type discs, with dac being repressed over a wider region than B and rn. Lowering EGFR activity in Egfrts discs to a level sufficient only for loss of al, results in expression of B and rn in the center, but not dac. Raising the temperature still further results in extension of the dac domain to fill the center. Clonal analysis shows that Egfr acts autonomously to repress dac. Ectopic EGFR activity can also repress B, dac and rn but again predominantly in ventral regions (B is repressed mainly at later stages). Previous studies have shown that repression of dac in distal regions requires high levels of Wg and Dpp signalling, so all three pathways appear to be required to achieve this (Campbell, 2002).

A distal-to-proximal gradient of EGFR activity predicts a source of ligand(s) at the presumptive tip. Potential ligands are the TGF-alpha family members Spitz and Keren, and the neuregulin, Vn; the former require activation by the membrane protein Rhomboid (Rho), or the homolog Roughoid (Ru). Both vn and rho are expressed in the center of the leg disc in early third instars. Genetic studies show that they are redundant so that loss of either gene alone has no effect on tarsus development, but loss of both together along with ru, which shows partial redundancy with rho even though no expression can be detected, has marked effects on leg patterning and growth. Large ru rho vn triple mutant clones can result in truncations of the tarsus, although these are never as extreme as in Egfrts mutants, possibly because of the difficulty of removing all ligand-expressing cells at the center of early leg discs using this technique, or because the ru mutant used is not null. Wild-type tissue located at the tip of adult legs always correlates with rescue of tarsal development. In addition, misexpression of a secreted form of Spitz results in non-autonomous activation of al. Verification of high levels of EGFR signalling in the distal leg is revealed by expression of sprouty in this location; this is upregulated in many tissues by EGFR signalling (Campbell, 2002).

Although a quantitative response to EGFR activity is important, an additional factor that can influence patterning of the tarsus is timing, revealed by shorter temperature shifts with the Egfrts mutant. A 12-h shift to 33°C in the first quarter of the third instar also results in leg truncations, although these are less severe than those produced by the 24-h shifts described above. However, a similar 12-h shift later during the second quarter of the third instar results in legs with normal distal pattern elements (claw, tarsal segment V), but with fusions or deletions of intermediate segments II–IV, suggesting that distal regions are specified before more proximal ones. This phenomenon mirrors the temporal pattern of gene expression in these regions in wild-type discs: the distal markers al and B are expressed in very early third instars whereas the intermediate tarsal marker, rn, cannot be detected until several hours later. The early 12-h shift can prevent all al and B expression, correlating with the pattern defect. However, the later shift does not result in loss of rn expression (although the domain is smaller than in wild-type discs. It is possible that the patterning defect resulting from the later shift may be caused by the loss of other, as yet unidentified, EGFR targets at this time. Alternatively, growth and patterning of this region requires segmentation of the tarsus, which is controlled by Notch signalling, so it is possible that EGFR signalling may be involved in regulating this process in the tarsus. In addition, these shifts are associated with cell death, which may also contribute to the defective patterning (Campbell, 2002).

Targets of Activity

Proximal-distal leg development in Drosophila involves a battery of genes expressed and required in specific proximal-distal (PD) domains of the appendage. apterous is required for PD leg development, and the functional interactions between ap, Lim1 and other PD genes during leg development have been explored. A regulatory network formed by ap and Lim1 plus the homeobox genes aristaless and Bar specify distal leg cell fates in Drosophila (Pueyo, 2000).

The expression of Lim1 is very similar to that of the PD gene aristaless. al is expressed in the most distal part of the leg discs, and in two rings in the peripheral and medial regions. Confocal immunofluorescence staining has shown that the expression of Lim1 and al are coincident in the most distal parts of the leg and antenna disc. Furthermore, there are similarities between the al and Lim1 mutant phenotypes, since in al mutants the claw organ, the sternopleural bristles in the leg and the arista are missing or reduced. A possible functional relationship between the two genes was explored. Since al is expressed earlier than Lim1, one possibility is that Lim1 expression is regulated by al. The expression of Lim1 was examined in al mutants, and Lim1 was found to be lost in the presumptive tip of the leg where both genes are co-expressed. In contrast, al expression is normal in Lim1 mutants. These results suggest that the Lim1 locus is regulated by the al gene, one possibility being that the Lim1 gene is a direct downstream target of the Aristaless protein (Pueyo, 2000).

In both al and Lim1 mutants, the strongest pupal lethal alleles eliminate the claw and reduced the rest of the pretarsal organs, but do not eliminate the whole pretarsus in all legs. This could be due to a functional cooperation between al and Lim1, such that the presence of any one product in the absence of the other would still provide enough function for some organs of the pretarsus to occasionally develop. Alternatively, the remnant pretarsal organs observed in al and Lim1 mutants could be due to hypomorphy of the mutants available and no functional relationship needs to be implied between the two genes. It was reasoned that if a functional relationship does exist, a double mutant should show an enhanced phenotype: a double mutant Lim1R12.4;alice was generated, and found to be embryonic lethal. Therefore, al and Lim1 double mutants show a synergistic effect that might betray a functional relationship. This synergistic relationship is also shown in ectopic expression experiments. Ectopic expression of either Lim1 or al driven by the 30AGal4 line produces only small defects in the joint between tarsus four and five. However, simultaneous expression of both al and Lim1 in 30AGal4;UASLim1;UASal flies produces stronger defects, including partial or complete fusion of tarsus four and five (Pueyo, 2000).

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and Bar are mutually antagonistic, resulting in exclusion of Bar expression from the pretarsus. Although Al is necessary, it is not sufficient to repress Bar, indicating another factor is required. This factor has been identified as the product of the C15 gene, also termed clawless, a homeodomain protein that is a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al -- together, C15 and Al appear to directly repress Bar and possibly to activate Lim1. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl− border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap. Similar to aristaless, the maximal expression of C15 requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and C15 expression through Lim1 repression. Aristaless and C15 proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless (Campbell, 2005; Kojima, 2005).

Protein Interactions

Proximodistal patterning in Drosophila requires division of the developing leg into increasingly smaller, discrete domains of gene function. The LIM-HOM transcription factors apterous (ap) and Lim1 (also known as dlim1), and the homeobox genes Bar and aristaless (al) are part of the gene battery required for the development of specific leg segments. Genetic results show that there are posttranslational interactions between Ap, Bar and the LIM-domain binding protein Chip in tarsus four, and between Al, Lim1 and Chip in the pretarsus, and that these interactions depend on the presence of balanced amounts of such proteins. In vitro protein binding is observed between Bar and Chip, Bar and Ap, Lim1 and Chip, and Al and Chip. Together with evidence for interactions between Ap and Chip, these results suggest that these transcription factors form protein complexes during leg development. It is proposed that the different developmental outcomes of LIM-HOM function are due to the precise identity and dosage of the interacting partners present in a given cell (Pueyo, 2004).

Biochemical studies in vitro have shown that LIM-HOM transcription factors confer little transcriptional activation of target genes on their own. LIM-HOM proteins interact with a variety of proteins, including members of the bHLH family, the POU family and also other LIM family members, to control specific developmental processes. It has been suggested that these protein interactions confer specificity and modulate LIM-HOM activity. For example, Dlmo proteins reduce LIM-HOM activity, and Lbd proteins such as Chip modulate LIM-HOM activity by acting as a bridge between LIM-HOM proteins and Chip-binding cofactors, thus leading to the formation of heteromeric complexes. LIM-HOM protein activity functions in different contexts is the development of Drosophila (Pueyo, 2004).

Bar and ap genes are expressed in the fourth tarsal segment and are required for its proper development, whereas the al and Lim1 genes are expressed and required in the pretarsus. All of these genes encode putative transcription factors and display canonical regulatory relationships. Thus, al activates lim1 expression and then both genes cooperate to repress Bar expression in the pretarsus. Reciprocally, Bar represses al and lim1 expression while activating the expression of ap in tarsus four. After the refinement of their gene expression domains by these regulatory interactions, Bar directs tarsus five development, whereas cooperation between al and lim1 directs pretarsus development, and cooperation between Bar and ap directs tarsus four. The results of this study offer more evidence for the existence of this regulatory network, but also suggest an interesting role for direct protein interactions in its mechanism (Pueyo, 2004).

The cooperation between Bar and Ap on the one hand, and Al and Lim1 on the other, is likely to be carried out by transcriptional complexes involving Chip. The Chip protein is required for development of the tarsus four, five and pretarsus, and Gst (Glutathione-S-transferase-Chip fusion construct) experiments reveal Chip's ability to bind Ap, Bar, Lim1 and Al. However, the results also show that modulation of LIM-HOM protein activity by Chip alone does not explain distal leg development. For example, Ap function is not modulated primarily by Chip and Dlmo. The relative amount of Chip and Ap has to be grossly unbalanced before a phenotype is obtained in the leg, and dlmo is not expressed or required in leg development. Furthermore, the interaction between Ap and Chip does not confer the developmental specificity that allows LIM-HOM proteins to produce different outcomes in different parts of the leg. (1) Ap and Chip also interact in the wing and the CNS. (2) A chimaeric Lim3-Ap protein containing the LIM domains of Lim3 and the HOM domain of Ap does not behave as a dominant negative when expressed in tarsus four, and is even able to fulfil Ap function and rescue ap mutants. In the distal leg, developmental specificity seems to be achieved at the level of DNA binding and the transcriptional control of targets genes, mediated by partnerships between LIM-HOM and HOM proteins (Pueyo, 2004).

The evidence for this is presented first by dosage interactions between LIM-HOM and HOM proteins. Whereas there seems to be a relative abundance of endogenous Ap in tarsus four, an excess of Bar or Chip leads to a mutant phenotype, which is rescued by restoring the normal balance between Ap, Bar and Chip proteins in co-expression experiments. The effects observed could be explained simply by independent competition and the binding of Bar and Ap to Chip, leading, for example, to an excess of Bar-Chip complexes and a reduction of the pool of Chip available for Ap-Chip complexes. However, this hypothesis alone does not explain the additional dominant-negative effects of ectopic LIM-HOM and HOM proteins in tarsus four (Lim3, Islet and Al), which are also not mediated by transcriptional regulation but are nonetheless rescued by co-expression of appropriate endogenous proteins. For example, ectopic expression of UAS-islet or UAS-Lim3 in the ap domain produces loss of tarsus four without affecting Ap or Bar expression, and simultaneous co-expression of UAS-Bar partially suppresses this phenotype. If the sole effect of both UAS-Bar and UAS-Lim3 or UAS-islet were to quench Chip away from Ap, then simultaneous co-expression of Bar and Lim3 or Islet should worsen the phenotype, not correct it as observed. Moreover, ectopic expression of Islet or Lim3 proteins is not corrected by simultaneous co-expression of either UAS-Chip or UAS-ap. Altogether these results show instead that UAS-islet and UAS-Lim3 must interfere posttranslationally with Bar. The most direct explanation is that Islet and Lim3 have the ability to quench Bar protein into a non-functional state. Interestingly, the hybrid UAS-Lim3:ap does not behave as dominant negative but as an endogenous Ap protein in these experiments, since it does not produce a mutant phenotype on its own and it rescues UAS-Bar overexpression. This suggests that the LIM domains are not very specific when it comes to interaction with Bar, and points to the involvement of a common LIM-binding intermediary such as Chip. These results suggest that a protein complex involving Ap, Chip and Bar is the correct functional state of these proteins in tarsus four, and deviations from this situation into separate Bar-Chip, Ap-Chip, or Bar-Chip-Lim3 or Bar-Chip-Islet complexes leads to a mutant phenotype (Pueyo, 2004).

The notion of a protein complex involving Ap, Chip and Bar together is also supported by the Gst pull-down assays. The domain of Chip involved in Ap binding, the LIM interaction domain (LID), is not involved in Bar binding. However, the LID and the dimerisation domains of Chip are necessary to rescue the dominant-negative effect of UAS-Bar on tarsus four, suggesting a requirement for the formation of a complex with a LIM-HOM protein such as Ap. In agreement with this view, the Ap protein, and the LIM domains of Ap alone, are able to retain Bar protein in a Gst assay (Pueyo, 2004).

In the pretarsus, Al and Lim1 are possibly engaged in a partnership with Chip similar to that suggested for Ap, Chip and Bar. Synergistic cooperation between Al and Lim1 is required to direct pretarsus development and to repress Bar expression and function. Their cooperation entails a close functional relationship because a proper balance of Al, Lim1 and Chip is required, as is shown by the loss of pretarsal structures in UAS-Chip or UAS-Lim1 flies. Ectopic expression of LIM-HOM proteins in the pretarsus also disrupts pretarsal development without affecting Lim1 and Al expression. The possibility of direct protein interactions between Al, Lim1 and Chip is also suggested by the reciprocal ability of Al to interfere posttranscriptionally with Bar and Ap in tarsus four, and by the binding of Chip to Lim1 and to Al in in vitro experiments (Pueyo, 2004).

Comparison of tarsal development with other developmental processes illustrates how LIM-HOM proteins are versatile factors to regulate developmental processes. It had been observed that the outcome of LIM-HOM activity depends on their developmental context. This context can now be analysed as being composed of the presence, concentration and relative affinities of other LIM-HOM proteins, Ldb adaptors, and other cofactors such as LMO proteins and HOM proteins. It is proposed that the different developmental outcomes of LIM-HOM protein function could be due to the precise identity and dosage of cofactors available locally (Pueyo, 2004).

Ectopic expression experiments distort these contexts and lead to non-functional or misplaced LIM-HOM activities. In the wing, a finely balanced amount of functional Ap protein is modulated by Dlmo and Chip. Over-abundance of Chip stops the formation of functional tetramers in the wing but not in the CNS, where the relative amount of Ap, which is not modulated by Dlmo, is limiting for the formation of Ap-Chip functional complexes. In tarsus four, the Ap-Chip-Bar partnership is affected by experimentally induced over-abundance of Chip, presumably also because ectopic Ap-Chip tetramers typical of the CNS and the wing, and Bar-Chip complexes typical of tarsus five, are produced. Similarly, an excess of Bar might be interpreted by the cells as being a wrong developmental outcome, since high levels of Bar in the absence of Ap direct tarsus five development. Overexpression of Ap rescues this Bar dominant-negative effect, by restoring the relative amounts of Bar and Ap, which are determinant and limiting for tarsus four development. Finally, the dominant-negative effects produced by overexpression of either Chip or Lim1 in the pretarsus could either prevent the formation of Al-Chip-Lim1 complexes, or could favor the existence of Lim1-Chip complexes typical of the CNS (Pueyo, 2004).

The wing and the CNS models have postulated that Ap function is carried out by an Ap-Chip tetramer; however, the molecular scenario might be more complex. A new component of Ap-Chip complexes, named Ssdp, has been identified and is required for the nuclear localisation of the complex. Thus it is possible that an Ap-Chip tetramer also contains two molecules of Ssdp. In addition, different types of Chip-mediated transcriptional complexes and different regulators have been identified in other developmental contexts, such as in sensory organ development and thorax closure, in which the GATA factor Pannier forms a complex with Chip and with the bHLH protein Daughterless. Heterodimers of this complex are negatively regulated by a protein interaction with Osa. Thus, although the current results indicate that in different segments of the leg there exist specific interactions between LIM-HOM, Chip and HOM proteins, the involvement of further elements in these multiprotein complexes is not excluded (Pueyo, 2004).

The results support a partnership between HOM and LIM-HOM proteins in the specification of distinct segments of the leg, and the results are compatible with Ap-Chip-Bar, Bar-Chip and Lim1-Chip-Al forming transcriptional complexes. Although the characterisation of the target sequences, followed by further biochemical and molecular assays, is necessary to study the transcriptional mechanism of these interactions, it has been shown that LIM-HOM proteins can interact specifically and directly with other transcription factors to regulate particular genes. For instance, mouse Lim1 (Lhx1) interacts directly with the HOM protein Otx2. In addition, the bHLH E47 transcription factor interacts with Lmx1, and both synergistically activate the insulin gene. This interaction is specific to Lmx1, since E47 is unable to interact with other LIM-HOM proteins such as Islet. Moreover, Chip is able to bind to other Prd-HOM proteins, such as Otd, Bcd and Fz, to activate downstream genes. Chip also complexes with Lhx3 and the HOM protein P-Otx, increasing their transcriptional activity. The current results reinforce the notion of Chip as a multifunctional transcriptional adaptor that has specific domains involved in each interaction (Pueyo, 2004).

Experiments in Drosophila have demonstrated a conservation of LIM-HOM activity at the functional and developmental level in the CNS between Drosophila and vertebrates. In addition, xenorescue experiments have shown that the mechanism of action of Ap and its vertebrate homolog Lhx2 is very conserved in Drosophila wings, whereas ectopic expression of dominant-negative forms of chick Lim1, Chip, Ap and Lhx2 mimic both Ap and Lhx2 loss-of-function phenotypes. The developmental role of Ap, Bar and Al in the fly leg, and their putative molecular interactions may also have been conserved because their vertebrate homologs Lhx2, Barx and Al4 are also co-expressed in the limb bud. It is expected that the interactions between the LIM-HOM and Prd-HOM proteins shown here represent a conserved mechanism to specify different cellular fates during animal development (Pueyo, 2004).

Interaction between Clawless/C15 and Aristaless

The aristaless/clawless cooperation found in Bar repression in the pretarsus may possibly stem from the interactions between Al and Cll. GST pull-down assay was first conducted in vitro to confirm this possibility. Cll was tagged with GST, and a possible binding of Cll to Al was monitored by Western blotting of the eluents from a GST column with anti-Al antibody. GST-Cll was prepared using E. coli cells, and Al was synthesized using reticulocyte lysates. Al signals were detected only when a mixture of GST-Cll and Al was applied to and then eluted from the GST column, indicating that Al and Cll are capable of binding to each other in the absence of DNA (Kojima, 2005).

A polymerase chain reaction-based approach, the systematic evolution of ligands by exponential enrichment (SELEX), was undertaken to determine a possible consensus DNA sequence for the binding of the Al/Cll complex. The nucleotide sequence alignment of 48 fragments obtained after five rounds of enrichment revealed a consensus sequence of 5′-(T/C)TAATTAA(T/A)(T/A)G-3′, which differs from the consensus sequences for the vertebrate homologs of Al (TAATNNNATTA; Alx and Cart proteins) and those for Cll homologs (CGGTAA(T/G)(T/C)(G/C)G; Hox11/tlx proteins (Kojima, 2005 and references therein).

Protein-DNA interactions were examined using the electrophoretic mobility shift assay (EMSA). A double-stranded oligonucleotide containing the SELEX consensus sequence was used as a probe. No or weak retardation bands were detected for Cll or All alone. In contrast, a very strong retardation signal was observed for a combination of Al and Cll. A few base substitutions in the consensus sequences results in a significant reduction in or the abolishment of retardation signals. Thus, the Al/Cll complex is significantly different in target-sequence specificity from Al and Cll, and only the Al/Cll complex can strongly bind to the SELEX-determined consensus sequence (Kojima, 2005).

It may thus be concluded that, in the pretarsus, Al and Cll form a complex capable of binding to specific sequences, which cannot be well recognized solely by Al or Cll, and that the resultant complex plays a central role in al/cll-dependent gene regulation in the future pretarsus. However, it should be noted that the possibility cannot be formally excluded that Al and Cll separately bind to their own consensus sequences and function cooperatively in the pretarsus (Kojima, 2005).

Thus al and cll seem to act cooperatively through the formation of the complex between their protein products. To determine whether vertebrate Al and Cll homologs possess similar properties, possible interactions between the Al/Cll consensus sequence and either one of vertebrate Al homolog, Cart1, or a Cll homolog, Hox11L1 (also called as Tlx2), were assessed. Cart1 is capable of binding to the Al/Cll consensus binding site to some extent, but Hox11L1 can not at all. A considerably strong signal is detected when a mixture of Cart1 and Hox11L1 is subjected to gel retardation. Moreover, strong retardation signals are detected for a mixture of Al and Hox11L1 and that of Cart1 and Cll. Thus, the formation of an Al/Cll-type complex may be an evolutionally conserved feature of Al-type and Hox11/tlx-type homeodomain protein family members (Kojima, 2005).



aristaless is expressed in specific larval head regions. First transcripts are seen in stage 10 embryos, just behind the cephalic furrow in the maxillary and labial segment primordia. Shortly thereafter, transcripts are detected in a more anterior location, probably the prospective antenna or mandibular segment. Later transcripts are seen in the posterior labial and posterior maxillary segments.

In the epidermis, a segmentally repeated pattern appears at the beginning of the extended germ band stage, continuing into stage 16. This consists of three thoracic and eight abdominal lateral patches. al is also expressed in the intestinal track (Schneitz, 1993).


aristaless transcript distribution in third-instar imaginal discs closely corresponds to the anlagen of the tissues that later become visibly affected in adult al mutants (Schneitz, 1993). The presumptive tips of both leg and wing, and the distal extreme of the proximodistal axis, are characterized by the close association of cells expressing wg, dpp and aristaless (Campbell, 1993). Ectopic al expression can induce a duplication of the proximodistal axis in the wing, suggesting that al may be directly involved in axis specification (Campbell, 1993).

Effects of Mutation or Deletion

The isolation of complete null alleles of aristaless has not yet been achieved. Viable aristaless mutations show affected pattern elements at both ends of the proximodistal axis of adult antennae, legs, and wings. Aristae are absent or reduced, and the thorax has absent or divergent hairs and bristles. Both the posterior mesonotum and scutellum are reduced in size. In adults carrying strong alleles, there is reduction or absence of tarsal claws (Schneitz, 1993).


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aristaless: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 23 July 2014

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