Interactive Fly, Drosophila

Effects of Mutation or Deletion (part 2/2)

Serrate and the establishment of leg segments (part 2/2)

The legs of Drosophila are cylindrical appendages divided into segments along the proximodistal axis by flexible structures called joints, with each leg having 9 segments. The separation between segments is already visible in the imaginal disc because folds of the epithelium and cells at segment boundaries have a different morphology during pupal development. The joints form at precise positions along the proximodistal axis of the leg; both the expression patterns of several genes in the leg and the results of regeneration experiments suggest that different positions along the proximodistal axis have different identities. Two signaling molecules, wingless (wg) and decapentaplegic (dpp) play a central role in patterning the leg discs. These genes are activated in complementary anterior dorsal (dpp) and anterior ventral (wg) sectors in response to the secreted protein Hedgehog, which is only expressed in posterior cells. The asymmetry of dpp and wg expression is maintained by mutual repression: dpp and wg act antagonistically to regulate several genes involved in generating differences along the dorsoventral axis. It is therefore likely that the proximodistal patterning system initiated by wg and dpp determines the localization of presumptive joints in developing leg discs, but the identity of the gene products mediating this process is unknown (de Celis, 1998 and references).

Although the mechanism underlying joint formation is not understood, the fusion of segments caused by some Notch alleles indicates a requirement for Notch signaling. In the leg imaginal disc most segments form concentric rings, with the most distal in the center of the disc. The exceptions are the distal femur and proximal tibia, which are indistinguishable in the larval imaginal disc and only separate during pupariation. This separation occurs through the formation of lateral invaginations that fuse creating two epithelial tubes constricted at the femur/tibia joint. When Notch activity is compromised in N^ts1 larvae during early and late third instar stage, the legs that develop are misshapen, with some fusion between femur/tibia (early) and tarsal (late) segments (de Celis, 1998).

To distinguish which elements of the Notch pathway are required during leg development, clones of homozygous mutant cells were generated, using lethal alleles in fng, Dl and Su(H) as well as a deficiency of the E(spl) complex. Lethal Ser alleles can survive into adults and they have a low frequency of joint fusions. The phenotype of Dl and Su(H) mosaics are similar to each other and, like Notch, result in a failure to make joints when mutant cells are in the position where a joint should have formed. Again, the wild-type cells near the clones can still form joints, but the length of the leg is reduced when the mutant clones are large and span more than one segment. In contrast, mutant cells homozygous for a deficiency that removes the E(spl)bHLH genes form normal joints even when they span more than one segment and are characterized by the differentiation of a vast array of ectopic sensory organs. These develop without intervening epidermal cells, indicating that E(spl) is required for the lateral inhibition mechanism that allows the spacing between sensory organs. The larger clones cause a slight reduction in the overall size of the leg (12% in area and 8% in length), but it is likely that these effects are due to the differentiation of ectopic sensory organs rather than direct effects on growth. Cells mutant for fng also result in fusions between segments. However, these effects are position dependent. Thus, with clones spanning the boundary between the femur and tibia the phenotypes are indistinguishable from those of Notch and Su(H), resulting in a fusion of these two segments and shortening of the leg, whereas in more distal segments defects in the joint can only be detected between the proximal two tarsal segments. The fact that fng is important in leg segmentation suggests that boundaries similar to the wing dorsal-ventral boundary are being created in at least some of the presumptive joints (de Celis, 1998).

In the developing wing the localized activation of Notch can be detected by the activation of certain target genes such as E(spl) and vestigial. Furthermore, the domains of expression of Dl and Ser are important in creating this localized activation of Notch. The expression of Ser, Dl, fng, Notch and E(spl)m beta were therefore examined during leg development. Heterogeneities in the expression of all these genes are detected in the third instar imaginal disc, where Dl and E(spl)m beta RNA are expressed in narrow concentric rings. In evaginating leg discs (0-4 hours APF) and in pupal legs, when the separation between leg segments becomes more evident, E(spl)m beta expression is localized to a ring of distal cells in each leg segment, suggesting that larval expression of E(spl)m beta also defines the distal end of each segment. The expression of fng is also restricted, and is only detected in several broad rings localized to the presumptive tibia and first tarsal segment, and in two groups of distal cells in the fifth tarsal segment that could correspond to the presumptive claws. At this stage, no heterogeneity could be detected in the expression of Notch RNA, but by 24 hours after puparium formation the levels are higher in the places where the joints are being formed, which appear to be the same cells where E(spl)m beta is expressed. At these later stages, Dl also accumulates in rings of cells located at the distal end of each segment and at the separation between the femur and tibia, as well as in many clusters of cells that correspond to developing sensory organs. Expression of E(spl) genes is dependent on Notch activity and hence the localization of E(spl)m beta mRNA to rings of cells in the imaginal and pupal leg disc indicates that there are high levels of Notch activation in the distal-most set of cells in each segment. To determine more precisely the relationship between the E(spl)m beta-expressing cells and the expression of other components of the Notch pathway, a reporter gene was generated in which 1.5 kb of genomic DNA upstream of E(spl)m beta was used to drive expression of a rat cell surface protein, CD2. As a landmark for the segment boundaries an enhancer trap in the bib gene, bib lacZ was used, which is expressed at higher levels in single-cell wide rings at the distal end of each leg segment during both larval and pupal development. The expression of E(spl)m beta-CD2 is localized to a narrow ring, 1-2 cells wide, which coincides with the cells expressing bib lacZ and with cells that have higher levels of lacZ expression in the N lacZ1 enhancer trap line. The expression of N lacZ1 at the dorsoventral boundary and at vein-intervein boundaries is dependent on Notch activity itself. Thus the coincident Notch, E(spl)m beta and bib expression indicates that high levels of Notch activation during imaginal leg development are restricted to the most distal cells of each segment. The accumulation of Notch ligands is also localized within the developing leg segments, with the highest levels of Dl and Ser detected in a narrow stripe of cells localized proximally to those expressing bib lacZ both in the larval imaginal disc and at pupal stages (de Celis, 1998).

Overall the effects produced by ectopic Dl and Ser are similar: the altered morphology of the resulting legs includes both fusion of segments and ectopic joints. However there are positional differences in the way the ligands exert their effects. Thus, the strongest effects of mis-expressing Dl are observed in the tarsal segments, where joint formation is perturbed resulting in foreshortened fused tarsi. This resembles Notch loss-of-function phenotypes suggesting that the levels or position of Dl expression are interfering with normal Notch activity. In addition, an abnormal structure forms at the junction between the first and second tarsal segments, which seems to consist of a partial perpendicular joint. The strongest effects of Ser mis-expression are suggestive of dominant negative effects, since the tibia is foreshortened and forms abnormal joints with the femur and tarsi. In addition, incomplete ectopic joints can be observed at low frequency in distal tarsal segments. Thus, the phenotypes indicate that both activation and repression of Notch occurs when high levels of Notch ligands are expressed. It is likely that the differential effects of misexpression of Dl and Ser are related to the distribution of fng, because the strongest dominant negative effects of Ser occur in the tibia, where fng expression is maximal, and those of Dl occur in distal tarsal segments, where fng is absent or expressed at low levels. Similar effects occur when the ligands are expressed in the wing using the GAL4 system, where the outcome is in part determined by interactions between Notch and Fng (de Celis, 1998).

The expression of the Serrate and Delta genes patterns the segments of the leg in Drosophila by a combination of their signaling activities. Coincident stripes of Serrate and Delta expressing cells activate Enhancer of split expression in adjacent cells through Notch signaling. These cells form a patterning boundary from which a putative secondary signal leads to the development of leg joints. Elsewhere in the tarsal segments, signaling by Dl and N is necessary for the development of non-joint parts of the leg. It is proposed that these two effects result from different thresholds of N activation, which are translated into different downstream gene expression effects. A general mechanism is proposed for creation of boundaries by Notch signaling (Bishop, 1999).

The legs of Drosophila are jointed appendages, as in all other arthropod animals. In Drosophila and most insects, the structure of the legs is remarkably constant as follows: every leg carries articulations, or joints, which divide the leg into parts, or segments; these segments are called, from proximal (or close to the body wall) to distal: coxa, trochanter, femur, tibia and five tarsal segments, plus a pre-tarsus, or claw organ, at the tip of the leg. The joints differ from other parts of the leg in that they are devoid of bristles and include a flexible intersegmental membrane and interlocking parts composed of thickened cuticle. The details of the morphology of these parts varies from joint to joint and only the joints between the tarsal segments are identical and composed of a 'socket' in the proximal part of the joint and an interlocking 'ball' in the distal part. The other joints do not include a 'ball and socket' structure but a variety of condyles and cavities (Bishop, 1999 and references).

Joints are not obvious in the developing legs until the time of pupation, when the everting leg discs show a series of constrictions, that different authors have identified with the presumptive positions of the final joints. However, during subsequent metamorphosis, the legs inflate and lose these constrictions although different cell contours can still be seen at positions that seem to correlate with future joints. The lack of markers other than morphological ones has not allowed an exploration of joint development in more depth. A marker that seems to correlate faithfully with joints has been used, an enhancer trap inserted into the disconnected (disco) gene. The disco gene encodes a protein required for axonal migration and leg development (Heilig, 1991) and in the legs, its expression is associated with joint development. disco expression is present in the developing leg imaginal disc at 120 hours after egg laying. Although it is expressed throughout the presumptive leg region, disco expression is upgraded in a series of rings around the center of the disc. During the first 4 hours after puparium formation (APF), disco expression can be seen to become more strongly modulated in rings, which correspond with the presumptive leg segments. At around 12 hours APF, lacZ expression is restricted to these rings, which in a lateral view of the developing leg appear as stripes about 6 cells wide situated next to but proximal to constrictions in the presumptive tarsal region. Staining of adult flies carrying this marker shows disco expression specifically restricted to the joints (Bishop, 1999 and references).

Null alleles of Ser are lethal but a few mutant flies are formed inside the pupal cases. These animals show a variety of developmental defects, amongst them, leg deformities. The legs lack joints between all segments although sometimes a remnant constriction can still be seen. No other leg area is affected and the segment boundaries are still present, as shown by the apical bristles in tarsi and tibia. This mutant phenotype correlates with the pattern of expression of Ser. Using an antibody against the Ser protein, expression is seen to appear in rings in third instar discs and later is found close to the presumptive joint areas in pupal legs. Stripes of Ser expression are seen proximal to constrictions in everting legs. Double staining with disco expression shows overlapping expression of disco and Ser. However, in the bell-shaped distribution of disco, cells with maximum levels of disco are located at the distal edge of the stripe of Ser expressing cells. These results show that Ser is required for the development of joints, and Ser expression adjacent to joint areas suggests that Ser could be directing joint development. If this were the case it would be expected that ectopic expression of Ser would lead to the ectopic development of joints, and indeed this is the case. A klumpfuss-Gal4 line, which is expressed in the legs outside the joints, was used to drive ectopic expression of a UAS-Ser construct. klu-driven expression of Ser leads to the ectopic development of joint-like cuticle, characterised by loss of bristles, cuticle thickenings and inpocketings. In leg regions like the tibia, where klu expression has defined boundaries, ectopic constrictions tend to appear. The transformation of interjoint leg regions towards joints is corroborated by the accompanying ectopic expression of disco. Reciprocally, in Ser mutants, disco expression is lost after 12 hours APF (Bishop, 1999 and references).

Dl expression has been revealed using a Dl-lacZ reporter allele and an antibody against the Dl protein. Dl is expressed throughout the presumptive leg at third instar, but with upgraded expression in rings. In pupal legs these rings can be seen to locate proximal to constrictions, and in adult legs lacZ expression is found at low levels throughout the leg, but it is stronger proximal to the joints. The expression of Ser, disco and Dl in pupal legs has been compared and the stripes of high Dl expression are found to coincide with cells expressing Ser. To study the requirements for Dl a viable temperature-sensitive mutant combination of alleles was used that produces mutant leg phenotypes. Following exposure to the restrictive temperature during the third instar and pupal periods, when Dl is expressed near the presumptive joints, the Dl mutant legs are shortened. The tarsal segments are particularly reduced and have seemingly disappeared, but on close inspection it can be seen that the tarsal apical bristles are still present and sometimes some remnant joint structures as well. Because Dl and Ser are co-expressed, the joint defects in Dl mutants could be indirectly due to a loss of Ser expression in Dl mutants. This was examined and it was found that the Ser stripes are still present in Dl mutant legs. Reciprocally, Dl expression is still present in Ser mutants, showing that the expression of Ser and Dl are not directly dependent on each other and that their mutant joint phenotypes reflect independent requirements. These results are interpreted as showing that although joint areas require Dl for their development, the strongest requirement for Dl is in the regions located between segmental boundaries. This implies that the requirements for Dl are more extensive than those of Ser, and this is corroborated by their different requirements for disco expression. In Ser mutant legs, the stripes of disco expression form but are not maintained properly. However, in Dl mutants, the stripes of disco are not formed correctly and instead wider and fewer rings remain, a pattern similar to that of Ser expression in Dl mutants. This disco expression in Dl mutants is interpeted as a corroboration of the main requirement for Dl being in the intersegmental regions. Failure of these regions to develop causes the absence of non-disco expressing cells between disco stripes so that fewer but wider disco stripes appear in the mutants. In spite of the differences in the requirements for Dl and Ser, there is an overlap in function in that they are both required for the development of joints. It is possible that this overlap explains why the requirements of Dl in the joints are weaker than those in the interjoints. It was decided to study whether this overlapping requirement is mediated by N (Bishop, 1999).

N mutant flies show a marked reduction in leg length with all areas of the leg segments being affected. Joints are completely lost but also often apical bristles. The overall length of the segments, and especially of the tarsal region, is more reduced than in Ser or Dl mutants because both joint and interjoint tissue is missing. Thus, the N mutant phenotype looks like a composition of the Ser and Dl mutant phenotypes. When the expression of disco is revealed in N mutants, a combination of Ser and Dl phenotypes is also seen. disco stripes do not resolve properly, as in Dl mutants, and then they are subsequently lost, as in Sermutants. Expressing a dominant-negative form of N in the interjoint regions results in shortened legs, due to the loss of interjoint tissue, but the joints are still present and sometimes fused. These leg phenotypes thus resemble those produced in interjoint regions by loss of Dl function. Expression of a truncated and constitutively activated form of N in the fourth tarsal segment becoming hyper-jointed: double ball joints are formed. In addition, the interjoint region is reduced, either as a consequence of its conversion to extra joint tissue, or to an inability to develop the interjoint cell fates that have low, but not high, levels of N activation. Altogether these results suggest that the overlap of Ser and Dl expression and requirements at the joints are mediated by N activation. As a marker of N activity, the expression of members of the E(spl) complex has been monitored. Using reporter constructs with the regulatory regions of E(spl), which reproduce the endogenous E(spl) expression in the leg discs, it can be seen that E(spl)m8 expression is related to joints while m5 and presumably m6 are not. Expression of the E(spl)m8 reporter construct in third instar discs is initially strong in regions undergoing PNS development. In the legs, these correspond to the chordotonal organs in the femur and the tibia. In addition, expression near the presumptive joints is seen to appear, and then resolve in the pupa into one-cell wide stripes proximal to the leg constrictions, in positions that correlate with cells with maximum levels of disco expression. A similar although much weaker pattern of expression of E(spl)mdelta is seen as revealed by the mAb323 antibody. Another marker of N activity is the expression of N itself, which becomes upregulated in cells where N signaling is being received. Using an anti-N antibody, upregulated expression of N is seen immediately proximal to constrictions in pupal legs. This upregulation is restricted to a single row of cells at this position, thus confirming that Ser and Dl are triggering N signaling in these cells (Bishop, 1999).

The results presented suggest a model in which the co-expression of Ser and high levels of Dl in a stripe of cells proximal to the future presumptive joints activate N in cells adjacent but distal to this stripe. Could this specificity be due to the presence of other factors that would be interfering with Ser and Dl signaling in cells located inside the Ser-Dl stripe or proximal to it? In the DV boundary of the wing, the membrane protein encoded by the gene fringe (fng) has been postulated to modulate N signaling by interfering with Ser signaling. In the developing legs, fng is expressed in stripes or rings around the positions of presumptive joints. Using a UAS-fng construct, fng was misexpressed. Uniform tibial and tarsal fng expression only affects the joints, which are reduced or disappear, a phenotype reminiscent of Ser mutants. Thus, fng activity in the leg seems to be restricted to a repression of joint development around presumptive joint areas. It is possible that fng expression in the wild type is repressing N signaling in cells located in the Ser-Dl stripe or proximal to it, providing the polarity in the joint-promoting function of Ser and Dl (Bishop, 1999).

These results suggest a model in which the co-expression of Ser and high levels of Dl in a stripe of cells activate N in cells adjacent but distal to this stripe. Activation of N promotes expression of members of the E(spl) complex and leads to joint formation and disco expression. Loss of Dl eliminates first the regions between disco/Ser-expressing rings, but also, secondly, joints. Since loss of interjoint regions is also seen both in N mutants and following expression of a dominant-negative form of N, it is postulated that Dl expression in the interjoint regions produces low levels of activation of N that do not lead to E(spl) expression but which allow cell survival and/or cell proliferation. Joint loss in Dl mutants is presumably less severe than interjoint loss because Ser and Dl expression could be synergistic and partially redundant. The combined and potentially synergistic effects of Ser and Dl would produce a high level of activation of N that would lead to expression of members of the E(spl) complex, upregulation of N expression, and to joint development and disco expression. Thus, it is believed that combinations of signaling by Ser and Dl could produce different levels of activation of N, which in turn are translated into different downstream effects. As noted in other systems these downstream effects of N signaling should be mediated by more factors than just E(spl), since E(spl) mutant legs have been reported as having a wild-type phenotype (Bishop, 1999 and references).

The width of the final joint region is wider than the single row of cells activated by the membrane-tethered Ser and Dl proteins and visualised by E(spl) expression. In principle it is possible that the cells of the whole final joint all descend from the E(spl) expressing cells, but previous studies have shown that only one or two cell divisions occur in the legs after puparium formation. Thus it is likely that in the E(spl) expressing cells another cell signaling molecule is activated, which in a secondary event would define a wider joint presumptive region, just as N-induced expression of the secreted signaling wingless protein defines the presumptive wing margin. A reflection of this putative second signaling event in the joints can be seen in the expression of disco. disco expression is dependent on Ser but it is wider than the single row of cells where N is activated and thus it cannot be directly reflecting N signaling at the joint. However, the 'bell-shaped' distribution of disco might reflect this putative secondary signaling event, with a maximum in cells at the edge of the Ser-Dl stripe. The nature of the joint-promoting putative secondary signal is unknown at the moment, but one possible component is the product of the four-jointed (fj) gene. The fj protein is a putative signaling molecule that is expressed and required at the joints. fj expression has recently been shown to depend on fng and N signaling during eye development, and it is lost in N mutant legs (Bishop, 1999 and references).

An autonomous negative effect of Dl and Ser does not explain why cells adjacent but proximal to the Ser-Dl stripe do not seem to be signaled. A possible explanation would be either an asymmetric distribution of Ser and Dl, forming gradients like those seen in the late third instar wing margin and in ectopic expression situations, or a downregulation of N expression as has been noted in the developing wing veins. The Ser and Dl stripes in legs show no apparent asymmetry but N distribution, although ubiquitous and initially uniform, becomes upregulated in cells distal to the Ser-Dl stripes. Low availability of N protein could have an effect on the intensity of N signaling, but since upregulation of N is in itself a consequence of N signaling, some other factor must polarize the signaling initially. Another explanation would rely on the action of a repressor acting upon cells proximal to the stripe. The phenotypes obtained after ectopic expression of fng are consistent with such a role for fng, as postulated in the wing. The expression of fng in the leg, which has been described as complementary to that of E(spl), that is, present in non-signaled cells but excluded from joint forming ones, is also consistent with this hypothesis. Such a function of fng could also repress Ser and Dl signaling in the stripe without recourse, or in addition, to putative autonomous dominant negative effects of Ser and Dl. However, other factors could also be involved, such as the cell polarity pathway. Mutant phenotypes for dsh and other members of the cell polarity pathway produce ectopic joints with reversed polarity, which appear just proximal to the position of Ser and Dl stripes. Furthermore, in dsh mutants ectopic N activation is seen proximal to the Ser-Dl stripe. Since the Dsh protein has been shown to interact with N, and Dsh has been postulated to inhibit N signaling in this manner, the cell polarity pathway could be involved in repressing Ser and Dl signaling to cells proximal to the Ser and Dl stripe (Bishop, 1999 and references).

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