Effects of Mutation or Deletion

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Notch and leg morphogenesis (part 2/2)

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).

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).

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, the Notch signaling pathway has been shown to play a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. While Dl expression overlaps fngand Ser, in some cases, it appears to extend into regions of the disc where neither fng nor Ser is expressed (Rauskolb, 1999).

fng mutant clones also result in fused joints and shortened legs. fng is required with the formation of all joints except the tibia-tarsal (ta1: basitarsus) joint. In most cases, the formation of the joints appears to be an autonomous property of wild type cells, while the failure to form joint structures is an autonomous property of cells mutant for Notch, Dl, Ser or fng. However, some exceptions have been observed in which joint formation is inhibited within wild type cells that border mutant clones or mutant cells appear to contribute to joint structure (Rauskolb, 1999).

These studies further show that Notch activation is both necessary and sufficient to promote leg growth. Target genes regulated both positively and negatively downstream of Notch signaling have been identified that are required for normal leg development. The nubbin gene (nub) encodes a POU-domain protein that is expressed in a series of concentric rings in late discs. The strongest mutant alleles, which are not null, result in shortened and gnarled legs. Notch mutant clones cause loss of Nub expression. Conversely, ectopic expression of Nub is induced within clones of cells expressing activated Notch. These observations indicate that nub is positively regulated downstream of Notch activation in the leg. Although in most cases the influence of Notch signaling on nub appears to be autonomous, exceptions to this have been observed. These exceptions indicate that regulation of nub expression by Notch signaling may be indirect. In addition, other factors must modulate the ability of Notch to induce nub expression, because tarsal segments 1-4 do not express Nubbin, and ectopic Notch activation in this region fails to induce Nub (Rauskolb, 1999).

When fng is ectopically expressed, Nub expression can be induced along the inside of clone borders. Similarly, joint structures in the adult can be induced along fng-expression borders and can be inhibited within patches of fng-expressing cells. These observations are consistent with prior studies of fng action during Drosophila wing and eye development, in that Notch activation is positioned along the borders of fng expression (Rauskolb, 1999).

The four-jointed (fj) gene encodes a type 2 transmembrane protein and is also expressed in concentric rings within the developing leg imaginal disc. In fj mutants, growth of the femur, tibia, and first three tarsal segments is reduced, and the ta2-ta3 segment border is absent. The rings of fj expression in leg imaginal discs are complementary to the rings of Nub expression. Consistent with this complementarity, fj expression is inhibited in cells expressing activated Notch; in cells neighboring ectopically expressing Ser or Dl, and in cells along the borders of ectopic fng expression. By contrast, fj expression is activated within cells expressing Ser or Dl. These observations indicate that fj is negatively regulated downstream of Notch signaling in the leg. Thus, Notch signaling subdivides each leg segment into distinct domains of gene expression (Rauskolb, 1999).

While the requirements for odd-skipped (odd) function during leg development have not yet been described, this gene is of special interest because it is required for embryonic segmentation in Drosophila and is expressed in a segmentally repeated pattern both in the embryo and in leg discs. odd expression, like nub, is induced within clones of cells expressing activated Notch in many regions of the leg disc, though not in ta1-ta4. The observation that three different genes expressed in segmentally repeated patterns all respond to Notch signaling, together with the severe effects of Notch mutant clones, indicates that Notch acts at a crucial step in a leg segmentation hierarchy. Together, these observations outline a regulatory hierarchy for the segmentation and growth of the leg. The Notch pathway is also deployed for segmentation during vertebrate somitogenesis, which raises the possibility of a common origin for the segmentation of these distinct tissues (Rauskolb, 1999).

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