BIOLOGICAL OVERVIEW

Distal-less is the first genetic signal for limb formation to occur in the developing zygote. Limbs are defined to include antennae, labium, legs and wings. Limbs form from imaginal discs, the small sacs of epithelial cells that grow and develop during the larval stage. The signal from Distal-less affects the formation of imaginal discs, distinguishing the site of imaginal disc development from the general body wall (B. Cohen, 1993), thus setting the stage for limb development.

What signals are required for the induction of Distal-less? In the developing fly Wingless (WG) is arrayed at compartment boundaries in the dorsal to ventral stripes of each parasegment. These stripes intersect cells secreting Decapentaplegic (DPP), arrayed in a single anterior to posterior stripe. It is at the points where WG and DPP stripes intersect that leg imaginal discs are specified and Distal-less is induced (B. Cohen, 1993).

A third signaling molecule, Hedgehog, is also required for Distal-less induction. HH is secreted from the posterior compartments of imaginal discs. These three secreted signaling molecules (HH, WG and DPP) specify the distal-axis of imaginal discs. Each is required for Distal-less induction (Diaz-Benjumea, 1994).

One must be careful in applying as a generalization, the above model to the origin of all discs. The involvement of three secreted signals is readily apparent for the origin of leg discs, but wing discs do not exhibit wingless expression until the second larval instar (Couso, 1993). At this stage wg expression is detected first in the anterior compartment, and Wingless is involved in the specification of the wing primordium. A primary target of Wingless in the specification of the wing primordium is pdm-1 which has an important role is specifying the proximo-distal axis of the wing disc. The interaction between the three secreted proteins and Distal-less in the second larval instar wing disc has not been documented, but it is clear that Dll performs a secondary and late function required for the normal patterning of the wing (Ng, 1996).

Thus Distal-less induces ventral appendage development in Drosophila. It appears that Dll suppresses dorsal appendage development. Lack of Dll function causes a change in the identity of ventral appendage cells (legs and antennae) that often results in the loss of the appendage. Ectopic Dll expression in the proximal region of ventral appendages induces nonautonomous duplication of legs and antennae by the activation of wingless and decapentaplegic. Ectopic Dll expression in dorsal appendages produces transformation into corresponding ventral appendages; wings and halteres develop ectopic legs and the head-eye region develops ectopic antennae. In the wing, the exogenous Dll product induces this transformation by activating the endogenous Dll gene and repressing the wing determinant gene vestigial. It is proposed that Dll induces the development of ventral appendages and also participates in a genetic address that specifies the identity of ventral appendages and discriminates the dorsal versus the ventral appendages in the adult. However, unlike other homeotic genes, Dll expression and function is not defined by a cell lineage border (Gorfinkiel, 1997).

The combination of Wg and Dpp induces the formation of the P/D axis in the leg of Drosophila. It was originally suggested that the Wg/Dpp combination may establish an organizer at the distal tip that controlled patterning along the P/D axis and that this organizer is characterized by expression of the homeobox gene aristaless. Even if such an organizer does exist then it is shown that al is not absolutely required for its activity because removing al at the tip using a null allele does not prevent formation of the P/D axis, although it does prevent the formation of the structures normally found at the tip of the leg. However, there is an absolute requirement for Dll activity in the formation of the P/D axis in the part of the leg that is more distal than the most proximal segment (the coxa). Yet, Dll protein does not show a graded distribution -- this presents a paradox between where Dll is expressed and where its activity is required -- late in development Dll protein can be detected only in the tarsus and distal tibia, but the genetic data reveal that Dll function is also required cell autonomously in more proximal regions, the femur and all of the tibia (Campbell, 1998).

Thus, studies of Dll expression in developing legs have suggested that the protein is restricted to the presumptive tarsus and distal tibia. However, mosaic analysis indicates a requirement for Dll gene function in the presumptive femur and proximal tibia early in development. If Dll is expressed in the more proximal parts of the leg early but then lost from this region, this would account for these observations. To show that Dll is expressed in more proximal regions earlier in development, a cell line was generated in which Dll expression switches on expression of lacZ and provides an inherited marker for Dll expression. Analysis of third instar leg discs from this line reveals that clones expressing beta-gal can be detected anywhere in the leg disc (i.e. including regions outside of the central Dll protein-positive domain), but at much higher frequency in the center of the disc, suggesting Dll is expressed early in development in all the leg disc cells. As an alternative approach the phenomenon of perdurance of protein products (in this case green fluorescent protein) was utilized to investigate whether evidence of earlier Dll transcriptional activity could be detected in the more proximal regions of the leg in which Dll transcription and Dll protein are absent later in development. In late third instar leg discs, the domain of high level GFP expression is generally coincident with Dll protein revealed by antibody staining, but much weaker staining can also be detected in the presumptive proximal tibia/femur. At earlier stages the level of GFP expression outside the Dll protein domain is much stronger, suggesting that the weak staining later (and the stronger staining earlier) is due to perdurance of GFP. Weak lacZ expression can also be detected in the presumptive proximal tibia/femur and again is likely due to perdurance. By the time leg eversion occurs, the low level expression of GFP is generally no longer evident in the femur and proximal tibia. However, in the adult, GFP expression is clearly expressed in most of the cells in the proximal tibia and at high levels in single cells associated with each bristle in the femur. There is a variable amount of expression elsewhere in the femur. Dll is required in the distal regions of the leg for the expression of tarsal-specific genes including al and bric-a-brac. Dll mutant cells in the leg sort out from wild-type cells suggesting one function of Dll here is to control adhesive properties of cells (Campbell, 1998).

The results of this detailed clonal analysis with a Dll null allele can be summarized as follows and are in general agreement with previous studies. Before about the early third instar there is an autonomous requirement for Dll in cells of the leg more distal than the coxa, apart from the dorsal femur, but later in development this requirement is limited to a distal domain corresponding to the tarsus and distal tibia. This domain corresponds to the region where Dll protein can be detected at these stages with antibodies. Dll is expressed in all cells of the presumptive leg, but does not distinguish between expression in the embryo or later during larval life. It should be noted that at late larval stages the domain showing high levels of Dll expression corresponds to the region where Dll function is required, so it is not unreasonable to suggest that similar high levels of expression will correspond to the regions where Dll function is required at earlier stages. If the above view is correct, the leg may be loosely divided into three regions along the P/D axis: (1) proximal - no Dll expression; (2) intermediate - Dll expressed and required early, but not later, and (3) distal - continuous Dll expression and requirement. This would then provide a clear explanation for the paradox raised by the observation that the apparent sites of Dll expression does not explain all the mutant phenotypes. Additionally, it is possible that one source of positional information along the P/D axis is the length of time a cell expresses Dll. Two exceptions to this simple hypothesis are the femur and the trochanter. (1) There is a differential requirement for Dll in the dorsal and the ventral femur (i.e. at the same P/D level) and at present there is no explanation for this, although it is possible that it is related to the situation in the embryo where dorsal Dpp appears to act as a repressor rather than activator of Dll expression. (2) The trochanter, which is proximal, appears to require Dll activity even late in development. This late requirement may correspond to the proximal ring of Dll expression in the leg discs. It should also be noted that expression in this domain appears to be independent of wg and dpp. An additional peculiarity is that although Dll mutant tissue can differentiate normally sized bristles, it fails to form bracts at the base of these bristles even when the clones are generated late in development in proximal locations. This probably reveals a later function for Dll during pupal development because Dll expression extends more proximally during pupal life, and in the adult Dll appears to be expressed in a support cell of each bristle. Not all of these develop bracts, but Dll may be required in these cells to allow the development of this structure (Campbell, 1998).

If the model suggesting cell fate along the P/D axis as specified by Wg and Dpp directly proves to be correct, then one static, simplified version of this model could hold that different cell fates are established above strict concentration thresholds of Wg/Dpp, which in turn would correspond to precise distances from the sources of these molecules. However, one possible problem with this simplified model is growth: as the imaginal disc grows in size, the distance of any one cell from the source will vary so that for such a strict model to produce precise patterning, all cell fates may have to be established simultaneously. Relevant data suggest this is not the case. To account for growth, it has been proposed that different target genes may require Wg and Dpp for different periods of time before expression becomes independent of these signals. Following growth, this could result in overlapping domains of target genes, and these domains could be established at different times in development. However, an alternative way of viewing this model is to propose that different cell fates are established not simply on the basis of how much Wg and Dpp they receive but by how long they receive it. Early in development most of the presumptive leg cells will receive a specific level of Wg and Dpp, but as the disc increases in size, the presumptive proximal cells at the edge of the disc will begin to receive less Wg and Dpp, as they become situated further and further from the sources: they will also experience this specific level of Wg and Dpp for a shorter period of time than more centrally located, presumptive distal cells. Consequently, the length of time a cell receives this specific level of Wg and Dpp may provide positional information along the P/D axis: the longer it receives it the more distal it becomes. It is proposed that the present results with Dll may provide some evidence for such a dynamic version of this model. These results suggest presumptive intermediate level cells express Dll early in development but it is lost later, whilst presumptive distal cells show continuous expression. If it is assumed that a cell expresses Dll above a certain threshold of Wg/Dpp, then its expression may be lost during development at the edge of its expression domain when these cells become situated further from the sources of Wg and Dpp, as the disc grows in size. One problem with this model is that maintenance of Dll expression does not appear to require continuous Wg and Dpp signaling. Dll expression is not lost in clones of a Dpp receptor, thick veins, or a Wg signal transducer, dishevelled, even when these are made during the second instar, i.e. at a time when the present results suggest Dll is still transiently expressed in some cells. There are at least two possible explanations for this: (1) the above model is correct but it is impossible to determine timing of gene function by making clones because this ignores the possibility of perdurance of gene products, or (2) the model is incorrect, but this may be because it assumes that Wg and Dpp are the only limiting factors controlling Dll expression (and patterning along the P/D axis): there may be an additional signal, possibly derived from the presumptive tip, which is also required for Dll expression. A temporal mechanism for axis formation is more evident in vertebrate appendages where positional identity along the P/D axis appears to be determined by such a mechanism: the longer a cell spends in the progress zone, the region behind the tip of the developing limb, the more distal it becomes. Consequently, the P/D axis is determined in a proximal to distal sequence. In Drosophila, there is contradictory evidence as to the order in which segments are specified along the P/D axis and further studies are required to resolve this question (Campbell, 1998 and references).

One function of Distal-less is marked by its absence. Homeotic genes Ultrabithorax and abdominal-A act on known elements of a distal enhancer of Distal-less to repress Dll expression in the abdomen, thereby effectively blocking induction of appendages in the posterior segments (Vachon, 1992).

One aspect of Distal-less research, perhaps the most exciting, has been almost completely neglected. distal-less is expressed in the optic lobe, targeted by Wingless (Kaphingst, 1994). The vertebrate homolog of Distal-less is expressed in premigratory and migratory neural crest cells and in the forebrain (Holland, 1996 and references). The role of Distal-less in Drosophila neurogenesis should be an exciting area of investigation. Whereas amphioxus (cephalochordata within the phylum Chordata) has only one Distal-less gene, craniates have five or six, which probably evolved from a single ancestral gene. The presence of a single Distal-less-related gene in amphioxus (phylum Urochordata) and invertebrates, and multiple members of this gene family in craniates is yet another indication that the size of developmental gene families increased abruptly early in craniate evolution. This increase may well have permitted the evolution of the complex features distinguishing the craniates from their invertebrate ancestors (Holland, 1966 and references).

GENE STRUCTURE

Bases in 5' UTR - greater than 741 bp

Introns - seven

Bases in 3' UTR - 7537 bp

PROTEIN STRUCTURE

Structural domains

DLL has a centrally located homeodomain (Vachon, 1992).

date revised: 20 December 97 

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