Appendages are thought to have arisen during evolution as outgrowths from the body wall of primitive bilateria. In Drosophila, subsets of body wall cells are set aside as appendage precursors through the action of secreted signaling proteins that direct localized expression of transcription factors. The Drosophila homeodomain protein Distal-less is expressed in the leg primordia and required for formation of legs, but not wings. The homeodomain protein Nubbin is expressed in the wing primordia and required for formation of wings, but not legs. Given that insect legs and wings have a common developmental and evolutionary origin, we sought to identify genes that underlie the specification of all appendage primordia. We present evidence that the zinc-finger proteins encoded by the elbow and no ocelli genes act in leg and wing primordia to repress body wall-specifying genes and thereby direct appendage formation.
The progenitors of the adult body regions in Drosophila are set aside in the embryonic ectoderm as discrete groups of cells known as imaginal discs. The disc primordia contain the progenitors of both the adult body wall and the appendages; the wing imaginal disc for example forms the wing and the dorsal thorax (reviewed by Cohen,1993). Subdivision of the imaginal discs into presumptive appendage and body wall territories results from the activity of conserved signaling proteins (summarized in Fig. 1B). The combined activities of Wingless (Wg) and Decapentaplegic(Dpp) specify proximal and distal domains in the leg imaginal discs, thereby defining the appendage fields (Campbell and Tomlinson, 1995; Diaz-Benjumea et al., 1994; Lecuit and Cohen, 1997). In the wing disc, signaling by the EGFR ligand Vein(Vn) antagonizes Wg activity to limit the appendage-forming region to the ventral part of the imaginal disc (Wang et al., 2000; Zecca and Struhl,2002a; Zecca and Struhl,2002b), whereas the appendage-forming region is centered in the leg disc.
Wg and Dpp signaling activities restrict the expression of the homeodomain protein Homothorax and the zinc-finger transcription factor Teashirt to the presumptive body wall by repressing them in distal domains(Abu-Shaar and Mann, 1998; Wu and Cohen, 1999; Wu and Cohen, 2000; Wu and Cohen, 2002). Wg and Dpp also activate expression of genes required for appendage formation,including Distal-less (Dll) in the distal leg primordium(Cohen and Jürgens,1989a; Diaz-Benjumea et al.,1994; Lecuit and Cohen,1997) and vestigial (vg) and nubbin(nub) in the distal wing primordium (wing pouch)(Ng et al., 1995; Ng et al., 1996; Wu and Cohen, 2002). Thus,different transcription factors appear to be used in leg and wing primordia to confer distal identity.
Given that there is a common evolutionary origin of legs and wings(Averof and Cohen, 1997), and that legs and wings derive from a common imaginal disc primordium in the Drosophila embryo (Cohen et al.,1993), we might expect that Wg and Dpp would act through the same transcription factor(s) to specify the appendage-forming region in the leg and wing discs. In this context we became interested in the zinc-finger proteins encoded by the elbow (el) and no ocelli(noc) loci. We present evidence that Elbow and Noc proteins are expressed in the presumptive distal cells of both leg and wing imaginal discs,where they are required to repress the expression of the body wall genes homothorax (hth) and teashirt (tsh) and promote appendage formation.
Materials and methods
ap-gal4 (Calleja et al.,1996); dpp-gal4, UAS-wg, UAS-sgg, and UAS-brinker are described in FlyBase. el1 is described by Davis et al.(Davis et al., 1997). EP(2)2039 directs Gal4-dependent expression of El protein (not shown)and produces a wing defect with scallopedGal4 (not shown). The enhancer trap line el-gal4 has a Gal4-containing P-element 950 bp upstream of the transcriptional start of el. NocGal4 contains a Gal4-containing P-element 285 bp upstream of the transcriptional start of noc. The loss-of-function mutant el3.3.1 was recovered by EMS-induced reversion of this phenotype. el3.3.1 contains a stop codon in exon 2, leading to truncation of the protein at Q262. nocΔ64is a 848 bp deletion within the Noc ORF(Dorfman et al., 2002). The double mutant chromosome was generated by inducing male-specific recombination in a S1 nocΔ64/Lrm el3.3.1EP(2)2039; Δ2,3 Sb genotype(Preston et al., 1996). Recombinants lacking both dominant markers were stocked and analyzed by PCR for the presence of the noc deletion and the el point mutation. The integrity of the recombination region was verified by PCR (data not shown). UAS-el and UAS-noc were constructed by cloning full-length cDNAs into pUAST (Cheah et al.,1994; Dorfman et al.,2002).
El and Noc protein expression was visualized using polyclonal antibodies raised against full-length proteins in rat (El) or guinea pig (Noc). The protein expression patterns were faithfully reflected by the nocGal4 and elGal4 enhancer trap lines, which were useful for double-labeling experiments.
Genotypes of larvae used for genetic mosaic analysis
hs-FLP (I);nocΔ64FRT40/arm-lacZ FRT40.
hs-FLP (I); el3.3.1nocΔ64FRT40/arm-lacZ FRT40.
hs-FLP (I); el3.3.1nocΔ64FRT40/M(+) arm-lacZ FRT40.
f36ahs-FLP (I);el3.3.1nocΔ64FRT40/P(f+) FRT40.
f36ahs-FLP (I);el3.3.1nocΔ64FRT40/P(f+) M(+) FRT40.
Clones were generated by giving a 1 hour heat shock at 38°C at the indicated stages.
Genotypes of larvae used for ectopic expression of el and noc
apγαλ4/+; uas-el uas-noc.
el and no ocelli expression in appendage primordia
The elbow (el) and no ocelli (noc) genes are located in close proximity on chromosome 2L and encode closely related zinc finger proteins (Fig. 1A). The predicted El and Noc proteins are 50% identical. We became interested in the functions of El and Noc because of their expression patterns in the developing wing and leg imaginal discs. In second instar larvae El and Noc proteins are co-expressed in the presumptive distal regions of the leg and wing imaginal discs where the appendages are specified(Fig. 1C,D). In second instar leg discs, El and Noc expression coincided with Dll(Fig. 1C), which marks the leg-forming part of the disc at this stage(Cohen and Jürgens,1989a; Diaz-Benjumea et al.,1994; Lecuit and Cohen,1997). At the earliest stages, the El and Noc domain appears slightly broader than the Dll domain, perhaps reflecting a slight delay in the onset of Dll expression relative to El and Noc. A similar result was observed in second instar wing discs, where the El and Noc expression domain included the nascent Nub domain, where Hth was beginning to be repressed(Fig. 1D). Later in development the expression patterns of El and Noc change(Fig. 1E,F), suggesting that they are used again under different regulatory control to fulfill secondary functions (see below).
The early onset and position of El and Noc was consistent with their genes being early targets of Wingless (Wg) and Decapentaplegic (Dpp), which specify the appendage primordia. Expression of GSK3/Shaggy, a repressor of the Wg signaling pathway (Diaz-Benjumea and Cohen, 1994), or Brinker, a repressor of the Dpp signaling pathway(Campbell and Tomlinson, 1999; Jazwinska et al., 1999; Minami et al., 1999), under control of dpp-Gal4, repressed El and Noc expression(Fig. 2A,C,D and not shown). Likewise ectopic expression of Wg caused ectopic expression of El and Noc(Fig. 2B and not shown). These observations indicate that Wg and Dpp signaling are required for induction of el and noc expression in early leg and wing discs. The late expression of el and noc in the wing disc(Fig. 1F) is also under Dpp and Wg control, with Wg signaling inducing their expression and Dpp repressing it(data not shown).
el and noc define the appendage-forming domain of the imaginal discs
We sought to analyze the functions of el and noc by producing mutants. A mutant allele of el was produced in an F1 screen by EMS-induced reversion of a phenotype caused by el overexpression (using an EP-element inserted at the ellocus, see Materials and methods). el3.3.1 truncates the protein before the zinc-finger domain and is probably a null allele(Fig. 1B). Nonetheless, flies homozygous for the el3.3.1 mutation were viable without obvious defects in phenotype from wild type (data not shown). For noc, we made use of a small deletion that removes exon 1 of the gene and is embryonic lethal (Dorfman et al.,2002) (Fig. 1B). Clones of cells homozygous for the nocΔ64allele develop without any observable defect in legs and wings (data not shown). As el and noc encode homologous proteins, this suggested that they might function redundantly in imaginal disc development. Consequently, it was necessary to generate an el noc double mutant chromosome and examine clones of cells lacking both. Although the two loci are located only 83 kb apart, this was possible using P-element-mediated male recombination (Preston and Engels,1996; Preston et al.,1996), because the el3.3.1 mutant was induced on a chromosome carrying an EP-element. P-transposase induced recombinants between the EP2039 el3.3.1 and the nocΔ64 FRT chromosome were recovered and tested for the presence of both alleles by PCR. The double mutant chromosomes were analyzed by PCR to ensure that no additional alterations were induced adjacent to the P-element in the course of recombination. Antibody labeling confirmed that the El and Noc proteins were not expressed in clones of el3.3.1 nocΔ64 double mutant cells (not shown).
To assess their role in early wing development, clones of el3.3.1 nocΔ64 double mutant cells were generated in early second instar larvae and their distribution was compared with their wild-type twins in the wing disc (e.g. Fig. 3A). The homozygous mutant cell and its wild-type “twin” are products of a single cell division, so the two clones are normally recovered at equal frequency and in close proximity to one another after a period of growth. This was the case for el and noc double mutant clones in the presumptive body wall portion of the wing disc (Fig. 3B, 26/26 pairs). When the clone pair was close to the edge of the endogenous Nub expression domain, mutant clones were recovered outside the Nub domain while their twin clones were found in the Nub expression domain (8/8 pairs). In some cases the mutant clone was separated from its twin(Fig. 3A), suggesting that the mutant clone had been displaced to the edge of the Nub domain. In other cases,the mutant clones appear to have been lost. Only five double mutant clones were recovered for 31 wild-type twins within the Nub domain. These observations suggested that el and noc double mutant clones sorted out from the Nub domain.
Although few clones of el and noc double mutant cells were recovered in the wing pouch, those that were examined had lost expression of Nub and showed ectopic expression of Tsh, which is normally limited to the body wall (Fig. 3C; Hth was also misexpressed; not shown). These clones rounded-up and appeared to extrude from the wing epithelium, suggesting that affinity differences were causing them to sort out. In some cases clones of mutant cells were recovered as vesicles of mutant tissue between the wing surfaces (data not shown). We next produced discs with very large areas of el and noc mutant tissue, using the Minute technique to give mutant cells a relative growth advantage (Morata and Ripoll,1975). Despite the large size of the double mutant clones, they were always located outside the domain of Nub expression, in the Hth expression domain (Fig. 3D,E). In some cases the Nub-expressing area was very small, but all mutant cells expressed Hth. We examined adult flies from these crosses to evaluate the effects on the adult wing. Although few larvae with many large clones survived to adulthood, those that did displayed a nearly complete loss of wing tissue(Fig. 4B,D). In such cases all the remaining wing elements were composed of heterozygous cells expressing one wild-type copy of el and noc. In the thorax, clones of mutant tissue were recovered without defects (not shown).
The behavior of el and noc double mutant clones was similar in the leg imaginal disc. When induced early in development, double mutant clones were recovered at normal frequencies in the proximal region of the disc (Fig. 5A,B; 21/21 pairs). Double mutant clones were not recovered in the distal-most part of the disc (0 clones for 6 twins). When induced in late second instar, only four double mutant clones were recovered distally. These clones ectopically expressed Hth, rounded up and sorted out from the epithelium (e.g. Fig. 5E). These observations again suggest that El/Noc activity is required downstream of Wg and Dpp to repress Hth expression in the leg disc. In contrast to the wing, ectopic expression of Tsh or Hth does not repress Dll expression in distal leg(Abu-Shaar and Mann, 1998; Wu and Cohen, 2000), and consequently Dll expression was not lost in the el and nocdouble mutant clones (not shown). Ectopic expression of Tsh and Hth can in some cases cause ectopic expression of Dll(Wu and Cohen, 2002), as in the example in (Fig. 5E).
Double mutant clones given a growth advantage were excluded from the presumptive tarsus (Fig. 5C,D). The relationship between proximal and distal domains of gene expression differs somewhat in the leg from that in the wing. Lineage tracing experiments have shown that cells born in the body wall portion of the leg disc(tshGal4 expression domain) normally contribute significantly to forming the femur and tibia segments of the leg, but do not contribute to the tarsus (Weigmann and Cohen, 1999). To do so the cells must lose proximal gene expression (e.g. hth) and acquire distal gene expression [e.g. Dll and/or Dac (Wu and Cohen, 1999)]. In this context it is interesting that eland noc double mutant clones occupied large proximal areas and extended distally into femur and tibia segments(Fig. 5C,D), but were not recovered in the tarsal segments of the adult leg(Fig. 5A). Apparently, during third instar, el and noc mutant cells can migrate distally and contribute to formation of femur and tibia. This correlates with a distal-ward retraction of the Dll expression domain(Campbell and Tomlinson, 1998). The finding that el and noc mutant cells do not contribute to wing at all may suggest that comparable distal migration of mutant tissue does not happen in the wing. Lineage tracing of proximal cells in the wing has shown that cells born proximally in the hinge region normally do not contribute to the wing blade (K. Weigmann and S.M.C., unpublished data)
Few larvae in which we generated large el and noc double mutant clones survived to adulthood and we did not recover adult flies in which all derivatives of the leg disc were mutant. The most severe defects recovered corresponded to nearly complete loss of distal leg tissue, including reduction of femur and tibia (Fig. 4A,C). Although El and Noc activity are not strictly required in femur- or tibia-producing cells, the defects observed in these segments in discs with large areas of mutant tissue presumably reflect a requirement at earlier stages. As for Dll the domain in which El and Noc are required may retract distally as the disc develops(Campbell and Tomlinson, 1998; Cohen et al., 1989; Cohen and Jürgens, 1989a; Gorfinkiel et al., 1997). Dll is required early for formation of tibia and femur as well as tarsus, even though Dll mutant clones induced later can be recovered in tibia and femur.
These observations suggest that the combined activity of El and Noc is required to prevent Tsh and Hth expression in the presumptive appendage-forming region of the early leg and wing imaginal discs. As neither gene produces a defect when mutated alone, it appears that either protein is sufficient to mediate repression of Hth and Tsh. We have attempted to test this by Gal4-dependent ectopic expression of UAS-noc or UAS-el constructs. To our surprise, ectopic expression of El or Noc or both together, resulted in a reduced expression of Nub and up-regulation of Tsh (data not shown). As these effects are the same as those produced by the loss-of-function mutant clones,we infer that overexpression of either protein causes a paradoxical dominant negative effect, reducing net activity. While unexpected, this is not unprecedented. For example, when overexpressed, dLDB/CHIP, a cofactor of Apterous, behaves as a dominant negative, apparently by disrupting the stoichiometry of a multi-protein complex that is required for transcription factor activity (Fernandez-Funez et al.,1998; Milan and Cohen,1999).
Taken together our observations suggest that the early activities of el and noc are required for specification of both, wings and legs in Drosophila. In the wing disc, repression of Hth and Tsh is required to allow Wg and Dpp to induce Nub expression in the early wing primordium. In the leg Wg and Dpp repress Hth and Tsh and independently induce Dll and Dac (Abu-Shaar and Mann,1998; González-Crespo et al., 1998; Wu and Cohen,1999; Wu and Cohen,2000). We suggest that Wg and Dpp act through el and noc to define the appendage-forming region of the early wing disc by repression of body-wall specific genes. Cells lacking El/Noc activity appear to adopt proximal identity by default, by virtue of not being able to repress Hth and Tsh.
Late functions of el and no ocelli
Transformation of el and noc mutant cells toward body wall identity occurred when clones were induced in first or early second instar. Clones induced later did not alter their proximal-distal identity,indicating that the role of el and noc in repressing body wall-specific genes is transient. This is consistent with the alteration in their patterns of expression as development proceeds(Fig. 1E,F). In the wing disc,El and Noc expression shifts to a ring near the base of the wing pouch and to a wedge shaped stripe along the dorsal-ventral boundary. Consistent with these changes, clones induced during early third instar lost proximal (hinge)structures (Fig. 6B,C). These phenotypes resemble those seen in the wings of the homozygous viable el1 mutant (compare Fig. 6C and D)(Davis et al., 1997). el1 is a deletion that removes approximately 25 kb of DNA from the region between the el and noc genes(Ashburner et al., 1999; Davis et al., 1997), but does not affect the coding region of either gene. By antibody labeling, we found that the el1 deletion causes loss of El and Noc protein expression in the wing pouch, while their expression in the hinge region seems to be broader than in wild-type discs (Fig. 7). On this basis we conclude that el1 deletes a common regulatory element that controls expression of el and noc in the wing pouch. As such el1 should be considered a regulatory allele of both loci and not just as an allele of el (Davis et al.,1997). The broader ring of El and Noc expression in the hinge might explain the adult hinge phenotype(Fig. 6).
We also noted that el noc double mutant clones had a non-autonomous effect on growth of the wing, which was similar to that described previously for nub mutant clones in the wing(Cifuentes and Garcia-Bellido,1997; Ng et al.,1995). It is possible that the effects observed in el nocdouble mutant clones resulted from loss of Nub expression in the proximal part of the wing, where Nub is directly required for patterning proximal wing elements and indirectly for normal growth of the wing pouch.
In the leg, large el and noc double mutant clones extended from coxa, through femur and tibia and caused failure of leg segmentation (Fig. 6E,F). These defects resembled those produced by hth or exd mutant clones, which fail to maintain an affinity border between body wall and leg(González-Crespo and Morata,1996; Wu and Cohen,1999). Thus the el and noc genes appear to serve distinct functions in proximal wing and proximal leg later in development that differ from their primary early role in appendage specification.
Insect legs and wings have a common developmental and evolutionary origin. The appendage primordia are specified by the Wg and Dpp signaling proteins,which subdivide leg and wing imaginal discs into presumptive body wall(proximal) and presumptive appendage regions (distal) during the second instar. Initially the entire discs express both Tsh and Hth, and their repression by Wg and Dpp signaling is required for subdivision of the disc into proximal and distal domains (Abu-Shaar and Mann, 1998; Dong et al.,2001; González-Crespo et al., 1998; Wu and Cohen,1999; Wu and Cohen,2000). We have provided evidence that the el and noc genes serve as mediators of the function of the Wg and Dpp signaling systems in specification of the appendage field within the imaginal discs. El and Noc are induced by Wg and Dpp and are required to repress the proximally expressed proteins Hth and Tsh. Previous work had identified Dll as a gene required for appendage formation in leg and antenna,and nub as a gene required for wing. This report identifies El and Noc as a pair of zinc-finger proteins that function in both ventral and dorsal appendages. However, there are interesting differences in the way that they do so, when examined in detail.
Dll expression is required for the formation of all leg and antenna elements in the ventral (leg) discs, and until this work Dll was the earliest known marker for the distal region leg disc(Cohen et al., 1989; Cohen and Jürgens, 1989a; Diaz-Benjumea et al., 1994; Gorfinkiel et al., 1997; Panganiban et al., 1994). Previous work has shown that repression of Hth and Tsh by Dpp and Wg was not required for expression of Dll in the leg, nor could Dll repress Hth and Tsh(Wu and Cohen, 1999). Thus an essential mediator of the effects of Wg and Dpp was missing. Our results present evidence that El and Noc serve this function, as their removal leads to ectopic expression of Hth and Tsh. Removal of El and Noc does not cause loss of Dll expression, so we conclude that Wg and Dpp act independently to induce El and Noc expression and Dll to define the distal region of the leg disc.
The situation differs slightly in the wing. Repression of Tsh is the earliest marker for specification of the distal wing region(Wu and Cohen, 2002),preceding the onset of Hth repression or of Nub induction(Azpiazu and Morata, 2000; Casares and Mann, 2000; Ng et al., 1996). Loss of Tsh and Hth are required to allow Nub expression. We observed ectopic expression of Hth and Tsh and loss of Nub in clones lacking El and Noc activity. Thus in the wing, expression of the distal marker Nub cannot be demonstrated to be independent of El and Noc (because ectopic Hth can repress Nub, but not Dll). The vestigial gene is also important for wing development and has been proposed to be a wing specifying gene(Kim et al., 1996; Simmonds et al., 1998; Williams et al., 1991). However, Vestigial is expressed all along the DV boundary of the wing, both in the wing primordium and in the body wall. This leads us to suggest that while Vestigial is essential for wing development, its expression cannot be taken as a molecular marker for wing identity per se, particularly at early stages [as discussed previously (Wu and Cohen,2002)]. For this reason we have not focussed on analysis of the relationship between El, Noc and Vestigial in this report.
Is the repression of trunk genes needed to specify appendage as opposed to the body wall in wing and leg discs? In the wing disc the answer appears to be yes; repression of `trunk genes' like hth is necessary to make the remaining part of the disc competent to form the appendage. However, in the leg the situation is more complex. Coexpression of Dll and Hth does not disrupt proximal-distal axis formation, but leads to homeotic transformation of leg tissue into antennal tissue (Casares and Mann, 1998; Dong et al.,2001). Hth is not repressed and limited to proximal areas in the antenna. However, loss of el and noc activities in the leg disc leads to loss of distal leg tissue without any evident transformation into antennal tissue. Thus, El and Noc may regulate the expression of other`trunk genes', whose restricted expression are required to make the remaining leg and antenna disc competent to form the appendage.
The regional requirements for El and Noc highlight another interesting difference between leg and wing disc development. el noc double mutant cells were excluded from contributing to the tarsal region of the leg but not from contributing to the femur and tibia. As summarized above, lineage tracing has shown a considerable net flux of cells from the proximal(Tsh-expressing domain) into femur and tibia(Weigmann and Cohen, 1999). While there is no boundary of lineage restriction separating these domains,cells must be able to change from expressing the proximal marker Hth to expressing the distal marker Dll in order to move from one territory to the other (Wu and Cohen, 1999). The wing in contrast does not appear to normally exhibit this large net flux of cells from proximal to distal (K. Weigmann and S.M.C., unpublished data)and the el noc double mutant cells were excluded from contributing to the entire wing region. Our clonal analysis has suggested that el nocdouble mutant cells attempt to sort out toward proximal territory, or if that fails they can be lost from the disc, apparently by sorting out perpendicular to the epithelium. These observations suggest that El and Noc activity may contribute to the production of proximal-distal differences in cell affinities and thereby may help to maintain segregation of these cell populations during development.
We thank Lidia Pérez for technical assistance, Ann-Mari Voie for generation of transgenic flies and members of the lab for comments on the manuscript. M.W.'s work was carried out in Claude Desplan's lab at NYU. R.D.'s work was carried out in Benny Shilo's lab at the Weizmann Institute. We thank them for their contributions and their support. Anonymous reviewers helped to improve the manuscript.