Expression of achaete-scute homologues in discrete proneural clusters on the developing notum of the medfly Ceratitis capitata, suggests a common origin for the stereotyped bristle patterns of higher Diptera

The peripheral nervous system of insects is composed largely of sensory bristles. These are often randomly arranged over the body surface but are sometimes organized into stereotyped patterns which, in dipteran flies, are of use in classification. The arrangement of bristles in Drosophila melanogaster has been shown to result from a precise temporospatial regulation of the genes of the achaete-scute complex (AS-C) that encode related transcriptional regulators of the basic-helix-loop-helix (bHLH) protein family (Villares and Cabrera, 1987; Ghysen and Dambly-Chaudière, 1988; Alonso and Cabrera, 1988; Gonzalez et al., 1989). Expression of these genes provides cells with neural potential. In D. melanogaster, there are eleven large bristles, or macrochaetes, on each heminotum and these occupy stereotyped positions that do not vary between individuals. They arise from small clusters of cells expressing two AS-C genes, achaete and scute, in the larval imaginal disc, called proneural clusters, that prefigure the sites of each of the future bristles (Romani et al., 1989; Cubas et al., 1991; Skeath and Carroll, 1991). Expression is then progressively refined to single precursor cells. The achaete-scute genes share cis-regulatory enhancer sequences that are scattered over about 100 kb of DNA and respond to local positional cues conveyed by trans-acting factors that regulate their dynamic spatial and temporal expression patterns (Ruiz-Gomez and Modolell, 1987; Gomez-Skarmeta et al., 1995). Therefore the positions of the eleven stereotyped macrochaetes is the result of complex regulation of achaete-scute expression.

Some of the transcriptional regulators of achaete-scute are known; they are expressed in distinct, but broad domains over the epithelium of the notal disc and are thought to define a “prepattern” (Stern, 1954; Simpson, 1996; Modolell and Campuzano, 1998). Amongst these, the transcriptional activator Pannier is required for the bristles of the medial half of the notum and has been shown to bind to the dorsocentral enhancer to activate transcription at the site of the dorsocentral cluster (Ramain et al., 1993; Haenlin et al., 1997; Garcia-Garcia et al., 1999). It is not known how the discrete proneural clusters of achaete-scute transcripts arise from the broad bands of expression of the prepattern genes.

There are a large number of species of Diptera and many of these, in particular those of the Acalyptrata (that includes the family Drosophilidae) have different, but equally stereotyped, bristle patterns. As is the case for Drosophila, many of these patterns are phylogenetically old (McAlpine, 1989a; Grimaldi, 1987). The question therefore arises as to how the different patterns are made and to what extent the underlying genetic mechanisms have been conserved. Here we describe the isolation and transcriptional expression patterns of achaete-scute and pannier homologues in the medfly Ceratitis capitata, belonging to the family Tephritidae. A common ancestor for the Tephritidae and the Drosophilidae probably goes back more than 100 million years (McAlpine, 1989a). A rich fossil record has shown that many families of Acalyptrata were well differentiated by Oligocene times. Fossils of drosophilid species have been described dating back 50 million years although no fossils of Tephritidae have been reported (Grimaldi, 1987; McAlpine, 1989a). Our results demonstrate that gene duplication within the AS-C preceded the separation of these two families. The bristle pattern of Ceratitis displays slight differences from that of Drosophila and we find that these are reflected in changes in the expression pattern of scute. Bristles not found in Drosophila arise from proneural clusters that have no Drosophila counterparts, implying the use of distinct regulatory elements. Our results provide evidence for a common origin for the stereotyped bristle patterns of cyclorraphous Brachycera (McAlpine, 1989a; Simpson et al., 1999). Finally, the expression domain of pannier suggests a conserved role in the regulation of achaete-scute.

Screening for homologues of the AS-C and pannier genes was performed under low stringency at 42°C with 20% formamide containing standard hybridisation buffer. Washes were carried out at 50°C with 2× SSC, 1% SDS.

A genomic Ceratitis library (from L. Zwiebel), was plated and nylon replica filters (PALL, Biodyne A) screened with a 180 bp PCR fragment containing the bHLH domain of Drosophila virilis achaete (from J. Modolell) (degenerated PCR-Primers: NARERN: 5′AAC/T GCI C/AGI GAG/A C/AGI AAT/C GC 3′, AVEYIR reverse: 5′CG/TT/G/A ATG/A TAT/C TCI ACI GC 3′). One phage containing asense (pBS-C1I1.1-3.2: 3166 bp and pBS-C1I1.1-2.2: 2203 bp) and several containing scute (pBS-C1I2.2-7.0: 3890 bp) were isolated. Complete coding sequences were subcloned into pBluescript.

The bHLH domain of lethal of scute was isolated independently by PCR with degenerated primers (as above) using either genomic Ceratitis DNA or a third larval instar cDNA λ-ZAP library (from C. Savakis and S. Brogna) as template (3 cycles: 94°C 2 minutes, 37°C 1 minute 30 seconds, 72°C 1 minute 30 seconds; 15 cycles: 94°C 1 minute 30 seconds, 42°C 1 minute 30 seconds, 72°C 1 minute 30 seconds, 30 cycles: 94°C 1 minute 30 seconds, 46°C 1 minute 30 seconds, 72°C 1 minute 30 seconds, 10 minutes 72°C). The Ceratitis genomic library was rescreened with the Ceratitis lethal of scute PCR probe under moderate stringency with 25% formamide containing hybridisation buffer at 42°C. Washes were performed at 55°C with 2× SSC, 1% SDS. One phage contained lethal of scute (C3Q2.1). A 0.9 kb cross-hybridising PstI fragment was subcloned in pBluescript (pBS-C3Q2). No sequences from the achaete gene were recovered.

The cDNA library was screened with a 498 bp SphI-BamHI fragment containing the GATA-DNA binding domain of Drosophila pannier (from P. Ramain). A single phage was isolated and in vivo excised (pBS-PCC1F1: 1953 bp). All sequences were submitted to GenBank (Ceratitis pannier: AF184154, Ceratitis scute: AF224282, Ceratitis asense: AF224716, Ceratitis lethal of scute: AF224717).

Inserts were sequenced by using Taq-DNA Polymerase and a cycle sequencing system (Big Dye Terminator, Applied Biosystems) and sequencing reactions were analysed on a 373 DNA sequencing machine (Applied Biosystems). Sequence analysis was done with the University of Wisconsin GCG computer package. For alignment of achaete sequences the ClustalX software (Thompson et al., 1997) was used to generate phylogenetic relationships.

All experiments were performed with the Benakeo strain of Ceratitis (from C. Savakis). Flies were kept at room temperature and fed with yeast/saccharose powder (1:1). Eggs were collected in water dishes, and transferred to standard Drosophila food which was stirred and kept moist. Larvae were maintained at 25°C and 60% humidity.

Digoxigenin-labelled RNA probes (DIG-UTP, Boehringer-Mannheim) were generated using the standard protocol of Boehringer Mannheim. The resulting RNA was resuspended in 100 μl preHyb solution (50% formamide, 5× SSC, 0.1% Tween-20, pH 6,0). RNA was transcribed from linearized DNA templates: scute pBS-C1I2.2-7.0 (T7 sense, T3 antisense), lethal of scute pBS-3Q2 (T7 sense, T3 antisense), asense pBS-C1I1.1-2.2 (T7 sense, T3 antisense) and pannier pBS-PCC1F1 (T7 antisense, T3 sense).

Embryos were collected at room temperature and staged at 25°C. They were dechorionised and fixed as for Drosophila embryos, devitellinized by hand, rinsed in PBS and stored in methanol at −20°C. White pupae were incubated at 25°C and wing discs and pupal thoraces dissected in PBS and fixed using a modified version of the protocol of Pattatucci and Kaufmann (1992) with 16% formaldehyde solution (Polyscience-EM grade) and compensatory modifications in the amounts of other components.

In situ hybridisations were performed using a protocol adapted from Wülbeck and Campos-Ortega (1997). Drosophila pannier and achaete were visualized as in Cubadda et al. (1997) and Haenlin et al. (1997).

Adult cuticles were cleared in 10% KOH, dehydrated and mounted in Euparal.

Drosophila has four AS-C genes: achaete (ac), scute (sc), lethal of scute (l’sc) and asense (ase), and homologues of three of these were isolated from Ceratitis. The complete sequence of the putative sc protein is shown in Fig. 1A. The bHLH domains of Sc, L’sc and Ase are highly conserved and display 95%, 91.5% and 90% identity with the Drosophila counterparts, respectively (Fig. 1B; Villares and Cabrera, 1987; Alonso and Cabrera, 1988; Gonzalez et al., 1989). Comparison of this domain to other AS-C members ranges from 67% to 76%. Stretches of identical amino acid sequences are also seen in the amino and carboxy terminal parts of all three genes. An acidic region at the carboxy terminal end of Sc is homologous to that of Drosophila Sc (shown in Fig. 1A; Villares and Cabrera, 1987; Botella et al., 1996). Asense contains a similar acidic region, but with a different amino acid sequence (54% identity to Drosophila), which, as in Drosophila, is located near the centre of the coding sequence (Gonzalez et al., 1989). A phylogenetic analysis indicates a more distant position for Ase and suggests these are true homologues of the Drosophila genes (Fig. 1C). Extensive screening has so far not enabled us to isolate an achaete homologue from Ceratitis.

We have isolated a single pannier (pnr) homologue that shows a high degree of amino acid conservation with the Drosophila gene in the two zinc finger motifs of the GATA domain, where there is 93% identity (Fig. 1D; Winick et al., 1993; Ramain et al., 1993). This domain is involved in both DNA-binding and protein-protein interactions (Haenlin et al., 1997). The Ceratitis protein is smaller, 324 amino acids compared to 540 in Drosophila; this is mainly due to deletions in the less-well-conserved carboxy terminal half. Furthermore stretches of identical amino acids (75% identity) encompass the GATA domain. The carboxy terminal part of Drosophila Pnr bears two alpha helices, only one of which is conserved in Ceratitis.

The arrangement of macrochaetes on the mesonotum of Ceratitis is compared with that of three other species in Fig. 2. Calliphora vicina displays an ancestral pattern of four longitudinal macrochaete rows labelled acrostichal, dorsocentral, intraalar and supraalar (see Discussion). There are no acrostichal macrochaetes in Drosophila melanogaster. In Ceratitis, there is a single acrostichal bristle near the scutellum called the prescutellar, one dorsocentral, one postalar, one intraalar, two supraalar, two notopleural and two scutellar bristles.

The three larval stages of Ceratitis take 6 days and the pupal period 9 days at 25°C. Development of the imaginal discs is similar to that described by Anderson (1963) for Dacus tryoni, a related species of Tephritidae. The wing pouch begins to evert 8 to 10 hours after pupariation (AP), fusion of the two heminota occurs at 24 to 30 hours, head eversion is complete at 38 hours and the pupal cuticle starts to be visible at about 36 hours AP.

In D. melanogaster, expression of ac and sc in the proneural clusters (PNC) and the singling out of bristles precursors occurs during the third larval instar and the first 3 hours AP (Cubas et al., 1991; Skeath and Carroll, 1991; Huang et al., 1991). In situ hybridization revealed that the Ceratitis sc gene is similarly transcribed in discrete PNCs in the wing-thoracic disc of Ceratitis that precede the formation of the sensory organ precursors. They develop, however, over a much shorter time window. Expression is barely detectable in late third instar larvae and, at pupariation, only a few patches on the future wing blade can be seen. The PNCs of the notum become distinguishable 2 hours AP, expression is strong at 5-6 hours AP (Fig. 3A,D), and it begins to fade slowly thereafter. As in D. melanogaster, expression is refined to one or two spaced cells per cluster, the future bristle precursors, but does not last long. It disappears soon after singling out of the precursors.

We were able to correlate most of the discrete PNCs in Ceratitis with their respective sensory organs, both through comparison of the similarly shaped discs of Drosophila, and by following the positions of the precursors at later stages with both sc and ase staining (Fig. 3B,E; see below). The positions of some PNCs are similar to those of D. melanogaster (Fig. 3B). The single dorsocentral bristle arises from a discrete PNC and the two scutellar bristles arise from a single PNC. The precise correspondence between the PNCs from which the supraalar and notopleural bristles develop, as well as the postalar bristle and several nearby sensilla trichoidea, has not been traced. In addition, there are two bristles on the mesopleurum of Ceratitis, the anepisternal and anepimeral, that probably form from the PNCs visible anterior and ventral to the wing blade (Fig. 3A,B). Interestingly, the prescutellar bristle arises from a discrete PNC that has no counterpart in D. melanogaster (Fig. 3A-D).

A number of clusters are also seen in the wing and hinge region (Fig. 3A,B). Some correspond to campaniform sensilla found in similar positions in D. melanogaster, and widely conserved throughout the Diptera (Fudalewicz-Niemzyk, 1963; Dickinson et al., 1997). A few others are not found in Drosophila. Ceratitis bears four rows of short bristles along the anterior wing margin, two central rows of closely packed short spur bristles, a ventral row of closely packed small bristles and a dorsal row of 12 widely spaced chemosensory bristles; there are 5 other chemosensory bristles on the costa, which are also widely conserved (Fudalewicz-Niemzyk, 1963; Dickinson et al., 1997). In addition, small spaced bristles are found along veins R1 and R4/5. In Ceratitis, sc expression along the anterior margin, in a more or less continuous band, starts much later than in Drosophila, at about 10 hours AP, when the notal PNCs are already disappearing and the wing pouch is evaginating (not shown).

In D. melanogaster, l’sc is expressed exclusively in the central nervous system (Martin-Bermudo et al., 1991). Surprisingly, in Ceratitis l’sc is also expressed in the wing-thoracic disc in PNCs with a spatial arrangement similar to those expressing sc (not shown). We have not studied their evolution in detail.

In D. melanogaster, ase is not expressed in the PNCs, transcripts appear exclusively in the bristle precursors shortly after their formation (Jarman et al., 1993; Dominguez and Campuzano, 1993; Brand et al., 1993). Expression of ase in Ceratitis is similarly restricted to the precursors and a large complement of precursors are visible with ase probes at 6 hours AP (not shown and Fig. 3H). Expression continues for some time until about 24 hours, after which it fades. The macrochaete precursors of the notum of Ceratitis are thus generated exclusively AP, but they all arise before fusion of the two heminota and deposition of the pupal cuticle. At 19 hours AP, expression of ase is seen strongly in about a dozen single spaced precursors along the wing margin that probably correspond to the chemosensory bristles (Fig. 3H).

A second period of sc expression starting at about 36 hours is correlated with the development of the microchaete precursors. By this time, the heminota have fused and the pupal cuticle has been laid down. Staining is first visible at low levels over the entire scutum. It then intensifies in small scattered individual patches some of which appear to include several cells. By 47 hours, many single cells are stained; they are the precursors of the microchaetes (Fig. 3F,G). The precise pattern of microchaetes on the notum of Ceratitis is somewhat variable from one individual to another as well as between two heminota of the same individual, although these bristles are distributed over similar regions of the notum (Fig. 3E). We counted between 193 and 212 microchaetes on the scutum of six heminota, and between 8 and 12 on the scutellum. In D. melanogaster the acrostichal microchaetes, situated between the midline and the dorsocentral bristles, are aligned into five longitudinal rows that arise sequentially from five stripes of ac-sc transcripts (Fig. 2; Usui and Kimura, 1993; Simpson et al., 1999). In Ceratitis, there are no such rows (Fig. 3E). The microchaete precursors appear over the notum in a random distribution, we could discern no specific order to their appearance. The scutellar microchaete precursors arise in a similar fashion shortly after those on the scutum. scute expression fades from the microchaete precursors some time after 52 hours AP.

When the microchaete precursors are forming, sc expression reappears in a single cell corresponding to each macrochaete (Fig. 3F). We do not know if this is the bristle precursor or one of the four cells of the bristle organ.

In situ hybridization revealed that both sc and l’sc are first expressed in the early cellular blastoderm in two dorsal spots in the head region (not shown) and subsequently in a series of dorsoventral stripes in the presumptive neuroectoderm (Fig. 4A). These are of alternating intensity and continuity; at gastrulation, they become rearranged to give lateral and ventral groups of stained cells on either side of the midline (Fig. 4B). These are the first PNCs, which stain with variable intensity, and as the germ band extends new intermediate PNCs, expressing l’sc but not sc, appear in alternate rows (not shown). Expression is then refined to segregating neuroblasts (Fig. 4C). We have not followed the successive waves of formation of neuroblasts but, by stage 11, they are arranged into three columns (M, I and L in Fig. 4) on either side of the midline. scute and l’sc are thus expressed in distinct but overlapping patterns, sc being confined to a subset of the l’sc-expressing cells. The expression patterns are similar to those in Drosophila but differ in detail (Martin-Bermudo et al., 1991; Cabrera et al., 1987).

As in Drosophila, ase is expressed in single neural precursors at the extended germ band stage (Fig. 4D; Gonzalez et al., 1989).

Transcripts of pnr could not be detected by in situ hybridisation in larval discs, they can be seen shortly AP in some cells in the dorsal part of the thoracic disc. At 2 to 4 hours AP, expression is strong in the dorsal half of the notum in a domain very similar to that described for D. melanogaster, which includes the PNCs of the acrostichal, dorsocentral and scutellar bristles (Fig. 5A,C). The lateral border of the pnr expression domain is coincident with the position of the dorsocentral PNC (Fig. 5B). Expression continues throughout pupal development, narrows to a straight band along the dorsal midline at 21 hours when the two heminotal sheets start extending prior to fusion (Fig. 5D) and undergoes further dynamic changes at later stages (not shown).

The structure of the ac-sc and related atonal gene families has been strongly conserved throughout evolution and these genes have been shown to participate in development of the nervous system in many animals (for review see Brunet and Ghysen, 1999). The Drosophila AS-C comprises four genes with highly related sequences (Villares and Cabrera, 1987; Alonso and Cabrera, 1988; Gonzalez et al., 1989). Slight differences in the function of the proteins can be detected but, by and large, they are functionally redundant (Balcells et al., 1988; Garcia-Alonso and Garcia-Bellido, 1988; Rodriguez et al., 1990; Brand et al., 1993; Dominguez and Campuzano, 1993; Hinz et al., 1994; Skeath and Doe, 1996). This suggests that they have arisen through gene duplication. The overall structural organization of the AS-C, including the arrangement of the four structural genes and many regulatory sequences, has been conserved in D. virilis, another member of the family Drosophilidae, thought to have diverged from D. melanogaster some 55 million years ago (Beamonte, 1990; Garcia-Garcia et al., 1999). Therefore any gene duplication event(s) must be of ancient origin.

In contrast to Drosophila, other invertebrate species appear to have fewer ac-sc homologues. A single homologue has been found in the buckeye butterfly, Precis coenia (Galant et al., 1998). Phylogenetic analysis shows that it is no more closely related to any one of the Drosophila or Ceratitis genes than to any other, perhaps indicating that gene duplication may have occurred more recently than the separation between Lepidoptera and Diptera (Fig. 1C). So far a single gene, which is more closely related to atonal, has been shown to control development of peripheral sense organs in Caenorhabditis elegans (Zhao and Emmons, 1995). A single homologue has also been found in hydra (Grens et al., 1995). This poses the interesting question of the origin of, and the necessity for, four ac-sc genes in Drosophila. Our results provide evidence that at least three of the four AS-C genes are conserved in Ceratitis capitata, a member of the family Tephritidae that probably diverged from Drosophilidae more than 100 million years ago. The spatiotemporal regulation of the AS-C genes is very complex in both Ceratitis and Drosophila, and would be difficult to achieve with a single copy. This could be one reason for gene duplication, although it would not explain the identical regulation of ac and sc in D. melanogaster. Increased complexity of the expression pattern may correlate with the greater morphological complexity seen in the peripheral nervous system of more derived species. Stereotyped bristle patterns are characteristic of the Schizophora, but more basal clades of Diptera tend to have simpler, non-stereotyped bristle patterns and it will be interesting to see how many AS-C homologues are present in these species (McAlpine, 1989a,b; Simpson et al., 1999). Vertebrates often have extra copies of genes that are found as single copies in invertebrates. The number is usually four and probably arose through fixation of extra chromosomal copies in a lineage where the diploid state was later re-established. Such genome duplications are thought to have promoted the diversity found amongst vertebrates.

We do not yet know whether the three genes are grouped into a complex with a similar organization to that of Drosophila, but there are clearly a number of similarities in the regulation of the different members. As in Drosophila, sc and l’sc share overlapping expression patterns in the embryo and, in Ceratitis, are also co-expressed in PNCs in the discs. This indicates that they probably share some of the same cis-regulatory sequences.

A subset of bristles are found at similar positions in Ceratitis and D. melanogaster, and it is noteworthy that these arise from PNCs situated at spatially similar positions in the imaginal disc. This suggests conservation of the corresponding enhancers, a hypothesis that awaits the isolation of regulatory sequences. The positions of many bristles are useful taxonomically, because of the similarities in bristle positioning between related species and the invariant nature of the patterns within species. Our observations reinforce earlier assumptions, based on morphological comparisons, of the “homology” of specific bristles (McAlpine, 1989a; Simpson et al., 1999). Bristles in Ceratitis not present in D. melanogaster, such as the prescutellar, anepisternal and anepimeral bristles, develop from novel, discrete PNCs, suggesting the existence of different cis-regulatory elements. Thus one mechanism driving evolutionary changes in bristle patterns may have been the loss and/or acquisition of regulatory elements in the AS-C. The expression of l’sc in PNCs in the discs of Ceratitis suggests, that, in this species, l’sc is under the control of enhancer elements, that, in Drosophila drive the two genes ac and sc (Gomez-Skarmeta et al., 1995).

The pattern on the scutum of many species of schizophoran flies is thought to be derived from a basic arrangement of four rows of bristles that appear to be in homologous positions in different flies (McAlpine, 1989a; Simpson et al., 1999; Fig. 2). This suggests that an ancestor, common to most of today’s species, already possessed these four rows. While the Calyptrata generally bear rows of macrochaetes extending the full length of the scutum, the Acalyptrata display only a subset of bristles from some or all rows, a feature that is thought to be derived (Sturtevant, 1970; Garcia-Bellido, 1981; McAlpine, 1989a; Simpson et al., 1999; Fig. 2). Therefore, another possibility is that the positional enhancer elements of the D. melanogaster AS-C originate from ancient regulatory elements whose function may have been to drive ac-sc expression in four stripe-like domains corresponding to the four rows of bristles. The fact that the two dorsocentral precursors in D. melanogaster arise sequentially from a single cluster may reflect their common origin from the same row (Cubas et al., 1991). The acrostichal row is absent in D. melanogaster, but is represented by a single bristle, the prescutellar, in Ceratitis (Fig. 2). The precursor for this bristle forms within a discrete dorsally located PNC, clearly separate from the DC cluster, consistent with the hypothesis that this bristle has a different origin.

Many families of Schizophora retain the prescutellar bristle, so if the prescutellar PNC relies on a discrete regulatory element this is likely to be ancient. It may not be present in D. melanogaster. However, this particular bristle is of special interest because it is conserved even within the family Drosophilidae itself. Indeed the two subfamilies of Drosophilidae, the Steganinae and the Drosophilinae, are classified on the basis of the presence or absence respectively, of this bristle (Grimaldi, 1990; Fig. 2). Absence of the prescutellar bristle, in the Drosophilinae, is attributable to a loss during the course of the evolutionary history of this taxon (Grimaldi, 1990). Interestingly, one genus of the Drosophilinae, Scaptodrosophila, does carry a prescutellar bristle, the presence of which is considered to be a secondary gain (Grimaldi, 1990). If so, then the information required for its differentiation, perhaps including a discrete regulatory sequence, may have been retained in a latent form in some species of Drosophilinae.

Formally, changes in bristle patterns could also be achieved through changes in the distribution of upstream trans-regulators of ac-sc; to examine this, we have isolated the pnr gene from Ceratitis. On the Drosophila notum, pnr is expressed in a broad longitudinal dorsal (medial) domain, where it is required for ac-sc expression and the development of macrochaetes (Ramain et al., 1993). Ceratitis pnr is expressed in an identical thoracic domain. In Drosophila, it has recently been demonstrated that Pnr activates ac-sc directly by binding to the dorsocentral enhancer, a characterised sequence of 1.4 kb situated close to the coding region of ac (Gomez-Skarmeta et al., 1995; Garcia-Garcia et al., 1999). Furthermore the position of the two dorsocentral bristles is defined by the lateral borders of expression of pnr and u-shaped (ush), a gene encoding a cofactor that downregulates Pnr activity (Cubadda et al., 1997; Haenlin et al., 1997; Garcia-Garcia et al., 1999). The lateral border of the pnr domain in Ceratitis is coincident with the site of formation of the dorsocentral bristle and, in addition, the sequence of the amino terminal zinc finger, required for association of Pnr with Ush, is 100% identical in Ceratitis pnr suggesting that regulation by Ush may have been conserved. It will be of interest to isolate a ush homologue in this species because, if the spatial expression of ush has been conserved, the prescutellar PNC would be situated within the ush expression domain.

In Ceratitis, as in Drosophila, the macrochaete precursors are formed before, and the microchaete precursors after, the pupal moult. The difference in morphology between the two classes of bristles may therefore reflect their origins at different instars (Simpson et al., 1999). In the acrostichal area of the scutum, the microchaetes of D. melanogaster are aligned into longitudinal rows (Fig. 2). Other species of Drosophilidae display a variable number of rows or no rows at all, merely a random distribution of evenly spaced microchaetes (Grimaldi, 1990). Cladistic analysis of this feature suggests that a random arrangement is more ancient so the rows are probably a derived feature (Grimaldi, 1990). Ceratitis displays a more ancestral, random distribution of acrostichal microchaetes, the number and position of which is variable between individuals. In D. melanogaster, stripes of ac-sc expression precede the rows of microchaetes (Simpson et al., 1999) but, in Ceratitis, we cannot detect any expression in discrete stripes. Instead scute transcripts are initially ubiquitously distributed and the precursors arise randomly. The Notch-mediated mechanism of lateral inhibition, together with ubiquitous ac-sc expression would be sufficient to generate a spaced pattern of randomly arranged bristles (Simpson et al., 1999). The random bristle arrangements that predominate in more basal groups of Diptera, may also result from ubiquitous ac-sc expression. If so, complex spatial transcriptional regulation of these genes, with its attendant cis-regulatory sequences, may be novel, more recent innovation.

Our results provide molecular evidence in favour of a similar genetic control for the stereotyped patterns of macrochaetes in two species of Acalyptrata. It appears that many aspects of the spatial regulation of the ac-sc genes have been conserved and that bristles at similar positions arise from proneural clusters situated at spatially equivalent positions in the imaginal disc. This suggests conservation of the cis-regulatory elements that drive ac-sc expression at these sites, a question that awaits isolation of these sequences in species other than Drosophila. Comparative morphology has suggested that such stereotyped patterns may have a common origin from an ancestral arrangement of four longitudinal rows on the scutum (McAlpine, 1989a,b; Simpson et al., 1999). It remains to be seen whether ac-sc transcripts are present in stripe-like domains in extant species bearing a more ancestral pattern of four rows.

We acknowledge the financial support of the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, as well as the Programme of the European Community (contract n° FMRX CT 96 0065) and Programme Génome du CNRS. C. W. is the recipient of a fellowship from the DFG. We warmly thank Drs C. Savakis, F. Kafatos, G. Saccone, S. Brogna, D. Artiaco, E. Giordano, L. Zwiebel, P. Ramain and J. Modolell for Ceratitis libraries, probes and help with establishing the fly colony, K. Usui and R. Woehl for help with a figure and the members of the sequencing facility of the IGBMC.