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scute expression in Calliphora vicina reveals an ancestral pattern of longitudinal stripes on the thorax of higher Diptera

Daniela Pistillo^*, Nick Skaer^* and Pat Simpson

Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, CU de Strasbourg, France
^* These authors contributed equally to this work

View larger version (74K): [in a new window]   Fig. 1. Amino acid sequence comparison of the three ac-sc genes isolated from Calliphora vicina (cv) with their orthologues in Drosophila (dm). (A) scute, (B) lethal of scute, and (C) asense. The highly conserved basic helix-loop-helix (bHLH) motif is in red. The line above the sequences indicates conserved positions with one of three symbols: (*) indicates positions that have a single, fully conserved residue. These positions have also been shaded blue. (:) and (.) represent strong and weaker degrees of conservation respectively, according to the Gonnet Pam250 matrix (Benner et al., 1994).

View larger version (123K): [in a new window]   Fig. 2. Early expression of scute (sc) and asense (ase) in the wing disc of Calliphora vicina. (A,B,D) expression of sc transcripts, (C,E) expression of ase transcripts, (F) visualisation of 22C10 protein. Enlargements of the thoracic domain of the discs in A-C, indicated by the rectangles, are shown directly below. By 3 hours after pupariation, sc transcripts, visualised by in situ hybridisation (A) are present in two broad longitudinal stripes in the medial half of the prospective notum. These correlate with the positions of the future acrostichal (AC) and dorsocentral (DC) bristle rows, and are interrupted by a mediolateral band at the site of the prospective transverse suture in which expression is absent. In the lateral domain, at the sites of the future intra-alar (IA), notopleural (NP) and supra-alar (SA) bristles, sc is expressed in a series of clusters. By 8 hours (D) to 10 hours (B) after pupariation, sc expression is restricted to the sites of the future bristle organs, where small groups of cells have accumulated higher levels of the transcripts. A clear expression domain is seen in the scutellum; it is separated by an area in which sc is absent that corresponds to the prospective scutellar suture. In D a schematic drawing of the relationship between sc expression at 8 hours after pupariation and the morphology of the future adult thorax (macrochaetes are shown in pink, microchaetes in blue). At this stage the disc remains a highly folded epithelium which later expands longitudinally at the midline, and laterally and ventrally at the lateral edge of the notum. This explains the compact aspect of the acrostichal and scutellar domains, and close proximity of the intra- and supra-alar domains at this stage. ase expression in single cells (C,E), and visualisation of neurons with the 22C10 antibody 10 hours after pupariation (F), show that the early expression of sc prefigures the sites at which bristle precursors are born.

View larger version (126K): [in a new window]   Fig. 3. Late expression of scute (sc) and asense (ase) in the pupal thorax of Calliphora vicina. After fusion of the two heminota along the midline, sc is re-expressed in the pupal thorax 29 hours after pupariation. Expression is initially widespread, and is excluded only from the sites of the prospective sutures, before becoming restricted to individual microchaete precursors by 31 hours after pupariation (A). scute is re-expressed in the macrochaete precursors (B: enlargement of the boxed area in A; macrochaete precursors are indicated by arrowheads) in the immediate vicinity of which down-regulation of sc in neighbouring cells is particularly clear. By 33 hours after pupariation, ase expression can be detected in microchaete precursors (C), and by 51 hours after pupariation, 22C10 staining reveals that axonogenesis has been initiated (D).

View larger version (45K): [in a new window]   Fig. 4. Expression of pannier (pnr) in the wing disc of Calliphora vicina. (A) The boundary of pnr expression in the medial half of the developing notum at 8 hours after pupariation (arrowheads). The lateral limit of pnr expression appears to correspond to the position of the future precursors of the dorsocentral row, visualised in B by 22C10 staining. This seems to be a conserved feature with other species of cyclorraphous Schizophora: a similar correlation has been described in Ceratitis capitata and Drosophila. (C) A schematic representation of how the expression domains of pnr (shown in pink) in the wing disc correlate with bristle positions on the imaginal notum of the three species. Note that dorsocentral (DC) bristles are always positioned at the limit of pnr expression, but that the bristle patterns differ. aDC, anterior DC; aSC, anterior scutellar; pSC, posterior scutellar.

View larger version (73K): [in a new window]   Fig. 5. Size-dependent variation in the number of bristles in the acrostichal row on the notum of Calliphora vicina. (A-E) Thoraces of adult Calliphora together with enlargements of the boxed areas which are shown directly beneath. All thoraces are to scale. Those from large flies (70 mg; A,B) regularly display more bristles than a ‘wild-type’ thorax (55 mg; C). Conversely, thoraces from smaller flies frequently have fewer bristles (40 mg; D,E). Variability in bristle number extends to both the presutural (E) and postsutural (A,B,D) domains. Comparison with the contra-lateral hemithorax reveals that an increase or decrease in the number of bristles is sometimes associated with a displacement of the ‘wild-type’ bristles (B,E), whilst at other times the changes are superimposed on the ‘wild-type’ pattern, leaving it unchanged (A,D). (F) Variation in bristle number between individuals of different sizes of Calliphora vicina. Individual flies were weighed and their bristle patterns examined; each bristle row was treated separately. The average, or ‘wild-type’, pattern is shown in Fig. 2D and Fig. 5C. The graph indicates departures from this pattern. Variation was discovered in all four scutal rows, and also in the scutellar row: both ‘additional’ and ‘missing’ bristles occurred relatively frequently. The highest variation was seen in the bristles of the scutellar and acrostichal rows, which were also the only rows in which variation correlated with the size of the individual.

View larger version (25K): [in a new window]   Fig. 6. A model for the evolution of proneural gene expression and bristle patterns within the higher Diptera. Representations of proneural gene expression in late larval/early pupal wing imaginal discs of different species are depicted on the right. The corresponding bristle pattern on the adult heminotum of each species is shown on the left. The acrostichal (AC) bristles and corresponding expression domains are shaded red, the dorsocentral (DC) blue, the intra-alar (IA) green, the supra-alar (SA) yellow, and the scutellar (SC) pink. Unshaded domains represent proneural expression not associated with bristles of the scutum and scutellum. The top diagram depicts a hypothetical ancestor of the cyclorraphous Schizophora. Proneural expression on the scutum is hypothesised to have been in four stripes, with a further stripe on the scutellum, giving rise to five rows of bristles each containing a variable number of spaced bristles. In Calliphora vicina, proneural expression corresponding to the AC, DC and SC bristle rows occurs in stripes, but that corresponding to the IA and SA rows is in proneural clusters. The number of bristles in the DC, IA and SA row is only very slightly variable. Bristles have a tendency to occupy more or less stereotyped positions. However, variability is quite common in the AC and SC rows, and a displacement of bristles from the stereotyped positions is also observed in the DC row. In Ceratitis capitata and Drosophila melanogaster expression of proneural genes occurs in clusters of cells that correspond to the positions of the bristles that occupy highly stereotyped positions (Cubas et al., 1991; Skeath and Carroll, 1991; Wülbeck and Simpson, 2000). The notal bristle pattern of Drosophila is extremely robust as changes are seen in less than 0.1% of individuals. Bristle rows are not present in many acalyptrates like Ceratitis and Drosophila, but the stereotyped arrangements may be derived from the pattern of rows in a common ancestor similar to that shown at the top, through secondary loss of bristles. Bristles are thus named AC, DC, IA or SA according to their presumed origin. The expression of proneural genes in clusters of cells in Drosophila is known to depend upon discrete cis-regulatory enhancer elements in the achaete-scute gene complex (Gomez-Skarmeta et al., 1995; Ruiz-Gomez and Modolell, 1987). One possibility is that these elements are derived from regulatory elements that allowed an expression of proneural genes in longitudinal stripes in an ancestor.