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
Author for correspondence at present address: Department of Zoology, Downing Street, Cambridge CB2 3EJ, UK (e-mail: pas49{at}cam.ac.uk)
Accepted 31 October 2001
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SUMMARY |
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Key words: Diptera, Calliphora vicina, Sensory organ, achaete-scute, pannier
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INTRODUCTION |
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Other species of Diptera, particularly those of the derived cyclorraphous Schizophora (that includes Drosophila), have different, but equally stereotyped bristle patterns. Many of these patterns are phylogenetically old, suggesting that they are stable over long periods of evolutionary time (McAlpine, 1981; Grimaldi, 1987
). Closely related species have similar arrangements of bristles, whereas evolutionarily more distant ones display more diverged patterns. Ceratitis capitata is separated from Drosophila by about 80 million years (McAlpine, 1981
). It displays a stereotyped bristle pattern with some bristles occupying similar positions to those of Drosophila (Wülbeck and Simpson, 2000
). The scute gene of Ceratitis is expressed in proneural clusters at the sites of each future bristle, suggesting a similar genetic organisation of the locus in this species (Wülbeck and Simpson, 2000
).
Throughout the cyclorraphous Schizophora there is a basic arrangement of bristles on the dorsal notum (McAlpine, 1981). There are four rows of bristles on the scutum: the acrostichal (AC), dorsocentral (DC), intra-alar (IA) and supra-alar (SA) rows. The pattern of most species of Schizophora can be superimposed upon this basic ground plan, even though some species display all rows and others have only a subset (McAlpine, 1981
). It has thus been postulated that the stereotyped bristle patterns of species such as Drosophila and Ceratitis are derived from an ancestral pattern similar to this ground plan (McAlpine, 1981
; Simpson et al., 1999
). The cyclorraphous Schizophora are subdivided into two subordinate groups, the Calyptrata and the Acalyptrata. Both Drosophila and Ceratitis are acalyptrates. When compared with the Acalyptrata, Calyptrata generally bear more macrochaetes and many species display all four rows extending the full length of the scutum. Calliphora vicina is one such species with a pattern resembling the hypothetical ancestral one. It is separated from Drosophila by at least 100 million years.
To investigate evolutionary changes in ac-sc expression, we have isolated homologues of these genes, and also pannier, from Calliphora vicina and examined their expression patterns. We find that sc is expressed in two longitudinal stripes that prefigure the development of the AC and DC rows of bristles. This result suggests that a stripe-like expression pattern of sc may be an ancestral feature and may have preceded the evolution of the small discrete proneural clusters characteristic of Ceratitis and Drosophila. In contrast, bristles of the IA and SA rows of Calliphora arise from domains of sc-expressing cells some of which resemble proneural clusters. These observations reinforce the hypothesis that the stereotyped patterns are derived from an ancestral pattern of four rows of bristles on the scutum and suggest that this pattern may have been the result of a regulated expression of sc in four stripes. We have also examined the expression pattern of the pannier homologue in Calliphora. We find that it is expressed in a conserved domain in the medial dorsal notum, consistent with a possibly conserved selector gene function. The implication of these results for the evolution of the cis-regulatory elements and the function of Pannier in the regulation of sc expression in the proneural clusters of Drosophila is discussed.
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MATERIALS AND METHODS |
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RT-PCR
Fragments of Calliphora scute (sc) (729 bp); pannier (pnr) (1194 bp), and Delta (Dl) (555 bp) were isolated by RT-PCR using the following degenerated primers (5' to 3', forward then reverse):
sc: AAYGCIMGIGARMGIAAYCG, CRTCRTCIGGIGTRCARTCYTC;
pnr: GAYTTYCARTTYGGIGARGG, GCIGYYTGIATIACRTTRTGYTG;
Dl: CCIGGIACITTYWSIYTIATIRTIGARGC, RCAIGTICCICCRTTIVCRCAIGG.
cDNA was generated from mRNA extracted from a 0- to 24-hour embryo collection using Superscript II reverse transcriptase (Gibco BRL). This was then used as a template. PCR was performed according the following general scheme: 94°C 1 minute; annealing temperature 1 minute 30 seconds; 72°C 2 minutes; 35 cycles; 10 minutes 72°C. PCR products were cloned into pGem T easy vector (Promega).
RACE
The 1194 bp fragment of pnr recovered by RT-PCR was extended by 5' RACE PCR using the 5'/3' RACE kit from Roche. A composite sequence of 1533 bp was generated.
Low stringency screening
Homologues for lethal of scute (lsc; also known as l(1)sc) and asense (ase) were isolated by low stringency screening performed at 42°C in buffer containing 20% formamide, 5x SSPE, 0.5% SDS, 5x Denharts solution. Washes were carried out at 50°C with 2x SSC, 0.5% SDS. A genomic Calliphora library (from M. Bownes) was plated and nylon replica filters (Amersham, Hybond-NX filters) were screened with a fragment containing the bHLH domain of Drosophila virilis achaete (ac) (from J. Modolell). Several phages containing either lsc or ase were isolated. Complete coding sequences were subcloned into pBluescript vector (Stratagene).
High stringency screening
To recover the full sequence of sc, the Calliphora genomic library was screened at high stringency with the 680 bp fragment recovered by RT-PCR using Amersham Hybond-NX filters and conditions according to the manufacturer.
All sequences were submitted to GenBank. Accession numbers: asense, AY061875; lethal of scute, AY061876; scute, AY061877; pannier, AY061878; Delta, AY061879.
Sequence analysis
Sequences were compared using the ClustalX software. Alignments were performed using default ClustalX parameters, and percentage identities calculated from the resulting alignments (Thompson et al., 1997).
Rearing of Calliphora
Flies were kept at room temperature and fed with sucrose. Eggs were laid in fresh meat and kept at room temperature. Larvae were fed on fresh meat and kept at room temperature. White pupae were collected and staged at 25°C.
Labelling of RNA probes
Digoxigenin-labelled RNA probes (DIG-UTP, Roche) were generated using the standard protocol of Roche. The resulting RNA was resuspended in 100:l preHyb solution (50% formamide, 5x SSC, 0.1% Tween-20, pH 6.0). RNA was transcribed from linearised DNA templates.
Tissue preparation and staining
In situ hybridisation
Wing discs and pupal thoraces were dissected in phosphate-buffered saline (PBS) and fixed using a modified version of the protocol of Pattatucci and Kaufmann (Pattatucci and Kaufmann, 1992) in a solution of 4% formaldehyde, 5% DMSO in PBS. In situ hybridisations were performed using a protocol adapted from Wülbeck and Campos-Ortega (Wülbeck and Campos-Ortega, 1997
).
Immunostaining
Wing discs and pupal thoraces were dissected in PBS, fixed in 4% formaldehyde/PBS for 20 minutes and stained. Mouse anti-22C10 and anti-HRP (horseradish peroxidase) primary antibodies were used at 1:200 dilution. Biotinylated anti-mouse secondary antibody was used and visualised using a standard ABC kit (Vector Chemicals). All preparations were mounted in 80% glycerol, 1x PBS.
Thoraces
Adult flies were collected 30-90 minutes after eclosion before the cuticle had tanned and darkened and stored in 70% ethanol, thereby allowing clearer visualisation of bristle patterns. Thoraces were dissected in 70% ethanol, transferred to 100% ethanol for 10 minutes and mounted under raised coverslips in Euparal (Fisher Chemicals).
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RESULTS |
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scute is expressed in stripes and clusters of cells in the developing notum
The bristle pattern of the dorsal notum of Calliphora is depicted in Fig. 2D. The four rows of large bristles on the scutum are labelled AC, DC, IA and SA for the acrostichal, dorsocentral, intra-alar and supra-alar rows, respectively. The transverse suture divides the scutum into pre-sutural and post-sutural domains. The scutellar suture also separates the scutum from the scutellum. The scutellum bears a single line of scutellar (SC) bristles round the lateral edge. The expression of sc in the developing notum was examined by in situ hybridisation. Expression starts at pupariation before the wing discs have started to evert and fuse along the midline. The general shape and morphology of the prospective notum is reflected in the shape of the discs, which bear strong similarity to the well-studied Drosophila discs (Usui and Simpson, 2000). Through examination of many different stages and comparison with Drosophila, we were able to determine the positions at which the various bristles arise. By 3 hours after puparium formation (h APF), two distinct longitudinal stripes of expression, aligned with the dorsal midline, are visible in the medial half of the future scutum at the positions of the future AC and DC bristles (Fig. 2A,D). Both stripes are interrupted by a band in which expression is absent; this corresponds to the future transverse suture (see Fig. 2D). A single stripe of expression along the posterior medial edge prefigures the row of SC bristles. In the lateral half of the notum expression appears in several broad domains and smaller clusters at the sites of the future IA and SA bristles (Fig. 2A,D). A stripe-like domain can be discerned but appears to include bristles of both IA and SA rows. In the lateral region, too, expression is lacking along the prospective transverse suture. The correspondence between the domains of expression and the future bristle rows is shown in Fig. 2D. Several clusters of expressing cells are also present in the region of the wing hinge and calypter, as well as on the wing blade, but we did not attempt to correlate these with specific sensory organs (Fig. 2A,B).
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By 8-10 hours APF, the stripes of sc expression are less coherent and later strong spots of high levels of expression are seen, which probably reflect the emergence of bristle precursors (Fig. 2B). At the same time ase starts to be expressed in single cells, the precursors of the bristles. By 10 hours, ase expression is widespread amongst the precursors (Fig. 2C,E). The rows of precursors arise from within each stripe or cluster of expression. However, ase expression is transient and the complete pattern of bristle precursors cannot be visualised at any one time. The order in which bristle precursors arise, as revealed by ase and high levels of sc, mirrors the progression of sc expression, and there is a general trend for precursors to arise in a posterior to anterior fashion. This holds true for the AC and DC domains, each of which have three presutural and three postsutural precursors. Exceptionally though, the final precursors to form in the post-sutural domain are the central ones of each triplet. These intercalate between the existing two, perhaps after growth of the epithelium by cell division generates more available space. This may also be the case for the three pre-sutural bristles in each row. By 16 hours APF sc expression has faded from the precursors.
22C10 is a marker of late precursors and the entire neural lineage, and is expressed later than sc and ase (Zipursky et al., 1984). The 22C10 antibody thus reveals neural precursors and staining for this marker reveals a similar pattern and time progression of precursor segregation (Fig. 2F). We also isolated a 555 bp fragment from the Delta gene of Calliphora. Delta has been shown to be downstream of Ac-Sc in Drosophila (Kunisch et al., 1994
; Parks et al., 1997
). In situ hybridisation with this probe, as well as staining with the cross-reacting antibody against horseradish peroxidase, a neural marker, confirmed the above sequence of events (not shown).
In addition to the large bristles (macrochaetes), the notum is also covered with numerous small bristles (microchaetes). At about 30 hours APF a second wave of sc expression takes place and is correlated with segregation of the precursors of the small bristles (Fig. 3A,B). Staining is fairly ubiquitous over the notum but is again excluded from the sutures. It is then refined to expression in single cells. sc expression at this stage reappears in the macrochaete precursors (Fig. 3A,B). By 33 hours APF ase staining is visible in single microchaete precursors (Fig. 3C), and by 50 hours 22C10 staining indicates that axonogenesis of the bristle neurons is taking place (Fig. 3D). We were unable to detect lsc transcripts during development of the imaginal notum.
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DISCUSSION |
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Our results indicate strong conservation of the roles of the different ac-sc genes. In Drosophila, lsc is essential for development of the central nervous system, and its loss results in lethality. We find that, as in Drosophila, lsc is expressed in the central nervous system of Calliphora during embryogenesis, but is not expressed in the developing notum. Similarly, expression of sc in proneural domains in the presumptive notum is conserved and expression of ase is restricted to sensory precursors. This suggests that specialisation of the functions of these three genes predates the separation of acalyptrate and calyptrate Schizophora.
Proneural clusters may have arisen from an ancestral pattern of longitudinal stripes of scute expression
An arrangement of four longitudinal rows of large bristles is characteristic of the scutum of a number of calyptrate flies and is thought to resemble an ancestral pattern or ground plan, from which the many different patterns seen in calyptrate and acalyptrate species are derived (McAlpine, 1981; Simpson et al., 1999
). Thus an alignment of bristles into four rows may have been the first patterning event in a series of steps that culminated in the stereotyped bristle arrangements characteristic of Drosophila. The single AC and DC bristles of Ceratitis capitata, an acalyptrate species, come from two separate proneural clusters, suggesting a different developmental origin for AC and DC bristles, consistent with the hypothesis that each of them may be derived from an independent row (Wülbeck and Simpson, 2000
). Drosophila does not bear any AC bristles, but does carry two DC bristles that interestingly arise from a single proneural cluster in a posterior to anterior sequence (Cubas et al., 1991
; Skeath and Carroll, 1991
). This cluster is controlled by a discrete cis-regulatory element, called the DC enhancer (Garcia-Garcia et al., 1999
; Gomez-Skarmeta et al., 1995
). The origin of this and the other positional enhancers is unknown. We have demonstrated that the row of DC bristles in Calliphora arises from a stripe of sc-expressing cells. It is thus tempting to speculate that a stripe-like domain may have preceded the cluster-shaped domain during the course of the evolutionary history of the lineage leading to Drosophila. If so, discrete regulatory elements may have been acquired to drive expression of sc in stripes on the scutum of a common ancestor of Calliphora and Drosophila. Identification of regulatory elements in Calliphora may help to resolve this hypothesis.
It is noteworthy that the two DC bristles of Drosophila are situated close to one another. If they are indeed derived from a complete longitudinal DC row present in an ancestor, through secondary loss of some of the bristles in the row, it seems likely that bristle loss would occur from the anterior downwards or from the posterior upwards, or both together. The DC enhancer may be derived from a single, discrete regulatory element that was responsible for a stripe of expression in the ancestor. If so, it is unlikely that bristles would be lost from the centre of the row, since this would entail a division of the stripe domain into two separate clusters of expression. We examined the distribution of DC bristles in 63 species of acalyptrate flies from 17 different families. 33% were found to have bristles missing from the anterior end of the row (see also Sturtevant, 1970), 8% from both anterior and posterior ends, and only one species (1.6%) had bristles missing from the middle of the row. The entire DC row was lacking in 1.6% of this sample. Similarly, examination of 52 species of calyptrate flies from 6 families, showed missing pre-sutural DC bristles in 9.6% of cases. The entire DC row was lacking in 7.7% of the animals.
The IA and SA bristles of Calliphora do not arise from stripes of sc expression but from apparent clusters. These resemble the proneural clusters of Drosophila and Ceratitis and are associated with a greater degree of determinacy of the positioning of these bristles (see below).
Stereotyped positioning of bristles along the anteroposterior coordinate of the scutum is a recent feature, whereas that along the mediolateral coordinate is of ancient origin
The pattern of four bristle rows appears to be an ancient, widespread one that has been retained regardless of considerable size differences between different species (McAlpine, 1981; Simpson et al., 1999
). This suggests that bristle positioning along the mediolateral coordinate of the scutum was fixed a very long time ago. In contrast, anteroposterior patterning, that is the stereotyped positioning of bristles within rows, seems to have been acquired more recently in derived species. It is a characteristic of many acalyptrate flies such as Drosophila and Ceratitis, but is not a consistent feature of more basal species that frequently display a variable number of bristles within the rows. Such variability is thought to be an ancestral feature.
Calliphora appears to be intermediate with respect to this morphological feature. The number of bristles in the AC row of Calliphora varies between individuals: large flies may have more and small flies fewer AC bristles. It is clear that the precise position of each bristle is, to some extent, variable, since the bristles are often displaced when compared with those on the contra-lateral side (Fig. 5B,E). The AC bristles arise from a stripe of sc expression, so spacing of the bristle precursors could be simply achieved through Notch-mediated lateral inhibition (Wigglesworth, 1940; Kimble and Simpson, 1997
; Simpson, 1990
). The distance between bristles is a function of the range of Notch signalling, so in larger animals there would be room for more precursors. This view is supported by the order in which the precursors arise within the post-sutural AC and DC rows. Each row has three post-sutural bristles, with precursors for the posterior and anterior-most forming first, followed by the central precursor. Formation of the central precursor may only be possible after growth of the epithelium has provided sufficient space between the other two precursors. It is also noticeable that missing post-sutural bristles are invariably those located between the two early forming bristles, which are never lost, and that supernumerary bristles are also added in the middle.
However, in other individuals the positions of the wild-type bristles do not change, and additional ones are superimposed on top of the wild-type pattern. This observation leads us to postulate that there may be another mechanism(s), in addition to the regulation of sc transcription, which helps to position the precursors. In Drosophila, one or two precursors are selected from each proneural cluster by means of Notch-mediated lateral inhibition (Wigglesworth, 1940; Hartenstien and Posakony, 1990
; Heitzler et al., 1996a
; Heitzler and Simpson, 1991
). However the choice is often biased to a cell at a specific position within the cluster (Cubas et al., 1991
; Simpson, 1997
; Skeath and Carroll, 1991
); it is not known how this is achieved.
The function of pannier and the origin of cis-regulatory elements in the achaete-scute complex
The pnr gene of Calliphora was found to be expressed in a conserved domain, similar to that of Drosophila and Ceratitis, that covers the medial half of the notum. This suggests that pnr has retained its selector gene function (Calleja et al., 2000) in all three species. The bristle patterns and the domains of sc expression within the pnr expression domain differ, however, between the three species (Fig. 4). So, if the function of pnr has been conserved, other factors must have changed in order to account for these differences. It is not entirely understood how the broad domain of Pnr in Drosophila is translated into three small clusters of ac-sc expression, but this requires the activity of three discrete cis-regulatory elements, as well as modulation of Pnr function by at least one cofactor, the product of the u-shaped (ush) gene (Heitzler et al., 1996b
; Ramain et al., 1993
; Garcia-Garcia et al., 1999
; Gomez-Skarmeta et al., 1995
; Haenlin et al., 1997
; Cubadda et al., 1997
). Homologues of ush have not been isolated in Ceratitis and Calliphora, but the AC bristles of these species are situated within the domain where ush is expressed in Drosophila. Changes in the regulation of genes encoding cofactors for Pnr, such as ush, is thus a possible mechanism for evolutionary changes in bristle patterns.
Conclusions
Our observations suggest a model for the changes in gene regulation that may have occurred during evolution of the stereotyped bristle patterns of higher flies (Fig. 6). An ancestor of the Schizophora would have had a pattern of four longitudinal rows of large bristles on the scutum (McAlpine, 1981; Simpson et al., 1999
). The bristle precursors would be spaced apart by lateral inhibition and the number of bristles in each row, and hence their position, would be variable. Four stripes of sc expression may have allowed the development of these rows, and they may have been a result of the activity of four discrete cis-regulatory elements. The two medial stripes would have been in the domain of pnr expression, and Pnr would have regulated activity of the two corresponding enhancers. During the course of evolution of the lineage leading to acalyptrate flies there has been a tendency to reduce the number of bristles through secondary loss (Grimaldi, 1987
; Simpson et al., 1999
; Sturtevant, 1970
). At the same time the anterior-posterior positioning of individual bristles has become stereotyped. The cis-regulatory elements responsible for the stripes of sc expression may have been retained during this process and perhaps have been modified to drive expression in small proneural clusters. This development may have been accompanied by modulation of the activity of Pnr brought about by changes in the expression of regulatory cofactors such as Ush.
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ACKNOWLEDGMENTS |
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