Redundant mechanisms mediate bristle patterning on the Drosophila thorax

Kazuya Usui1, Claire Goldstone, Jean-Michel Gibert2, and Pat Simpson3

Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK

Edited by Eric H. Davidson, California Institute of Technology, Pasadena, CA and approved September 11, 2008 (received for review May 2, 2008)

The thoracic bristle pattern of Drosophila results from the spatially heterologous promoter and in the absence of normal cis- restricted expression of the achaete-scute (ac-sc) in clusters regulation, indicates that factors acting downstream of Sc are of cells, mediated by the activity of many discrete cis-regulatory sufficient to position the bristles. We discuss the possibility that sequences. However, ubiquitous expression of sc or asense (ase) patterning by sr, emc, and h is ancestral, and that the positional achieved with a heterologous promoter, in the absence of endog- cis-regulatory sequences might be of recent origin. enous ac-sc expression, and the activity of the cis-regulatory elements, allows the development of bristles positioned at wild- Results type locations. We demonstrate that the products of the genes Uniform Scute and Asense Expression Rescues Macrochaetes at Wild- stripe, hairy, and extramacrochaetae contribute to rescue by an- type Locations in achaete-scute Mutant Flies. ase is a target of Ac-Sc tagonizing the activity of Sc and Ase. The three genes are ex- and encodes a closely related to Ac and Sc that displays pressed in specific but overlapping spatial domains of expression stronger proneural properties than either Ac or Sc when mis- that form a prepattern that allows precise positioning of bristles. expressed in the epithelium (10, 11, 24). We used the Gal4-UAS The redundant mechanisms might contribute to the robustness of method to drive simultaneous ubiquitous expression of sc and the pattern. We discuss the possibility that patterning in trans by ase. This combination allows significant bristle rescue. In (1)ac3 antagonism is ancestral and that the positional cis-regulatory sc10-1 animals lack activity of both ac and sc, and are devoid of sequences might be of recent origin. bristles (Fig. 1B) (2, 25). In males of the genotype In (1)ac3 sc10-1/Y; HS-Gal4ϾUAS-sc/ϾUAS-ase, bristles were rescued in achaete-scute ͉ extramacrochaetae ͉ hairy ͉ redundancy ͉ stripe many flies after heat shock. In the absence of heat shock, no rescue was observed. he arrangement of large bristles (macrochaetes) on the Eleven macrochaetes are present at stereotypical positions on Tmesonotum of Drosophila is a well-canalized phenotype. The the wild-type hemi-notum (Fig. 1A). Their positions on rescued formation of bristle precursors is preceded by expression of flies were assessed by using the underlying muscles and sutures proneural genes of the achaete-scute (ac-sc) family that encode as reference points. Remarkably, all rescued macrochaetes (n ϭ related basic helix–loop–helix (bHLH) transcriptional regula- 158) were found exclusively at wild-type locations (Fig. 1 D–G). tors that confer neural potential to cells (1–3). The ac-sc genes No ectopic bristles at abnormal locations were observed. An are activated in small proneural clusters of cells at the sites of average of 6.58 (n ϭ 24) macrochaetes was rescued. Each of the formation of the precursors (4, 5). This precise spatial regulation 11 wild-type bristles was recovered, but the frequency of rescue relies on the modular cis-regulatory promoter of ac-sc (6). Each varied significantly (Fig. 1C). bristle organ is constructed from a single , the sensory organ precursor (SOP) that is singled out from a proneural cluster by Uniform Scute and Asense Expression Gives Rise to Macrochaetes at Notch-mediated lateral inhibition (7, 8). Precursors arise at repro- Ectopic Locations in ac-sc; emchsrMutant Flies. Bristle rescue was ducible positions from each proneural cluster (4, 5), and selection assessed in In (1)ac3 sc10-1 flies that were, in addition, mutant for of specific SOP cells may underlie the stereotypical nature of the the hypomorphic, viable alleles emcpel, h1, and sr1 (In (1)ac3 final pattern. Uniform expression of sc or asense (ase), achieved sc10-1/Y; HS-Gal4ϾUAS-sc/ϾUAS-ase; emcpel h1 sr1). In the ab- with a heterologous promoter in the absence of endogenous ac-sc sence of heat shock, no rescue was observed. After heat shock, expression, can rescue bristles that are positioned at wild-type in those flies that displayed rescue, many more macrochaetes locations (9–11). This implies the existence of a patterning mech- were present than in the previous experiment (Fig. 2A). The anism that acts downstream of ac and sc . average number was 12.33 (n ϭ 24). Fig. 2A shows the locations The genes extramacrochaetae (emc), hairy (h), and stripe (sr) of 296 rescued bristles. Some were located at wild-type positions. encode three factors able to antagonize the function of ac-sc. emc encodes a HLH protein devoid of a basic domain, that sequesters Ac-Sc preventing them from binding DNA This paper results from the Arthur M. Sackler Colloquium of the National Academy of (12–15). It has been shown to prevent development of additional Sciences, ‘‘ Networks in Animal Development and Evolution,’’ held February 15–16, 2008, at the Arnold and Mabel Beckman Center of the National Academies of Sciences and bristles at ectopic locations and to play a part in the positioning Engineering in Irvine, CA. The complete program and audio files of most presentations are of the SOP cell (16–19). h encodes a bHLH protein that acts as available on the NAS web site at: http://www.nasonline.org/SACKLER_Gene_Networks. a transcriptional repressor of ac (20, 21). stripe encodes an early Author contributions: K.U. and P.S. designed research; K.U., C.G., and J.-M.G. performed growth response (egr)-like that is required research; K.U. and P.S. analyzed data; and P.S. wrote the paper. for the development of tendon cells (22) and is able to prevent The authors declare no conflict of interest. selection of SOPs from proneural clusters (23). Here, we provide This article is a PNAS Direct Submission. evidence that the spatially restricted distributions of Emc, H, and 1Present address: 1-2-7, Higashimonzen, Kawasaki-ku, Kawasaki-shi, Kanagawa-ken, Ja- Sr contribute to the pattern by preventing bristle formation pan, 210-0812. outside proneural clusters and by influencing the selection of 2Present address: Department of Zoology and Animal Biology, Science III, University of specific precursor cells from proneural clusters. In animals Geneva, 30 Quai Ernest Ansermet, 1211 Gene`ve 4, Switzerland. devoid of endogenous ac-sc activity, uniform expression of sc 3To whom correspondence should be addressed. E-mail: [email protected]. and ase does not rescue bristles at wild-type locations when the This article contains supporting information online at www.pnas.org/cgi/content/full/ levels of activity of emc, h, and sr are reduced. The fact that 0804282105/DCSupplemental. bristles are rescued at correct positions with the use of a © 2008 by The National Academy of Sciences of the USA

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Fig. 2. Rescue of bristles by ubiquitous scute and asense expression in animals mutant for achaete-scute, extramacrochaetae, hairy, and stripe.(A) Schema of the mesonotum showing positions at which bristles were found in flies of the genotype In (1)ac3 sc10-1/Y; HS-Gal4ϾUASsc/UAS-ase; emcpel h1 sr1 (red crosses). A total of 296 bristles were plotted. (B–D) nota of In (1)ac3 sc10-1/Y; HS-Gal4ϾUASsc/UAS-ase; emcpel h1 sr1 flies illustrating the bristle phenotypes. Black arrows indicate ectopic bristles. White arrows in B point to Fig. 1. Rescue of bristles by ubiquitous scute and asense expression in the displacement of the dorsocentral bristles that do not align on the left and animals devoid of endogenous achaete-scute expression. (A) Notum of a right heminota. wild-type fly showing the positions of the 11 macrochaetes on the mesono- tum. (B) Notum of a fly of the genotype In (1)ac3 sc10-1/Y devoid of bristles. (C) Schema of the mesonotum showing positions at which bristles were rescued was ubiquitous, but there were 2 strong bands of expression, one 3 10-1 Ͼ in flies of the genotype In (1)ac sc /Y; HS-Gal4 UASsc/UAS-ase (red crosses). on the anterior notum and one immediately posterior to the A total of 158 bristles were plotted by using the muscle attachment sites (green) as landmarks. Numbers indicate the percentage of rescue for each areas where the dorsocentral and postalar bristles develop (Fig. bristle. (D–G) nota of In (1)ac3 sc10-1/Y; HS-Gal4ϾUASsc/UAS-ase flies illustrat- 3A). The single expression domain of h was situated mainly over ing bristle rescue. aDC, anterior dorsocentral; pDC, posterior dorsocentral; the position of the presumptive scutal-scutellar suture and aSC, anterior scutellar; pSC posterior scutellar; pPA, posterior postalar; aPA, overlapped the posterior domain of high emc expression (Fig. 3 anterior postalar; pSA, posterior supraalar; aSA, anterior supraalar; pNP, B and C). sr was expressed in several broad, longitudinal domains posterior notopleural; aNP, anterior notopleural; PS, presutural. Hu, humeral in the anterior medial part of the notum and overlapped the bristles of the pronotum, not part of the mesonotum. anterior domain of emc expression (Fig. 3 D and E). Double staining with neulacZ showed that the SOPs arose at positions where there was little or no expression of these genes (Fig. 3 E Others were found close to, but slightly displaced from, their and F). Expression of emc, sr, and h is independent of ac-sc (4, normal positions (Fig. 2B). In addition, extra macrochaetes were 16, 18; K.U., unpublished observations). observed at ectopic locations mostly located close to the posi- To investigate possible redundancy between emc, h, and sr,we tions of extant bristles (Fig. 2 C and D). For example, 4 heminota examined the bristle phenotype of single and double mutants as had 3 instead of 2 dorsocentral bristles. The 3 bristles were well as the triple mutant. We used the hypomorphic alleles aligned and sometimes equally spaced such that none of them emcpel, h1, and sr1. Over half of the h1 mutant flies displayed an occupied the exact position of either of the 2 wild-type ones additional anterior scutellar bristle (Fig. 4E). Furthermore, the (arrows in Fig. 2D). In 8 flies where 2 dorsocentral bristles are dorsocentral, scutellar, and postalar bristles were slightly mis- present on each heminotum, the 4 bristles were correctly aligned placed (data not shown). There were few ectopic bristles in sr1 in only one of them. In the other cases, the 2 anterior (or the 2 mutant flies (Fig. 4F). The dorsocentral and supraalar bristles posterior) bristles were not aligned, such that one was shifted were, however, sometimes slightly misplaced (data not shown). either anteriorly or posteriorly (arrows in Fig. 2B). This is a consistent feature of clones mutant for a null allele [Fig. 2A in (23)]. Double mutant h1 sr1 flies had slightly more bristles A Prepattern of Synergistically Acting Antagonists. The expression than each of the single mutants (Fig. 4G). There was little patterns of sr and emc in the wing/thoracic disk have been overlap of expression of h and sr. Flies mutant for emcpel described previously (12, 16, 18, 26). We compare the domains displayed additional macrochaetes mainly in the anterior notum of expression of emc and h with sr, and show that they are [Fig. 4A, supporting information (SI) Fig. S1]. The bristles expressed in distinct but overlapping domains. Expression of emc preferentially clustered at sites devoid of sr and h expression. We

Usui et al. PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 ͉ 20113 Downloaded by guest on September 27, 2021 cis-regulatory elements. For example, the 2 dorsocentral bristles AB arise from a cluster driven by the dorsocentral enhancer (DCE) C (27). Many of the ectopic bristles seen in emc mutants arise at positions normally devoid of proneural clusters, where Ac and Sc accumulate ectopically. For instance, flies mutant for emcpel display additional, anteriorly located dorsocentral bristles result- ing from an expanded domain of ac-sc expression (4, 16, 18). This expanded domain could result from a change in the activity of the DCE such that it now drives ac-sc expression in a larger area. Alternatively, it could be because of ectopic expression of ac-sc D E that does not result from the activation of this regulatory element. We found that the activity of the DCE, visualized with the lacZ reporter gene, was unchanged in emcpel, h1,orsr1 mutants or in the double and triple mutants (Fig. 5 A–F). Double labeling with a marker for bristle precursors showed that ectopic bristles in emcpel arise from cells clearly situated outside the cluster of cells expressing lacZ (Fig. 5 G and H). Thus, the expanded domain of ac-sc expression in the dorsocentral region of the mutants arises independently of the activity of the DCE. Discussion Patterning of Macrochaetes Downstream of Proneural Gene Expres- F G sion. In wild-type flies, ac-sc expression is restricted to the proneural clusters where cis-regulatory sequences enhance tran- scription. Outside of these domains basal expression is insuffi- cient to allow autoregulation (18, 28) and accumulation of Ac-Sc. Hairy and Emc both help prevent expression outside proneural clusters by affecting basal transcription (Hairy) and autoregu- lation (Emc) (18–21); sr may also contribute. Bristle patterning is then a matter of correctly positioning the bristle precursors from within the proneural clusters. In the rescue experiment, the endogenous ac and sc genes were nonfunctional, and so their cis-regulatory sequences cannot contribute to bristle patterning. Proneural protein was Fig. 3. Expression patterns of stripe, hairy, and extramacrochaetae in the presumptive mesonotum of the wing disk. (A and B) The expression patterns expressed ubiquitously from a heterologous promoter, and the of emc (green) and h (red) are visualized by ␤ galactosidase staining of emc, pattern results from both the prevention of bristle formation lacZ, and an antibody against Hairy. (C) The merge of A and B shows an overlap at locations outside the normal positions of the proneural of expression of emc and h in a band across the posterior notum. (D) Triple clusters and the selection of precursors at the correct sites from staining showing the expression of emc, h, and sr. Expression of sr (blue) is the regions where ac-sc are usually expressed (see also ref. 9). visualized from Gal4-srϾUAS-GFP, that of emc (green) from ␣ galactosidase It is likely that h,* sr, and emc are all contributing to both of staining of emc, lacZ, and that of h (red) from ␣-Hairy. Expression of emc and these processes in the rescue experiment. First, they were sr overlaps in the anterior notum. (E) Triple staining showing expression of sr expressed at appropriate sites to antagonize Sc/Ase activity at (green), h (red), and A101, lacZ (blue), a marker of the sensory organ precur- locations outside those of the proneural clusters. The domains sors. The precursors arise at positions where the levels of Sr and H are low. (F) Schematic drawing of the expression domains of sr (blue), emc (green), and h of sr expression correspond to sites where tendons develop and (red) as well as the positions where precursors of the bristles arise (brown flight muscles attach; the absence of macrochaetes here is a circles). White circles represent other sensilla. This diagram indicates regions conserved, ancestral feature of the Diptera (23). Expression of of high levels of ; emc is ubiquitously expressed, and h is h was greatest over the position of the presumptive scutal- expressed in the form of a gradient, as indicated by the paler colors. Anterior scutellar suture, a region also devoid of bristles throughout the is up, and the dorsal midline is to the left in all images. Diptera. High levels of emc transcripts overlapped with those of sr and h but also covered additional sites. Second, ectopic noted a synergy between emc and both h and sr: The double bristles in the double and triple mutants are often located outside mutants displayed significantly more bristles than the sum of the the sites of the proneural clusters. We show that this phenotype is single mutants. emcpel sr1 flies had many more bristles in the independent of the activity of at least one of the cis-regulatory anterior notum where both emc and sr were highly expressed elements of ac-sc, indicating that ectopic bristles can result from an (Fig. 4C). emcpel h1 flies had ectopic bristles at many locations increase in basal ac-sc levels mediated in trans. including the dorsocentral, sutural, and scutellar regions where Within a given proneural cluster, macrochaete precursors few ectopics were found in the single mutants and where both arise at reproducible positions in wild-type flies (4, 5). Selec- genes were expressed at high levels (Fig. 4B). This suggests that

the activity of emc and sr, and of emc and h, act redundantly in *Alone, of the three genes, h encodes a transcriptional repressor of ac (19, 20), but we the anterior and posterior notum, respectively. Finally, triple nevertheless think h is contributing to the positioning of bristles in the rescue experiment emcpel h1 sr1 mutant flies displayed large numbers of ectopic because of the pattern of rescue of the small bristles (microchaetes). Microchaetes cover bristles over many parts of the notum (Fig. 4 D and H). the scutum of wild-type animals, but are absent from the scutellum and midline. Signif- icant numbers of microchaetes were rescued, but, in contrast to emcϩ hϩ srϩ flies, rescue in the emchsrtriple mutant background allowed the formation of microchaetes on the Ectopic Dorsocentral Bristles in the Mutants Do Not Arise as a Result scutellum and dorsal midline, a phenotype that is exclusive to h mutants. Rescue of of Expansion of the Proneural Cluster Driven by the Dorsocentral microchaetes, which arise after pupariation, might be because of activation of the endogenous ase gene [as well as senseless (sens)] by the Sc and Ase provided by the Enhancer. In the wild type, bristle precursors arise from the transgene (23, 29). One possibility, therefore, is that Hairy can act through conserved expression of ac-sc in proneural clusters mediated by discrete binding sequences in the promoter of ase and/or sens.

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emc[pel] 13.0 emc[pel] h[1] 23.4 emc[pel] sr[1] 17.9

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Fig. 4. The bristle phenotype of single, double, and triple mutants of extramacrochaetae, hairy, and stripe.(A–G) Schematic drawings showing the positions of macrochaetes on the notum of flies mutant for the genotypes indicated. Red crosses mark the positions of bristles, which were plotted onto a standard diagram. Each diagram shows bristles from 50 thoraces. Numbers indicate the average number of bristles per heminotum (n ϭ 100). The positions of wild-type bristles are in pale blue. Pale green areas denote the domains of expression of sr. The medial domain of expression is lacking in sr1 mutants (shown in white in C, D, F, and G). (H) Photograph of a triple mutant.

tion of precursors requires lateral inhibition mediated by the proneural clusters. The dorsocentral and supraalar bristles arise on Delta (Dl)/Notch (N) signaling pathway (8, 30). Activation of the borders of sr expression domains. These bristles were slightly N leads to the repression of ac-sc. Cell-fate choice is reinforced displaced from their normal locations in sr mutants. Similarly, the by means of a transcriptional feedback loop within each cell postalar and anterior scutellar bristles arise just where the levels of linking high levels of activation of N with reduced production Hairy decreased markedly; more than half of h1 mutant flies of Dl (8, 31). Expression of Dl depends on Ac-Sc (32). Any displayed an additional anterior scutellar bristle, whereas in others, asymmetrically distributed factor that antagonizes Ac-Sc the anterior scutellar and postalar bristles were slightly displaced. might bias the choice of fate by reducing the levels of ac-sc Although emc plays the major role in bristle patterning, it is likely activity within some cells of a proneural cluster, both decreas- to act synergistically with sr and h in parts of the anterior and ing autoregulation and rendering the cells less efficient at posterior notum, respectively, where their expression domains signaling (Fig. 6). Such an effect has been demonstrated for overlap. As ac-sc are subject to autoregulation in the wild type, emc. The spatial distributions of transcripts of emc and of ac-sc factors that antagonize Ac-Sc, such as Emc, have an indirect effect are complementary over much of the notum (16, 18). Fur- on transcription that would compound the effects of transcriptional thermore, many SOPs arise at positions where the levels of emc regulators such as Hairy and vice versa (15, 18). Although auto- transcripts decrease sharply, and SOPs displaced from wild- regulation of ac-sc cannot play a role in the rescue experiment, type positions appear in emc mutants with decreased expres- autoregulation of the endogenous ase gene is likely. Because sion (16–18). A role for emc in the precise positioning of expression of both h and sr is spatially regulated, their synergistic bristles in the rescue experiment (and the wild type) is, thus, effects with emc would be local. Synergistic activity would explain likely. Several observations suggest that h and sr also contribute to the increase in ectopic bristles seen in the double and triple mutants. the positioning of some SOPs. Ectopic Sr in proneural clusters does not prevent expression of ac-sc, but their expression fails to refine Is Patterning by Antagonism Ancestral? By antagonizing ac-sc to single cells and no SOPs develop (23). This suggests that Sr activity, Emc, Hairy, Sr, and probably other yet undescribed interferes with autoregulation and/or inhibition. Thus it could factors are able to pattern the macrochaetes on the Drosophila influence SOP selection in those areas where its expression overlaps notum from uniform ac-sc expression. Bristles are rescued at

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D E F Dl N Fig. 6. Model for the function of extramacrochaetae, hairy, and stripe in biasing the choice of bristle precursor cell. The singling out of one bristle precursor from a cluster of cells expressing achaete-scute depends on Notch- mediated lateral signaling. Activation of Notch (N) leads to the repression of ac-sc. Cells in a proneural cluster are initially equivalent. Each cell expresses emc[pel] h[1] h[1] sr[1] emc[pel] h[1] sr[1] both the ligand, Delta (Dl), and the receptor, N, and can both send and receive the inhibitory signal. With time, only a single cell strongly expresses the ligand, G H and its neighbors have their receptors activated. This relies on a feedback loop within each cell linking high levels of activation of the receptor with reduced production of the ligand. A small difference between cells, in the level of any component of the loop, can be amplified via the loop itself, such that even- tually only a single cell will become a signaling cell. By lowering the levels/ activity of Ac-Sc in some cells of a cluster, the products of emc, h, and sr would pDC pDC reduce their capacity to signal and, thus, cause them to take up the epidermal fate.

WT emc[pel] notum appears to have increased in spatial complexity in this lineage, and the number of genes at the ac-sc complex has increased by duplication (37). It is conceivable that this may Fig. 5. Activity of the dorsocentral enhancer of achaete-scute in single, have provided material for the evolution of numerous cis- double, and triple mutants of extramacrochaetae, hairy, and stripe.(A–F) regulatory modules (38). If the modular promoter of the ac-sc genes Presumptive notum of wing/thoracic discs of the genotypes indicated. The of Drosophila is of recent origin and was superimposed onto an flies carry a transgene with the sequences of the dorsocentral enhancer ancestral patterning mechanism mediated by antagonism, the en- driving expression of lacZ in cells of the dorsocentral proneural cluster. An ␣-galactosidase antibody was used to visualize lacZ expression. The size and hancers could have evolved sequentially one-by-one. position of the cluster are unchanged in all genotypes. (G and H) Double Redundancy of developmental mechanisms has been widely staining for the dorsocentral enhancer, lacZ (␣-galactosidase, red), and the documented and contributes to the plasticity of development bristle precursors labeled with Gal4-neurϾUAS-GFP (green) in wild-type (G) and to the robustness of phenotype (39, 40). Patterning of the and emcpel mutant (H) discs. In the wild type, two DC bristles arise from the DC vulva in nematodes is a good example of this: Significant cluster. The posterior dorsocentral (pDC) is formed first and can be seen in G. redundancy has been documented despite conservation of pel In the emc mutant, the pDC is also visible inside the dorsocentral cluster, but morphology (41). It is not known how redundancy is built up in addition, two other bristle precursors, anterior to and aligned with the pDC, are seen (white arrow) that are clearly outside the dorsocentral cluster. These during evolution, nor whether it can be a source of evolution- ectopic precursors have formed even before the anterior dorsocentral bristle ary novelty. If the spatially regulated expression of ac-sc has appeared. Forty discs were examined, and precursors were consistently evolved later than patterning downstream of uniform expres- observed outside the dorsocentral proneural cluster in the mutant. sion, it might be possible to dissect this evolutionary process through identification of extant species in which patterning is exclusively dependent on the latter. correct locations independently of the activity of the cis- regulatory elements that normally regulate spatial expression Materials and Methods of ac-sc in proneural clusters. Restricting expression of ac-sc Fly Strains. The In (1)ac3 sc10-1 is associated with a loss of ac and to proneural clusters in the wild type, therefore, provides a sc activity: No detectable ac protein accumulation occurs (4) because of the In redundant input. We suggest that patterning by antagonism (1)ac3 breakpoint. sc10-1 induces a truncated sc protein because of an amber downstream of uniform ac-sc transcription might be ancestral. mutation in the coding sequence (2). Strains used for bristle counts were w; sr1 ϩ ϩ Uniform expression of ac-sc coupled with Notch-mediated kar ry, h1 kar ry, emcpel neuA101[lac ry ] kar ry, emcpel sr1 eca, w; emcpel h1, pel 1 1 1 1 lateral inhibition would be sufficient to generate the random w; emc h sr , h sr . The DC1.4 enhancer-lacZ reporter gene was used (27), and anti-galactosidase staining for its activity was carried out in the following pattern of spaced sensory organs seen in many hemimetabo- stocks: emcpel DC 1.4 lacZ/emcpel, h1 DC 1.4 lacZ/h1, sr1 DC lacZ/sr1, emcpel h1 DC lous insects. Indeed ubiquitous expression of sc precedes 1.4 lacZ/emcpel h1, h1 sr1 DC 1.4 lacZ/h1 sr1, emcpel h1 sr1 DC 1.4 lacZ/emcpel h1 development of the randomly positioned microchaetes (small sr1. Other strains for anti-galactosidase and antibody staining were: emcP5c bristles) on the notum of several species of Diptera (33, 34). lacZ Gal4-sr UAS-GFP/TM6b, Gal4-sr UAS-GFP/neuA101[lacϩ ryϩ]. DC 1.4 lacZ/ Patterning of the small bristles on the legs of Drosophila has Gal4-neur UAS-GFP, emcpel DC 1.4 lacZ/Gal4-neur UAS-GFP emcpel. For other also been shown to result from broad activation of ac coupled mutant strains, see FlyBase. to repression by h and Dl (35). The absence of macrochaetes at the sutures and sites of muscle attachment is an ancestral Bristle Rescue Experiment. Rescue of bristles in an acϪ scϪ background was feature correlated with conservation of the pattern of these performed by using In (1)ac3 sc10-1; HSGal4 UAS-Sc/UAS-AseHA and In (1)ac3 sc10-1; pel 1 1 characters throughout the Diptera (36), suggesting ancient HSGal4 UAS-Sc/UAS-AseHA; emc h sr stocks. Eggs were collected over a period of 3 days at 25 °C, then subjected to heat shock at 37 °C for 30 min, three times at regulation by sr and h (23). Finally, we have postulated that the 2-h intervals, and then left at 18 °C until just before eclosion. position-specific regulatory enhancers driving ac-sc in proneu- ral clusters may have originated in the lineage leading to Antibody Staining.Third instar larval wing disks were processed according to cyclorraphous Diptera. Expression of ac-sc homologues on the classical protocols. For ␤-Gal staining, we used a 1:200 dilution of the 40-1a

20116 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0804282105 Usui et al. Downloaded by guest on September 27, 2021 mouse primary antibody (Developmental Studies Hybridoma Bank) and a ACKNOWLEDGMENTS. We thank Manuel Calleja, Gine`s Morata, and Juan FEATURE ␣ Modolell for fly stocks; Emma Hatton-Ellis for help with photography; Emma

1:500 dilution of secondary antibody conjugated to Alexa-647 (goat -mouse SACKLER SPECIAL antibody, Molecular Probes, Invitrogen). For GFP staining, we used a 1:500 Hatton-Ellis and Barbara Negre for help with a genotype; Savita Ayyar for dilution of a rabbit ␣-GFP antibody conjugated to Alexa-488 (Molecular UAS-Ase; the members of our group for lively discussion and comments on the Probes). For Hairy, we used a 1:20 dilution (monoclonal from Insight Biotech- manuscript; and Rosalba Melchoirre and Ben Taylor for technical assistance. nology) and a 1:500 dilution of secondary antibody conjugated to Alexa-546 J.-M.G. was the recipient of a Marie Curie postdoctoral fellowship. This work (goat anti-mouse antibody). was supported by the Wellcome Trust [29156].

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