Redundant mechanisms mediate bristle patterning onthe Drosophila thoraxKazuya 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 spatiallyrestricted expression of the achaete-scute (ac-sc) genes in clustersof cells, mediated by the activity of many discrete cis-regulatorysequences. However, ubiquitous expression of sc or asense (ase)achieved with a heterologous promoter, in the absence of endog-enous ac-sc expression, and the activity of the cis-regulatoryelements, allows the development of bristles positioned at wild-type locations. We demonstrate that the products of the genesstripe, hairy, and extramacrochaetae contribute to rescue by an-tagonizing the activity of Sc and Ase. The three genes are ex-pressed in specific but overlapping spatial domains of expressionthat form a prepattern that allows precise positioning of bristles.The redundant mechanisms might contribute to the robustness ofthe pattern. We discuss the possibility that patterning in trans byantagonism is ancestral and that the positional cis-regulatorysequences might be of recent origin.

achaete-scute � extramacrochaetae � hairy � redundancy � stripe

The arrangement of large bristles (macrochaetes) on themesonotum of Drosophila is a well-canalized phenotype. The

formation of bristle precursors is preceded by expression ofproneural genes of the achaete-scute (ac-sc) family that encoderelated basic helix–loop–helix (bHLH) transcriptional regula-tors that confer neural potential to cells (1–3). The ac-sc genesare activated in small proneural clusters of cells at the sites offormation of the precursors (4, 5). This precise spatial regulationrelies on the modular cis-regulatory promoter of ac-sc (6). Eachbristle organ is constructed from a single cell, the sensory organprecursor (SOP) that is singled out from a proneural cluster byNotch-mediated lateral inhibition (7, 8). Precursors arise at repro-ducible positions from each proneural cluster (4, 5), and selectionof specific SOP cells may underlie the stereotypical nature of thefinal pattern. Uniform expression of sc or asense (ase), achievedwith a heterologous promoter in the absence of endogenous ac-scexpression, can rescue bristles that are positioned at wild-typelocations (9–11). This implies the existence of a patterning mech-anism that acts downstream of ac and sc transcription.

The genes extramacrochaetae (emc), hairy (h), and stripe (sr)encode three factors able to antagonize the function of ac-sc.emc encodes a HLH protein devoid of a basic domain, thatsequesters Ac-Sc proteins preventing them from binding DNA(12–15). It has been shown to prevent development of additionalbristles at ectopic locations and to play a part in the positioningof the SOP cell (16–19). h encodes a bHLH protein that acts asa transcriptional repressor of ac (20, 21). stripe encodes an earlygrowth response (egr)-like transcription factor that is requiredfor the development of tendon cells (22) and is able to preventselection of SOPs from proneural clusters (23). Here, we provideevidence that the spatially restricted distributions of Emc, H, andSr contribute to the pattern by preventing bristle formationoutside proneural clusters and by influencing the selection ofspecific precursor cells from proneural clusters. In animalsdevoid of endogenous ac-sc activity, uniform expression of scand ase does not rescue bristles at wild-type locations when thelevels of activity of emc, h, and sr are reduced. The fact thatbristles are rescued at correct positions with the use of a

heterologous promoter and in the absence of normal cis-regulation, indicates that factors acting downstream of Sc aresufficient to position the bristles. We discuss the possibility thatpatterning by sr, emc, and h is ancestral, and that the positionalcis-regulatory sequences might be of recent origin.

ResultsUniform Scute and Asense Expression Rescues Macrochaetes at Wild-type Locations in achaete-scute Mutant Flies. ase is a target of Ac-Scand encodes a protein closely related to Ac and Sc that displaysstronger proneural properties than either Ac or Sc when mis-expressed in the epithelium (10, 11, 24). We used the Gal4-UASmethod to drive simultaneous ubiquitous expression of sc andase. This combination allows significant bristle rescue. In (1)ac3

sc10-1 animals lack activity of both ac and sc, and are devoid ofbristles (Fig. 1B) (2, 25). In males of the genotype In (1)ac3

sc10-1/Y; HS-Gal4�UAS-sc/�UAS-ase, bristles were rescued inmany flies after heat shock. In the absence of heat shock, norescue was observed.

Eleven macrochaetes are present at stereotypical positions onthe wild-type hemi-notum (Fig. 1 A). Their positions on rescuedflies were assessed by using the underlying muscles and suturesas reference points. Remarkably, all rescued macrochaetes (n �158) were found exclusively at wild-type locations (Fig. 1 D–G).No ectopic bristles at abnormal locations were observed. Anaverage of 6.58 (n � 24) macrochaetes was rescued. Each of the11 wild-type bristles was recovered, but the frequency of rescuevaried significantly (Fig. 1C).

Uniform Scute and Asense Expression Gives Rise to Macrochaetes atEctopic Locations in ac-sc; emc h sr Mutant Flies. Bristle rescue wasassessed in In (1)ac3 sc10-1 f lies that were, in addition, mutant forthe hypomorphic, viable alleles emcpel, h1, and sr1 (In (1)ac3

sc10-1/Y; HS-Gal4�UAS-sc/�UAS-ase; emcpel h1 sr1). In the ab-sence of heat shock, no rescue was observed. After heat shock,in those flies that displayed rescue, many more macrochaeteswere present than in the previous experiment (Fig. 2A). Theaverage number was 12.33 (n � 24). Fig. 2 A shows the locationsof 296 rescued bristles. Some were located at wild-type positions.

This paper results from the Arthur M. Sackler Colloquium of the National Academy ofSciences, ‘‘Gene Networks in Animal Development and Evolution,’’ held February 15–16,2008, at the Arnold and Mabel Beckman Center of the National Academies of Sciences andEngineering in Irvine, CA. The complete program and audio files of most presentations areavailable on the NAS web site at: http://www.nasonline.org/SACKLER_Gene_Networks.

Author contributions: K.U. and P.S. designed research; K.U., C.G., and J.-M.G. performedresearch; K.U. and P.S. analyzed data; and P.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1Present address: 1-2-7, Higashimonzen, Kawasaki-ku, Kawasaki-shi, Kanagawa-ken, Ja-pan, 210-0812.

2Present address: Department of Zoology and Animal Biology, Science III, University ofGeneva, 30 Quai Ernest Ansermet, 1211 Geneve 4, Switzerland.

3To whom correspondence should be addressed. E-mail: pas49@cam.ac.uk.

This article contains supporting information online at www.pnas.org/cgi/content/full/0804282105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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Others were found close to, but slightly displaced from, theirnormal positions (Fig. 2B). In addition, extra macrochaetes wereobserved at ectopic locations mostly located close to the posi-tions of extant bristles (Fig. 2 C and D). For example, 4 heminotahad 3 instead of 2 dorsocentral bristles. The 3 bristles werealigned and sometimes equally spaced such that none of themoccupied the exact position of either of the 2 wild-type ones(arrows in Fig. 2D). In 8 flies where 2 dorsocentral bristles arepresent on each heminotum, the 4 bristles were correctly alignedin only one of them. In the other cases, the 2 anterior (or the 2posterior) bristles were not aligned, such that one was shiftedeither anteriorly or posteriorly (arrows in Fig. 2B).

A Prepattern of Synergistically Acting Antagonists. The expressionpatterns of sr and emc in the wing/thoracic disk have beendescribed previously (12, 16, 18, 26). We compare the domainsof expression of emc and h with sr, and show that they areexpressed in distinct but overlapping domains. Expression of emc

was ubiquitous, but there were 2 strong bands of expression, oneon the anterior notum and one immediately posterior to theareas where the dorsocentral and postalar bristles develop (Fig.3A). The single expression domain of h was situated mainly overthe position of the presumptive scutal-scutellar suture andoverlapped the posterior domain of high emc expression (Fig. 3B and C). sr was expressed in several broad, longitudinal domainsin the anterior medial part of the notum and overlapped theanterior domain of emc expression (Fig. 3 D and E). Doublestaining with neulacZ showed that the SOPs arose at positionswhere there was little or no expression of these genes (Fig. 3 Eand F). Expression of emc, sr, and h is independent of ac-sc (4,16, 18; K.U., unpublished observations).

To investigate possible redundancy between emc, h, and sr, weexamined the bristle phenotype of single and double mutants aswell as the triple mutant. We used the hypomorphic allelesemcpel, h1, and sr1. Over half of the h1 mutant flies displayed anadditional anterior scutellar bristle (Fig. 4E). Furthermore, thedorsocentral, scutellar, and postalar bristles were slightly mis-placed (data not shown). There were few ectopic bristles in sr1

mutant flies (Fig. 4F). The dorsocentral and supraalar bristleswere, however, sometimes slightly misplaced (data not shown).This is a consistent feature of clones mutant for a null allele [Fig.2A in (23)]. Double mutant h1 sr1 f lies had slightly more bristlesthan each of the single mutants (Fig. 4G). There was littleoverlap of expression of h and sr. Flies mutant for emcpel

displayed additional macrochaetes mainly in the anterior notum[Fig. 4A, supporting information (SI) Fig. S1]. The bristlespreferentially clustered at sites devoid of sr and h expression. We

A

B

E F G

D

C

Fig. 1. Rescue of bristles by ubiquitous scute and asense expression inanimals devoid of endogenous achaete-scute expression. (A) Notum of awild-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 rescuedin flies of the genotype In (1)ac3 sc10-1/Y; HS-Gal4�UASsc/UAS-ase (red crosses).A total of 158 bristles were plotted by using the muscle attachment sites(green) as landmarks. Numbers indicate the percentage of rescue for eachbristle. (D–G) nota of In (1)ac3 sc10-1/Y; HS-Gal4�UASsc/UAS-ase flies illustrat-ing bristle rescue. aDC, anterior dorsocentral; pDC, posterior dorsocentral;aSC, anterior scutellar; pSC posterior scutellar; pPA, posterior postalar; aPA,anterior postalar; pSA, posterior supraalar; aSA, anterior supraalar; pNP,posterior notopleural; aNP, anterior notopleural; PS, presutural. Hu, humeralbristles of the pronotum, not part of the mesonotum.

A B

C D

Fig. 2. Rescue of bristles by ubiquitous scute and asense expression inanimals mutant for achaete-scute, extramacrochaetae, hairy, and stripe. (A)Schema of the mesonotum showing positions at which bristles were found inflies of the genotype In (1)ac3 sc10-1/Y; HS-Gal4�UASsc/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-Gal4�UASsc/UAS-ase; emcpel h1 sr1 flies illustrating the bristlephenotypes. Black arrows indicate ectopic bristles. White arrows in B point tothe displacement of the dorsocentral bristles that do not align on the left andright heminota.

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noted a synergy between emc and both h and sr: The doublemutants displayed significantly more bristles than the sum of thesingle mutants. emcpel sr1 f lies had many more bristles in theanterior notum where both emc and sr were highly expressed(Fig. 4C). emcpel h1 f lies had ectopic bristles at many locationsincluding the dorsocentral, sutural, and scutellar regions wherefew ectopics were found in the single mutants and where bothgenes were expressed at high levels (Fig. 4B). This suggests thatthe activity of emc and sr, and of emc and h, act redundantly inthe anterior and posterior notum, respectively. Finally, tripleemcpel h1 sr1 mutant flies displayed large numbers of ectopicbristles over many parts of the notum (Fig. 4 D and H).

Ectopic Dorsocentral Bristles in the Mutants Do Not Arise as a Resultof Expansion of the Proneural Cluster Driven by the DorsocentralEnhancer. In the wild type, bristle precursors arise from theexpression of ac-sc in proneural clusters mediated by discrete

cis-regulatory elements. For example, the 2 dorsocentral bristlesarise from a cluster driven by the dorsocentral enhancer (DCE)(27). Many of the ectopic bristles seen in emc mutants arise atpositions normally devoid of proneural clusters, where Ac and Scaccumulate ectopically. For instance, f lies mutant for emcpel

display additional, anteriorly located dorsocentral bristles result-ing from an expanded domain of ac-sc expression (4, 16, 18). Thisexpanded domain could result from a change in the activity ofthe DCE such that it now drives ac-sc expression in a larger area.Alternatively, it could be because of ectopic expression of ac-scthat does not result from the activation of this regulatoryelement. We found that the activity of the DCE, visualized withthe lacZ reporter gene, was unchanged in emcpel, h1, or sr1

mutants or in the double and triple mutants (Fig. 5 A–F). Doublelabeling with a marker for bristle precursors showed that ectopicbristles in emcpel arise from cells clearly situated outside thecluster of cells expressing lacZ (Fig. 5 G and H). Thus, theexpanded domain of ac-sc expression in the dorsocentral regionof the mutants arises independently of the activity of the DCE.

DiscussionPatterning of Macrochaetes Downstream of Proneural Gene Expres-sion. In wild-type flies, ac-sc expression is restricted to theproneural 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 proneuralclusters by affecting basal transcription (Hairy) and autoregu-lation (Emc) (18–21); sr may also contribute. Bristle patterningis then a matter of correctly positioning the bristle precursorsfrom within the proneural clusters.

In the rescue experiment, the endogenous ac and sc geneswere nonfunctional, and so their cis-regulatory sequencescannot contribute to bristle patterning. Proneural protein wasexpressed ubiquitously from a heterologous promoter, and thepattern results from both the prevention of bristle formationat locations outside the normal positions of the proneuralclusters and the selection of precursors at the correct sites fromthe regions where ac-sc are usually expressed (see also ref. 9).It is likely that h,* sr, and emc are all contributing to both ofthese processes in the rescue experiment. First, they wereexpressed at appropriate sites to antagonize Sc/Ase activity atlocations outside those of the proneural clusters. The domainsof sr expression correspond to sites where tendons develop andf light muscles attach; the absence of macrochaetes here is aconserved, ancestral feature of the Diptera (23). Expression ofh was greatest over the position of the presumptive scutal-scutellar suture, a region also devoid of bristles throughout theDiptera. High levels of emc transcripts overlapped with thoseof sr and h but also covered additional sites. Second, ectopicbristles in the double and triple mutants are often located outsidethe sites of the proneural clusters. We show that this phenotype isindependent of the activity of at least one of the cis-regulatoryelements of ac-sc, indicating that ectopic bristles can result from anincrease in basal ac-sc levels mediated in trans.

Within a given proneural cluster, macrochaete precursorsarise at reproducible positions in wild-type f lies (4, 5). Selec-

*Alone, of the three genes, h encodes a transcriptional repressor of ac (19, 20), but wenevertheless think h is contributing to the positioning of bristles in the rescue experimentbecause of the pattern of rescue of the small bristles (microchaetes). Microchaetes coverthe scutum of wild-type animals, but are absent from the scutellum and midline. Signif-icant numbers of microchaetes were rescued, but, in contrast to emc� h� sr� flies, rescuein the emc h sr triple mutant background allowed the formation of microchaetes on thescutellum and dorsal midline, a phenotype that is exclusive to h mutants. Rescue ofmicrochaetes, which arise after pupariation, might be because of activation of theendogenous ase gene [as well as senseless (sens)] by the Sc and Ase provided by thetransgene (23, 29). One possibility, therefore, is that Hairy can act through conservedbinding sequences in the promoter of ase and/or sens.

A BC

D E

F G

Fig. 3. Expression patterns of stripe, hairy, and extramacrochaetae in thepresumptive mesonotum of the wing disk. (A and B) The expression patternsof emc (green) and h (red) are visualized by � galactosidase staining of emc,lacZ, and an antibody against Hairy. (C) The merge of A and B shows an overlapof expression of emc and h in a band across the posterior notum. (D) Triplestaining showing the expression of emc, h, and sr. Expression of sr (blue) isvisualized from Gal4-sr�UAS-GFP, that of emc (green) from � galactosidasestaining of emc, lacZ, and that of h (red) from �-Hairy. Expression of emc andsr overlaps in the anterior notum. (E) Triple staining showing expression of sr(green), h (red), and A101, lacZ (blue), a marker of the sensory organ precur-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(red) as well as the positions where precursors of the bristles arise (browncircles). White circles represent other sensilla. This diagram indicates regionsof high levels of gene expression; emc is ubiquitously expressed, and h isexpressed in the form of a gradient, as indicated by the paler colors. Anterioris up, and the dorsal midline is to the left in all images.

20114 � www.pnas.org�cgi�doi�10.1073�pnas.0804282105 Usui et al.

tion of precursors requires lateral inhibition mediated by theDelta (Dl)/Notch (N) signaling pathway (8, 30). Activation ofN leads to the repression of ac-sc. Cell-fate choice is reinforcedby means of a transcriptional feedback loop within each celllinking high levels of activation of N with reduced productionof Dl (8, 31). Expression of Dl depends on Ac-Sc (32). Anyasymmetrically distributed factor that antagonizes Ac-Scmight bias the choice of fate by reducing the levels of ac-scactivity within some cells of a proneural cluster, both decreas-ing autoregulation and rendering the cells less efficient atsignaling (Fig. 6). Such an effect has been demonstrated foremc. The spatial distributions of transcripts of emc and of ac-scare complementary over much of the notum (16, 18). Fur-thermore, many SOPs arise at positions where the levels of emctranscripts decrease sharply, and SOPs displaced from wild-type positions appear in emc mutants with decreased expres-sion (16–18). A role for emc in the precise positioning ofbristles in the rescue experiment (and the wild type) is, thus,likely. Several observations suggest that h and sr also contribute tothe positioning of some SOPs. Ectopic Sr in proneural clusters doesnot prevent expression of ac-sc, but their expression fails to refineto single cells and no SOPs develop (23). This suggests that Srinterferes with autoregulation and/or inhibition. Thus it couldinfluence SOP selection in those areas where its expression overlaps

proneural clusters. The dorsocentral and supraalar bristles arise onthe borders of sr expression domains. These bristles were slightlydisplaced from their normal locations in sr mutants. Similarly, thepostalar and anterior scutellar bristles arise just where the levels ofHairy decreased markedly; more than half of h1 mutant fliesdisplayed an additional anterior scutellar bristle, whereas in others,the anterior scutellar and postalar bristles were slightly displaced.

Although emc plays the major role in bristle patterning, it is likelyto act synergistically with sr and h in parts of the anterior andposterior notum, respectively, where their expression domainsoverlap. As ac-sc are subject to autoregulation in the wild type,factors that antagonize Ac-Sc, such as Emc, have an indirect effecton transcription that would compound the effects of transcriptionalregulators such as Hairy and vice versa (15, 18). Although auto-regulation of ac-sc cannot play a role in the rescue experiment,autoregulation of the endogenous ase gene is likely. Becauseexpression of both h and sr is spatially regulated, their synergisticeffects with emc would be local. Synergistic activity would explainthe increase in ectopic bristles seen in the double and triple mutants.

Is Patterning by Antagonism Ancestral? By antagonizing ac-scactivity, Emc, Hairy, Sr, and probably other yet undescribedfactors are able to pattern the macrochaetes on the Drosophilanotum from uniform ac-sc expression. Bristles are rescued at

emc[pel]

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emc [pel] h[1] sr[1] 26.2

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sr[1] 11.3 h[1] sr[1] 22.2

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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 positionsof macrochaetes on the notum of flies mutant for the genotypes indicated. Red crosses mark the positions of bristles, which were plotted onto a standarddiagram. Each diagram shows bristles from 50 thoraces. Numbers indicate the average number of bristles per heminotum (n � 100). The positions of wild-typebristles 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 inC, D, F, and G). (H) Photograph of a triple mutant.

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correct locations independently of the activity of the cis-regulatory elements that normally regulate spatial expressionof ac-sc in proneural clusters. Restricting expression of ac-scto proneural clusters in the wild type, therefore, provides aredundant input. We suggest that patterning by antagonismdownstream of uniform ac-sc transcription might be ancestral.Uniform expression of ac-sc coupled with Notch-mediatedlateral inhibition would be sufficient to generate the randompattern of spaced sensory organs seen in many hemimetabo-lous insects. Indeed ubiquitous expression of sc precedesdevelopment of the randomly positioned microchaetes (smallbristles) on the notum of several species of Diptera (33, 34).Patterning of the small bristles on the legs of Drosophila hasalso been shown to result from broad activation of ac coupledto repression by h and Dl (35). The absence of macrochaetesat the sutures and sites of muscle attachment is an ancestralfeature correlated with conservation of the pattern of thesecharacters throughout the Diptera (36), suggesting ancientregulation by sr and h (23). Finally, we have postulated that theposition-specific regulatory enhancers driving ac-sc in proneu-ral clusters may have originated in the lineage leading tocyclorraphous Diptera. Expression of ac-sc homologues on the

notum appears to have increased in spatial complexity in thislineage, and the number of genes at the ac-sc complex hasincreased by duplication (37). It is conceivable that this mayhave provided material for the evolution of numerous cis-regulatory modules (38). If the modular promoter of the ac-sc genesof Drosophila is of recent origin and was superimposed onto anancestral patterning mechanism mediated by antagonism, the en-hancers could have evolved sequentially one-by-one.

Redundancy of developmental mechanisms has been widelydocumented and contributes to the plasticity of developmentand to the robustness of phenotype (39, 40). Patterning of thevulva in nematodes is a good example of this: Significantredundancy has been documented despite conservation ofmorphology (41). It is not known how redundancy is built upduring evolution, nor whether it can be a source of evolution-ary novelty. If the spatially regulated expression of ac-scevolved later than patterning downstream of uniform expres-sion, it might be possible to dissect this evolutionary processthrough identification of extant species in which patterning isexclusively dependent on the latter.

Materials and MethodsFly Strains. The chromosome In (1)ac3 sc10-1 is associated with a loss of ac andsc activity: No detectable ac protein accumulation occurs (4) because of the In(1)ac3 breakpoint. sc10-1 induces a truncated sc protein because of an ambermutation in the coding sequence (2). Strains used for bristle counts were w; sr1

kar ry, h1 kar ry, emcpel neuA101[lac� ry�] kar ry, emcpel sr1 e ca, w; emcpel h1,w; emcpel h1 sr1, h1 sr1. The DC1.4 enhancer-lacZ reporter gene was used (27),and anti-galactosidase staining for its activity was carried out in the followingstocks: emcpel DC 1.4 lacZ/emcpel, h1 DC 1.4 lacZ/h1, sr1 DC lacZ/sr1, emcpel h1 DC1.4 lacZ/emcpel h1, h1 sr1 DC 1.4 lacZ/h1 sr1, emcpel h1 sr1 DC 1.4 lacZ/emcpel h1

sr1. Other strains for anti-galactosidase and antibody staining were: emcP5c

lacZ Gal4-sr UAS-GFP/TM6b, Gal4-sr UAS-GFP/neuA101[lac� ry�]. DC 1.4 lacZ/Gal4-neur UAS-GFP, emcpel DC 1.4 lacZ/Gal4-neur UAS-GFP emcpel. For othermutant strains, see FlyBase.

Bristle Rescue Experiment. Rescue of bristles in an ac� sc� background wasperformed by using In (1)ac3 sc10-1; HSGal4 UAS-Sc/UAS-AseHA and In (1)ac3 sc10-1;HSGal4UAS-Sc/UAS-AseHA;emcpel h1 sr1 stocks.Eggswerecollectedoveraperiodof 3 days at 25 °C, then subjected to heat shock at 37 °C for 30 min, three times at2-h intervals, and then left at 18 °C until just before eclosion.

Antibody Staining.Third instar larval wing disks were processed according toclassical protocols. For �-Gal staining, we used a 1:200 dilution of the 40-1a

emc[pel]

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Fig. 5. Activity of the dorsocentral enhancer of achaete-scute in single,double, and triple mutants of extramacrochaetae, hairy, and stripe. (A–F)Presumptive notum of wing/thoracic discs of the genotypes indicated. Theflies carry a transgene with the sequences of the dorsocentral enhancerdriving expression of lacZ in cells of the dorsocentral proneural cluster. An�-galactosidase antibody was used to visualize lacZ expression. The size andposition of the cluster are unchanged in all genotypes. (G and H) Doublestaining for the dorsocentral enhancer, lacZ (�-galactosidase, red), and thebristle precursors labeled with Gal4-neur�UAS-GFP (green) in wild-type (G)and emcpel mutant (H) discs. In the wild type, two DC bristles arise from the DCcluster. The posterior dorsocentral (pDC) is formed first and can be seen in G.In the emcpel mutant, the pDC is also visible inside the dorsocentral cluster, butin addition, two other bristle precursors, anterior to and aligned with the pDC,are seen (white arrow) that are clearly outside the dorsocentral cluster. Theseectopic precursors have formed even before the anterior dorsocentral bristlehas appeared. Forty discs were examined, and precursors were consistentlyobserved outside the dorsocentral proneural cluster in the mutant.

ac-scac-sc

SOPEPIDERMAL CELL

emc, h, sr

N

NDl

Dl

Fig. 6. Model for the function of extramacrochaetae, hairy, and stripe inbiasing the choice of bristle precursor cell. The singling out of one bristleprecursor from a cluster of cells expressing achaete-scute depends on Notch-mediated lateral signaling. Activation of Notch (N) leads to the repression ofac-sc. Cells in a proneural cluster are initially equivalent. Each cell expressesboth the ligand, Delta (Dl), and the receptor, N, and can both send and receivethe inhibitory signal. With time, only a single cell strongly expresses the ligand,and its neighbors have their receptors activated. This relies on a feedback loopwithin each cell linking high levels of activation of the receptor with reducedproduction of the ligand. A small difference between cells, in the level of anycomponent 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 wouldreduce their capacity to signal and, thus, cause them to take up the epidermalfate.

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mouse primary antibody (Developmental Studies Hybridoma Bank) and a1:500 dilution of secondary antibody conjugated to Alexa-647 (goat �-mouseantibody, Molecular Probes, Invitrogen). For GFP staining, we used a 1:500dilution of a rabbit �-GFP antibody conjugated to Alexa-488 (MolecularProbes). For Hairy, we used a 1:20 dilution (monoclonal from Insight Biotech-nology) and a 1:500 dilution of secondary antibody conjugated to Alexa-546(goat anti-mouse antibody).

ACKNOWLEDGMENTS. We thank Manuel Calleja, Gines Morata, and JuanModolell for fly stocks; Emma Hatton-Ellis for help with photography; EmmaHatton-Ellis and Barbara Negre for help with a genotype; Savita Ayyar forUAS-Ase; the members of our group for lively discussion and comments on themanuscript; and Rosalba Melchoirre and Ben Taylor for technical assistance.J.-M.G. was the recipient of a Marie Curie postdoctoral fellowship. This workwas supported by the Wellcome Trust [29156].

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