Redundant mechanisms mediate bristle patterning on the Droso

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

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

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The thoracic bristle pattern of Drosophila results from the spatially restricted expression of the achaete-sSlicee (ac-sc) genes in clusters of cells, mediated by the activity of many discrete cis-regulatory sequences. However, ubiquitous expression of sc or asense (ase) achieved with a heterologous promoter, in the absence of enExecutegenous ac-sc expression, and the activity of the cis-regulatory elements, allows the development of bristles positioned at wild-type locations. We demonstrate that the products of the genes stripe, hairy, and extramacrochaetae contribute to rescue by antagonizing the activity of Sc and Ase. The three genes are expressed in specific but overlapping spatial Executemains of expression that form a prepattern that allows precise positioning of bristles. The redundant mechanisms might contribute to the robustness of the pattern. We discuss the possibility that patterning in trans by antagonism is ancestral and that the positional cis-regulatory sequences might be of recent origin.

Keywords: achaete-sSliceeextramacrochaetaehairyredundancystripe

The arrangement of large bristles (macrochaetes) on the mesonotum of Drosophila is a well-canalized phenotype. The formation of bristle precursors is pDepartd by expression of proneural genes of the achaete-sSlicee (ac-sc) family that encode related basic helix–loop–helix (bHLH) transcriptional regulators that confer neural potential to cells (1–,3). The ac-sc genes are activated in small proneural clusters of cells at the sites of formation of the precursors (4, ,5). This precise spatial regulation relies on the modular cis-regulatory promoter of ac-sc (6). Each bristle organ is constructed from a single cell, the sensory organ precursor (SOP) that is singled out from a proneural cluster by Notch-mediated lateral inhibition (,7, ,8). Precursors arise at reproducible positions from each proneural cluster (,4, ,5), and selection of specific SOP cells may underlie the stereotypical nature of the final pattern. Uniform expression of sc or asense (ase), achieved with a heterologous promoter in the absence of enExecutegenous ac-sc expression, can rescue bristles that are positioned at wild-type locations (9–,11). This implies the existence of a patterning mechanism that acts Executewnstream 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 Executemain, that sequesters Ac-Sc proteins preventing them from binding DNA (12–,15). It has been Displayn to prevent development of additional bristles at ectopic locations and to play a part in the positioning of the SOP cell (,16–,19). h encodes a bHLH protein that acts as a transcriptional repressor of ac (20, ,21). stripe encodes an early growth response (egr)-like transcription factor that is required for the development of tenExecuten cells (22) and is able to prevent selection of SOPs from proneural clusters (,23). Here, we provide evidence that the spatially restricted distributions of Emc, H, and Sr contribute to the pattern by preventing bristle formation outside proneural clusters and by influencing the selection of specific precursor cells from proneural clusters. In animals devoid of enExecutegenous ac-sc activity, uniform expression of sc and ase Executees not rescue bristles at wild-type locations when the levels of activity of emc, h, and sr are reduced. The fact that bristles are rescued at Accurate positions with the use of a heterologous promoter and in the absence of normal cis-regulation, indicates that factors acting Executewnstream of Sc are sufficient to position the bristles. We discuss the possibility that patterning by sr, emc, and h is ancestral, and that the positional cis-regulatory sequences might be of recent origin.


Uniform SSlicee and Asense Expression Rescues Macrochaetes at Wild-type Locations in achaete-sSlicee Mutant Flies.

ase is a tarObtain of Ac-Sc and encodes a protein closely related to Ac and Sc that displays stronger proneural Preciseties than either Ac or Sc when mis-expressed in the epithelium (10, ,11, ,24). We used the Gal4-UAS method to drive simultaneous ubiquitous expression of sc and ase. This combination allows significant bristle rescue. In (1)ac3 sc10-1 animals lack activity of both ac and sc, and are devoid of bristles (Fig. 1B) (2, ,25). In males of the genotype In (1)ac3 sc10-1/Y; HS-Gal4>UAS-sc/>UAS-ase, bristles were rescued in many flies after heat shock. In the absence of heat shock, no rescue was observed.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Rescue of bristles by ubiquitous sSlicee and asense expression in animals devoid of enExecutegenous achaete-sSlicee expression. (A) Notum of a wild-type fly Displaying the positions of the 11 macrochaetes on the mesonotum. (B) Notum of a fly of the genotype In (1)ac3 sc10-1/Y devoid of bristles. (C) Schema of the mesonotum Displaying positions at which bristles were rescued in 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 landImpresss. Numbers indicate the percentage of rescue for each bristle. (D–G) nota of In (1)ac3 sc10-1/Y; HS-Gal4>UASsc/UAS-ase flies illustrating bristle rescue. aDC, anterior Executersocentral; pDC, posterior Executersocentral; aSC, anterior sSliceellar; pSC posterior sSliceellar; pPA, posterior postalar; aPA, anterior postalar; pSA, posterior supraalar; aSA, anterior supraalar; pNP, posterior notopleural; aNP, anterior notopleural; PS, presutural. Hu, humeral bristles of the pronotum, not part of the mesonotum.

Eleven macrochaetes are present at stereotypical positions on the wild-type hemi-notum (Fig. 1A). Their positions on rescued flies were assessed by using the underlying muscles and sutures as reference points. ReImpressably, all rescued macrochaetes (n = 158) were found exclusively at wild-type locations (Fig. 1 D–G). No ectopic bristles at abnormal locations were observed. An average of 6.58 (n = 24) macrochaetes was rescued. Each of the 11 wild-type bristles was recovered, but the frequency of rescue varied significantly (Fig. 1C).

Uniform SSlicee and Asense Expression Gives Rise to Macrochaetes at Ectopic Locations in ac-sc; emc h sr Mutant Flies.

Bristle rescue was assessed in In (1)ac3 sc10-1 flies that were, in addition, mutant for the hypomorphic, viable alleles emcpel, h1, and sr1 (In (1)ac3 sc10-1/Y; HS-Gal4>UAS-sc/>UAS-ase; emcpel h1 sr1). In the absence of heat shock, no rescue was observed. After heat shock, in those flies that displayed rescue, many more macrochaetes were present than in the previous experiment (Fig. 2A). The average number was 12.33 (n = 24). Fig. 2A Displays the locations of 296 rescued bristles. Some were located at wild-type positions. Others were found close to, but slightly disSpaced from, their normal positions (Fig. 2B). In addition, extra macrochaetes were observed at ectopic locations mostly located close to the positions of extant bristles (Fig. 2 C and D). For example, 4 heminota had 3 instead of 2 Executersocentral bristles. The 3 bristles were aligned and sometimes equally spaced such that none of them occupied the exact position of either of the 2 wild-type ones (arrows in Fig. 2D). In 8 flies where 2 Executersocentral bristles are present on each heminotum, the 4 bristles were Accurately aligned in only one of them. In the other cases, the 2 anterior (or the 2 posterior) bristles were not aligned, such that one was shifted either anteriorly or posteriorly (arrows in Fig. 2B).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Rescue of bristles by ubiquitous sSlicee and asense expression in animals mutant for achaete-sSlicee, extramacrochaetae, hairy, and stripe. (A) Schema of the mesonotum Displaying positions at which bristles were found in flies 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 bristle phenotypes. Black arrows indicate ectopic bristles. White arrows in B point to the disSpacement of the Executersocentral bristles that Execute not align on the left and right heminota.

A Prepattern of Synergistically Acting Antagonists.

The expression patterns of sr and emc in the wing/thoracic disk have been Characterized previously (12, ,16, ,18, ,26). We compare the Executemains of expression of emc and h with sr, and Display that they are expressed in distinct but overlapping Executemains. Expression of emc was ubiquitous, but there were 2 strong bands of expression, one on the anterior notum and one immediately posterior to the Spots where the Executersocentral and postalar bristles develop (Fig. 3A). The single expression Executemain of h was situated mainly over the position of the presumptive sSliceal-sSliceellar suture and overlapped the posterior Executemain of high emc expression (Fig. 3 B and C). sr was expressed in several broad, longitudinal Executemains in the anterior medial part of the notum and overlapped the anterior Executemain of emc expression (Fig. 3 D and E). Executeuble staining with neulacZ Displayed that the SOPs arose at positions where there was Dinky or no expression of these genes (Fig. 3 E and F). Expression of emc, sr, and h is independent of ac-sc (4, 16, 18; K.U., unpublished observations).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Expression patterns of stripe, hairy, and extramacrochaetae in the presumptive mesonotum of the wing disk. (A and B) The expression patterns of emc (green) and h (red) are visualized by β galactosidase staining of emc, lacZ, and an antibody against Hairy. (C) The merge of A and B Displays an overlap of expression of emc and h in a band across the posterior notum. (D) Triple staining Displaying the expression of emc, h, and sr. Expression of sr (blue) is visualized from Gal4-sr>UAS-GFP, that of emc (green) from α galactosidase staining of emc, lacZ, and that of h (red) from α-Hairy. Expression of emc and sr overlaps in the anterior notum. (E) Triple staining Displaying expression of sr (green), h (red), and A101, lacZ (blue), a Impresser of the sensory organ precursors. The precursors arise at positions where the levels of Sr and H are low. (F) Schematic drawing of the expression Executemains of sr (blue), emc (green), and h (red) as well as the positions where precursors of the bristles arise (brown circles). White circles represent other sensilla. This diagram indicates Locations of high levels of gene expression; emc is ubiquitously expressed, and h is expressed in the form of a gradient, as indicated by the paler colors. Anterior is up, and the Executersal midline is to the left in all images.

To investigate possible redundancy between emc, h, and sr, we examined the bristle phenotype of single and Executeuble mutants as well as the triple mutant. We used the hypomorphic alleles emcpel, h1, and sr1. Over half of the h1 mutant flies displayed an additional anterior sSliceellar bristle (Fig. 4E). Furthermore, the Executersocentral, sSliceellar, and postalar bristles were slightly misSpaced (data not Displayn). There were few ectopic bristles in sr1 mutant flies (Fig. 4F). The Executersocentral and supraalar bristles were, however, sometimes slightly misSpaced (data not Displayn). This is a consistent feature of clones mutant for a null allele [Fig. 2A in (23)]. Executeuble mutant h1 sr1 flies had slightly more bristles than each of the single mutants (Fig. 4G). There was Dinky overlap 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 bristles preferentially clustered at sites devoid of sr and h expression. We noted a synergy between emc and both h and sr: The Executeuble mutants displayed significantly more bristles than the sum of the single mutants. emcpel sr1 flies had many more bristles in the anterior notum where both emc and sr were highly expressed (Fig. 4C). emcpel h1 flies had ectopic bristles at many locations including the Executersocentral, sutural, and sSliceellar Locations where few ectopics were found in the single mutants and where both 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 the anterior and posterior notum, respectively. Finally, triple emcpel h1 sr1 mutant flies displayed large numbers of ectopic bristles over many parts of the notum (Fig. 4 D and H).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

The bristle phenotype of single, Executeuble, and triple mutants of extramacrochaetae, hairy, and stripe. (A–G) Schematic drawings Displaying the positions of macrochaetes on the notum of flies mutant for the genotypes indicated. Red crosses Impress the positions of bristles, which were plotted onto a standard diagram. Each diagram Displays bristles from 50 thoraces. Numbers indicate the average number of bristles per heminotum (n = 100). The positions of wild-type bristles are in pale blue. Pale green Spots denote the Executemains of expression of sr. The medial Executemain of expression is lacking in sr1 mutants (Displayn in white in C, D, F, and G). (H) Photograph of a triple mutant.

Ectopic Executersocentral Bristles in the Mutants Execute Not Arise as a Result of Expansion of the Proneural Cluster Driven by the Executersocentral Enhancer.

In the wild type, bristle precursors arise from the expression of ac-sc in proneural clusters mediated by discrete cis-regulatory elements. For example, the 2 Executersocentral bristles arise from a cluster driven by the Executersocentral enhancer (DCE) (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 Executersocentral bristles resulting from an expanded Executemain of ac-sc expression (4, ,16, ,18). This expanded Executemain could result from a change in the activity of the DCE such that it now drives ac-sc expression in a larger Spot. Alternatively, it could be because of ectopic expression of ac-sc that Executees 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, or sr1 mutants or in the Executeuble and triple mutants (Fig. 5 A–F). Executeuble labeling with a Impresser for bristle precursors Displayed 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 Executemain of ac-sc expression in the Executersocentral Location of the mutants arises independently of the activity of the DCE.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Activity of the Executersocentral enhancer of achaete-sSlicee in single, Executeuble, and triple mutants of extramacrochaetae, hairy, and stripe. (A–F) Presumptive notum of wing/thoracic discs of the genotypes indicated. The flies carry a transgene with the sequences of the Executersocentral enhancer driving expression of lacZ in cells of the Executersocentral proneural cluster. An α-galactosidase antibody was used to visualize lacZ expression. The size and position of the cluster are unchanged in all genotypes. (G and H) Executeuble staining for the Executersocentral enhancer, lacZ (α-galactosidase, red), and the bristle 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 DC cluster. The posterior Executersocentral (pDC) is formed first and can be seen in G. In the emcpel mutant, the pDC is also visible inside the Executersocentral cluster, but in addition, two other bristle precursors, anterior to and aligned with the pDC, are seen (white arrow) that are clearly outside the Executersocentral cluster. These ectopic precursors have formed even before the anterior Executersocentral bristle has appeared. Forty discs were examined, and precursors were consistently observed outside the Executersocentral proneural cluster in the mutant.


Patterning of Macrochaetes Executewnstream of Proneural Gene Expression.

In wild-type flies, ac-sc expression is restricted to the proneural clusters where cis-regulatory sequences enhance transcription. Outside of these Executemains basal expression is insufficient 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 autoregulation (Emc) (,18–,21); sr may also contribute. Bristle patterning is then a matter of Accurately positioning the bristle precursors from within the proneural clusters.

In the rescue experiment, the enExecutegenous ac and sc genes were nonfunctional, and so their cis-regulatory sequences cannot contribute to bristle patterning. Proneural protein was expressed ubiquitously from a heterologous promoter, and the pattern results from both the prevention of bristle formation at locations outside the normal positions of the proneural clusters and the selection of precursors at the Accurate sites from the Locations where ac-sc are usually expressed (see also ref. 9). It is likely that h,* sr, and emc are all contributing to both of these processes in the rescue experiment. First, they were expressed at appropriate sites to antagonize Sc/Ase activity at locations outside those of the proneural clusters. The Executemains of sr expression corRetort to sites where tenExecutens develop and flight muscles attach; the absence of macrochaetes here is a conserved, ancestral feature of the Diptera (23). Expression of h was Distinguishedest over the position of the presumptive sSliceal-sSliceellar suture, a Location also devoid of bristles throughout the Diptera. High levels of emc transcripts overlapped with those of sr and h but also covered additional sites. Second, ectopic bristles in the Executeuble and triple mutants are often located outside the sites of the proneural clusters. We Display that this phenotype is independent of the activity of at least one of the cis-regulatory elements of ac-sc, indicating that ectopic bristles can result from an increase in basal ac-sc levels mediated in trans.

Within a given proneural cluster, macrochaete precursors arise at reproducible positions in wild-type flies (4, ,5). Selection of precursors requires lateral inhibition mediated by the Delta (Dl)/Notch (N) signaling pathway (,8, ,30). Activation of N leads to the repression of ac-sc. Cell-Stoute choice is reinforced by means of a transcriptional feedback loop within each cell linking high levels of activation of N with reduced production of Dl (8, ,31). Expression of Dl depends on Ac-Sc (32). Any asymmetrically distributed factor that antagonizes Ac-Sc might bias the choice of Stoute by reducing the levels of ac-sc activity within some cells of a proneural cluster, both decreasing autoregulation and rendering the cells less efficient at signaling (Fig. 6). Such an Trace has been demonstrated for emc. The spatial distributions of transcripts of emc and of ac-sc are complementary over much of the notum (16, ,18). Furthermore, many SOPs arise at positions where the levels of emc transcripts decrease sharply, and SOPs disSpaced from wild-type positions appear in emc mutants with decreased expression (16–,18). A role for emc in the precise positioning of bristles in the rescue experiment (and the wild type) is, thus, likely. Several observations suggest that h and sr also contribute to the positioning of some SOPs. Ectopic Sr in proneural clusters Executees not prevent expression of ac-sc, but their expression fails to refine to single cells and no SOPs develop (23). This suggests that Sr interferes with autoregulation and/or inhibition. Thus it could influence SOP selection in those Spots where its expression overlaps proneural clusters. The Executersocentral and supraalar bristles arise on the borders of sr expression Executemains. These bristles were slightly disSpaced from their normal locations in sr mutants. Similarly, the postalar and anterior sSliceellar bristles arise just where the levels of Hairy decreased Impressedly; more than half of h1 mutant flies displayed an additional anterior sSliceellar bristle, whereas in others, the anterior sSliceellar and postalar bristles were slightly disSpaced.

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint 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-sSlicee 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 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, 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 Inequity between cells, in the level of any component of the loop, can be amplified via the loop itself, such that eventually 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 reduce their capacity to signal and, thus, cause them to take up the epidermal Stoute.

Although emc plays the major role in bristle patterning, it is likely to act synergistically with sr and h in parts of the anterior and posterior notum, respectively, where their expression Executemains overlap. As ac-sc are subject to autoregulation in the wild type, factors that antagonize Ac-Sc, such as Emc, have an indirect Trace on transcription that would compound the Traces of transcriptional regulators such as Hairy and vice versa (15, ,18). Although autoregulation of ac-sc cannot play a role in the rescue experiment, autoregulation of the enExecutegenous ase gene is likely. Because expression of both h and sr is spatially regulated, their synergistic Traces with emc would be local. Synergistic activity would Elaborate the increase in ectopic bristles seen in the Executeuble and triple mutants.

Is Patterning by Antagonism Ancestral?

By antagonizing ac-sc activity, Emc, Hairy, Sr, and probably other yet unCharacterized factors are able to pattern the macrochaetes on the Drosophila notum from uniform ac-sc expression. Bristles are rescued at Accurate locations independently of the activity of the cis-regulatory elements that normally regulate spatial expression of ac-sc in proneural clusters. Restricting expression of ac-sc to proneural clusters in the wild type, therefore, provides a redundant inPlace. We suggest that patterning by antagonism Executewnstream of uniform ac-sc transcription might be ancestral. Uniform expression of ac-sc coupled with Notch-mediated lateral inhibition would be sufficient to generate the ranExecutem pattern of spaced sensory organs seen in many hemimetabolous insects. Indeed ubiquitous expression of sc pDeparts development of the ranExecutemly positioned microchaetes (small bristles) on the notum of several species of Diptera (33, ,34). Patterning of the small bristles on the legs of Drosophila has also been Displayn to result from broad activation of ac coupled to repression by h and Dl (35). The absence of macrochaetes at the sutures and sites of muscle attachment is an ancestral feature correlated with conservation of the pattern of these characters throughout the Diptera (,36), suggesting ancient regulation by sr and h (23). Finally, we have postulated that the position-specific regulatory enhancers driving ac-sc in proneural clusters may have originated in the lineage leading to cyclorraphous Diptera. Expression of ac-sc homologues on the 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 have provided material for the evolution of numerous cis-regulatory modules (38). If the modular promoter of the ac-sc genes of Drosophila is of recent origin and was superimposed onto an ancestral patterning mechanism mediated by antagonism, the enhancers could have evolved sequentially one-by-one.

Redundancy of developmental mechanisms has been widely Executecumented and contributes to the plasticity of development and to the robustness of phenotype (39, ,40). Patterning of the vulva in nematodes is a Excellent example of this: Significant redundancy has been Executecumented despite conservation of morphology (,41). It is not known how redundancy is built up during evolution, nor whether it can be a source of evolutionary Modernty. If the spatially regulated expression of ac-sc evolved later than patterning Executewnstream of uniform expression, it might be possible to dissect this evolutionary process through identification of extant species in which patterning is exclusively dependent on the latter.

Materials and Methods

Fly Strains.

The chromosome In (1)ac3 sc10-1 is associated with a loss of ac and sc activity: No detectable ac protein accumulation occurs (4) because of the In (1)ac3 Fracturepoint. sc10-1 induces a truncated sc protein because of an amber mutation 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 following stocks: emcpel DC 1.4 lacZ/emcpel, h1 DC 1.4 lacZ/h1, sr1 DC lacZ/sr1, emcpel h1 DC 1.4 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 other mutant strains, see FlyBase.

Bristle Rescue Experiment.

Rescue of bristles in an ac− sc− background was performed by using In (1)ac3 sc10-1; HSGal4 UAS-Sc/UAS-AseHA and In (1)ac3 sc10-1; HSGal4 UAS-Sc/UAS-AseHA; emcpel h1 sr1 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 2-h intervals, and then left at 18 °C until just before eclosion.

Antibody Staining.

Third instar larval wing disks were processed according to classical protocols. For β-Gal staining, we used a 1:200 dilution of the 40-1a mouse primary antibody (Developmental Studies HybriExecutema Bank) and a 1:500 dilution of secondary antibody conjugated to Alexa-647 (goat α-mouse antibody, Molecular Probes, Invitrogen). For GFP staining, we used a 1:500 dilution of a rabbit α-GFP antibody conjugated to Alexa-488 (Molecular Probes). For Hairy, we used a 1:20 dilution (monoclonal from Insight Biotechnology) and a 1:500 dilution of secondary antibody conjugated to Alexa-546 (goat anti-mouse antibody).


We thank Manuel Calleja, Ginès Morata, and Juan MoExecutelell for fly stocks; Emma Hatton-Ellis for help with photography; Emma Hatton-Ellis and Barbara Negre for help with a genotype; Savita Ayyar for UAS-Ase; the members of our group for lively discussion and comments on the manuscript; and Rosalba Melchoirre and Ben Taylor for technical assistance. J.-M.G. was the recipient of a Marie Curie postExecutectoral fellowship. This work was supported by the Wellcome Trust [29156].


3To whom corRetortence should be addressed. E-mail: pas49{at}

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

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

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

This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, “Gene Networks in Animal Development and Evolution,” held February 15–16, 2008, at the ArnAged and Mabel Beckman Center of the National Academies of Sciences and Engineering in Irvine, CA. The complete program and audio files of most presentations are available on the NAS web site at:

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at

↵* Alone, of the three genes, h encodes a transcriptional repressor of ac (19, ,20), but we nevertheless Consider h is contributing to the positioning of bristles in the rescue experiment because of the pattern of rescue of the small bristles (microchaetes). Microchaetes cover the sSliceum of wild-type animals, but are absent from the sSliceellum and midline. Significant numbers of microchaetes were rescued, but, in Dissimilarity to emc+ h+ sr+ flies, rescue in the emc h sr triple mutant background allowed the formation of microchaetes on the sSliceellum and Executersal midline, a phenotype that is exclusive to h mutants. Rescue of microchaetes, which arise after pupariation, might be because of activation of the enExecutegenous ase gene [as well as senseless (sens)] by the Sc and Ase provided by the transgene (23, ,29). One possibility, therefore, is that Hairy can act through conserved binding sequences in the promoter of ase and/or sens.

© 2008 by The National Academy of Sciences of the USA


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