Secretion of the amino-terminal fragment of theHedgehog protein is necessary and sufficient for

hedgehog signalling in DrosophilaM.J. Fietz*, A. Jacinto, A.M. Taylor, C. Alexandre and P.W. Ingham

Molecular Embryology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

Background: The Drosophila segment polarity genehedgehog encodes a member of a family of secreted pro-teins that are involved in a variety of patterning processes,in both vertebrates and invertebrates. Some of these pro-cesses depend upon short-range or contact-dependentinteractions, whereas others seem to involve long-rangesignalling. Two different models have been proposed toaccount for the execution of these contrasting processesby the same proteins: one postulates that Hedgehog actsexclusively over short distances, its long-range influencesbeing effected through regulation of other signallingfactors; the second postulates that different aspects ofHedgehog activity are mediated by distinct forms of theprotein that are generated by autoproteolysis .Results: We have investigated these models by mutatingthe hedgehog coding region such that only the amino-

terminal or carboxy-terminal half of the protein issecreted. Deletion of the carboxy-terminal portion haslittle effect on the signalling activity of the protein,whereas abolishing the secretion of the amino-terminalhalf leads to a complete loss of signalling. In addition, wefind that increases in the level of expression within thenormal hedgehog transcriptional domain of either the wild-type protein or the carboxy-terminal-deleted formexpand the range of activity to a limited extent, but haveonly minor effects on cell identity.Conclusions: In Drosophila, all of the signalling activityof Hedgehog resides in the amino-terminal portion of theprotein, the secretion of which is essential for its function.The range of Hedgehog is limited by the close associationof the amino-terminal peptide with the cell surface butcan be extended by elevating the level of its expression.

Current Biology 1995, 6:643-650

Introduction

The expression of the segment polarity gene hedgehog(hh) is required for the normal patterning of both the lar-val body segments and the adult appendages of Drosophila[1]. In imaginal discs, the precursors of adult appendages,the influence of hh activity extends over many cell diam-eters [1,2]. Despite this long-range effect, there is goodevidence to suggest that the protein itself acts over onlyshort distances, regulating the expression of genes encod-ing other signalling factors, such as wingless (wg) anddecapentaplegic (dpp) [2-6].

An absence of hh activity during embryogenesis has simi-larly widespread effects on the patterning of individualsegments, as revealed in both the dorsal and the ventralcuticle patterns of hh mutant larvae [1,7]. As in the imag-inal discs, hh acts at short range to maintain the expres-sion of wg in cells adjacent to those expressing hh at eachparasegment boundary [8-11]. Although the control ofwg by hh provides a molecular basis for the long-rangeeffects of hh on ventral patterning, it has been argued thatthis cannot explain the dependence of the dorsal cuticularpattern on hh activity [7]. Instead, Heemskerk andDiNardo [7] suggest that hh has a second distinct role as amorphogen; according to this scenario, the hh-expressingcells at the parasegment boundary would act as a localized

source generating a graded distribution of Hh proteinacross the segment. Thus, the positional identity of dorsalectodermal cells would be directly specified by differentlevels of Hh.

The Hh protein undergoes post-translational modification[6,12] to yield three major species that differ in apparentmolecular mass: a 39 kD species, generated by cleavage ofthe signal peptide sequence; and 19 kD and 25 kD species,generated by the autoproteolysis of this secreted form intoamino-terminal and carboxy-terminal peptides, respec-tively [13]. These latter species have contrasting propertiesboth in vitro and in vivo: bacterially produced amino-ter-minal peptide binds to heparin, whereas the carboxy-ter-minal peptide does not, and when expressed in tissue cul-ture cells, the amino-terminal peptide remains associatedwith the cell surface while the carboxy-terminal form issecreted into the medium [13]. In line with this behaviourin vitro, the amino-terminal peptide appears closely associ-ated with the cells in which it is expressed in the Droso-phila embryo [6,13,14], whereas the carboxy-terminalspecies is more widely distributed across each segment[13]. These findings have led to the suggestion that differ-ent modes of hh function may be mediated by these twodifferent portions of the protein, the amino-terminal formmediating short-range effects and the carboxy-terminalpeptide effecting long-range signalling [13].

© Current Biology 1995, Vol 5 No 6

*Present address: Department of Biochemistry, University of Tasmania, Hobart, Tasmania, Australia. Correspondence to: P.W. Ingham.

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In this study, we have explored this model by generatingmutant forms of the Hh protein and assaying their activ-ity when expressed ectopically both in imaginal discs andin embryos. In addition, we have investigated the pro-posal that different levels of Hh activity act instructivelyto specify positional identity, by manipulating the levelsof Hh protein produced by cells at the parasegmentboundary.

Results and discussion

Construction and characterization of modified Hh proteinsTo investigate the relative contributions of the amino-and carboxy-terminal portions of the Hh protein to hhsignalling activity, we designed two mutated forms of thehh cDNA and expressed them in developing embryosand larvae. In the first of these, ACT, we aimed to gener-ate a form that resembles the endogenous 19 kD amino-terminal polypeptide. As the precise location of the auto-proteolysis cleavage site was not known (although it mustlie carboxy-terminal to residue 254 [13]), we introduceda stop codon at position 310 (see Materials and methods);this should result in the elimination of 161 residues (orapproximately 80 %) of the carboxy-terminal peptide (see,Fig. la). In the second construct, ASC, we aimed to gen-erate a form of the protein from which only the carboxy-terminal peptide is secreted. As autoproteolysis of Hhhas been shown to be independent of signal cleavage[13], we mutated the signal cleavage site, anticipating thatthis would allow secretion of the carboxy-terminal poly-peptide while anchoring the amino-terminal half of theprotein in the membrane (see Fig. la). Both modifiedcDNAs were cloned downstream of yeast upstream acti-vating sequences (UAS) in the transformation vectorpUAST [15] and transformed into flies.

To verify that the constructs give rise to the expectedproducts, protein extracts of embryos in which the con-structs are activated by GAL4 protein expressed undercontrol of patched enhancers (ptcGAL4; [16]) were ana-lysed by western immunoblot analysis (Fig. lb) using anantibody specific for the amino-terminal portion of theHh protein (see Materials and methods). In extracts ofembryos in which the wild-type hh cDNA is driven byptcGAL4, we detect two bands with this antibody (Fig.lb, lane 3), one corresponding to the 39 kD form of theHh protein generated by signal cleavage and a secondof around 19 kD corresponding to the amino-terminalpeptide generated by autoproteolysis. Interestingly, thissmaller peptide seems to be more abundant than the un-cleaved form, in contrast to the endogenous protein [13],suggesting that autoproteolysis occurs more efficientlywhen the protein is overexpressed.

Expression of ASC produces three novel bands (Fig. lb,lane 2); the largest of these migrates at around 46 kD,corresponding to the size of the full-length uncleaved Hhprotein. The smallest and most abundant band migratesat around 26 kD, the size expected of the amino-terminal

Fig. 1. (a) Schematic representation of the wild-type Hh proteinand the two mutant derivatives, ACT and ASC, showing theirexpected dispositions following signal cleavage (SC) and auto-proteolysis (AP). Crosses indicate the sites of mutation in the twomutant forms. (b) Western immunoblot of protein extracts fromembryos expressing wild-type or modified forms of the hh cDNA.Lane 1: UAShhACT; lane 2: UAShhASC; lane 3: UAShh. Theweak bands running at 19 kD in lanes 1 and 2 represent theproducts of the endogenous hh gene and do not derive from themutant constructs.

peptide generated by autoproteolysis in the absence of sig-nal cleavage (19+7 kD). From this we infer that the ASCconstruct also gives rise to the 25 kD carboxy-terminalpeptide that is not detected by our antibody. A third bandthat migrates with a relative molecular mass of around30 kD is present at much lower levels and is presumablygenerated by autoproteolysis at a more carboxy-terminalposition. The relative levels of the 46 and 26 kD bandscompared to those of the 39 and 19 kD bands producedby the wild type cDNA suggest that autoproteolysis ofthe ASC protein is slightly less efficient.

Signalling by the amino-terminal fragment of Hedgehog Fietz et al.

Fig. 2. Distribution and consequences ofectopically expressed wild-type andmutant forms of Hh protein in imaginaldiscs. Wing imaginal discs from (a)30A-UAShh, (b) 30A-UAShhACT and(c) 30A-UAShhASC third-instar larvaeshowing the expression of the exoge-nous and, at much lower levels,endogenous Hh proteins. (d-f) Thecellular distribution of each of theexogenous proteins shown in detail.

The ACT construct produces a single band that migrateswith a relative molecular mass of around 25 kD (Fig. 1lane 1) - the predicted size of the signal-cleaved formof the truncated protein. Signal cleavage of this proteinthus occurs efficiently, resulting in the secretion of theamino-terminal peptide fused to the first approximately50 residues of the carboxy-terminal peptide.

Secretion of the amino-terminal peptide is necessary andsufficient to reorganize the patterning of imaginal discsIn order to assay the signalling activity of the modifiedproteins, we examined the consequences of their ectopicexpression on the development of the wing imaginaldisc. First, we analysed the cellular distribution of eachmodified protein by indirect immunofluorescence ofwing discs in which the proteins are ectopically expres-sed in the anterior compartment, a region of the discdevoid of endogenous protein. Using our amino-termi-nal-specific antibody we detect products of both theASC and ACT constructs around the periphery of cells,a distribution similar to that seen with the wild typeprotein (Fig. 2d-f); thus, both mutated forms of theprotein seem to be correctly inserted into the plasmamembrane.

As described previously [5], ectopic expression of hhdriven by the GAL4 line 30A results in overgrowth ofthe anterior compartment (see Fig. 2a), ectopic activationof dpp expression and the duplication with reversedpolarity of anterior compartment structures - anteriormargin, wing blade and veins I, II and III (Fig. 3a,b).Similarly, ectopic expression of ACT results in increasedgrowth of the anterior compartment (Fig. 2b) associatedwith the inappropriate activation of dpp transcription inneighbouring cells (data not shown) and a concomitantduplication of anterior wing-blade structures originating

around the costal region of the wing (Fig. 3c). Theseeffects are similar to, though slightly less extreme than,those caused by ectopic expression of the wild-type hhcDNA under the same conditions, resembling moreclosely the duplications induced by the latter whencultured at a lower temperature (Fig. 3b).

Ectopic expression of the ASC protein, by contrast, hasno effect on imaginal disc growth (Fig. 2c) or dpp tran-scription (data not shown), or on the pattern of the dif-ferentiated wing (Fig. 3d). We conclude that the organ-izing activity of hh in the imaginal discs can be mediatedby the amino-terminal half of the protein acting at shortrange to activate dpp expression, and that secretion of thispeptide is essential for its activity.

Ectopic expression of the amino-terminal portion of Hhaffects the patterning of both ventral and dorsalembryonic ectodermIn order to analyse the signalling capacity of the modi-fied proteins in the developing embryo, we monitoredthe expression of wg in embryos in which each hh con-struct is activated by GAL4 expressed under the controlof ptc regulatory elements (ptcGAL4). This drives expres-sion of the proteins in a pattern complementary to thatof the endogenous hh gene. Expression of the wild-typeHh protein in this pattern results in the ectopic activa-tion of wg throughout the 'wg-competent' [10] domainin each parasegment (Fig. 4e), exactly as seen when hhis ubiquitously expressed under the control of a heat-inducible promoter [6,17,18]. Expression of ACT underptcGAL4 control has essentially identical effects on wgexpression to those of the wild-type Hh protein(Fig. 4c), whereas in embryos in which ASC is driven byptcGAL4, ug is expressed in a wild-type pattern (Fig.4a). As expected from the changes in wg expression,

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Fig. 3. Effects of ectopic expression of wild-type and mutant forms of Hh on the pattern of the wing. (a) Heminotum and wing of a30A-UAShh fly raised at 25 C. Note the duplicated notal structures (arrow) and duplication of anterior wing blade; (b) Heminotumof fly as in (a) but raised at 18 C. There is no duplication of notal structures; duplication of the anterior wing initiates at a more prox-imal position than in (a). (c) Heminotum and wing of a 30A-UAShhACT fly raised at 25 C. Note the duplicated notal structures(arrow) and the duplication of anterior wing blade. (d) Wild-type wing typical of a 30A-UAShhASC fly. The arrowheads in (a-c) markthe axis of duplication.

Fig. 4. Phenotypic effects of the ectopicexpression of wild-type and mutantforms of hh on wg expression and larvalpatterning. Embryonic wg expressionat stage 10 and larval ventral cuticlepatterns of (a,b) ptcGAL4-UAShhASC,(c,d) ptcGAL4-UAShhACT and (e,f)ptcGAL4-UAShh animals, respectively.Note the expansion of the wg domainin (c) and (e). This alters the specifica-tion of ectodermal cells, as revealedin the altered patterns of ventral denti-cles (d,f); note particularly the elimina-tion of the posterior rows of finerdenticles from each belt that are typi-cal of wild-type, and are unaffected inptcGAL4-UAShhASC animals (b).

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embryos in which the wild-type hh cDNA or the ACTconstruct are expressed under ptcGAL4 control exhibitchanges in the ventral (Fig. 4d,f) and dorsal (data notshown, but see Fig. 5) cuticular patterns that are iden-tical to those seen in embryos expressing hh ubiqui-tously. By contrast, the cuticular pattern of ptcGAL4ASC embryos is indistinguishable from wild-type (Fig.4b). Thus, ectopic expression of the amino-terminal halfof the protein, but not of the carboxy-terminal peptide,is sufficient to respecify the pattern of both ventral anddorsal cuticle.

It is possible that the effects of ACT on dorsal patterningare an indirect consequence of its effect on wg expression.To exclude this possibility, we used a heat-shock-indu-cible HSGAL4 construct to express the ACT constructtransiently at the stage at which hh activity is proposedto be directly required for the specification of dorsal hairidentity [7]. Embryos were heat shocked for 20 minutes,at 8 hours (+ 0.5 h) after fertilization, and their cuticles

were analysed on completion of embryogenesis. As acontrol, the wild-type hh cDNA was similarly inducedusing the HSGAL4 construct. In each case, transientubiquitous expression of the transgene has little effecton the ventral pattern (Fig. 5a-c), but causes a respecifi-cation of the dorsal pattern - the region of smoothcuticle designated cell type 2 by Heemskerk andDiNardo [7] is expanded at the expense of the thickhairs and fine hairs that characterise cell types 30 and 4° ,respectively (Fig. 5e-g). This effect appears stronger inthe case of the wild-type protein than for ACT; but, it isclear that transient expression of the amino-terminalportion of Hh is sufficient to respecify the identity ofdorsal ectodermal cells.

Elevated levels of transcription can increase the range of hhsignalling activityAlthough the amino-terminal Hh peptide normallyappears in close proximity to the cells from which it issecreted [6,13,14], it remains possible that some of this

Fig. 5. Larval cuticular patterns follow-ing transient ubiquitous expressionor elevated spatially-restricted expres-sion of Hh proteins. Ventral denticle(a-c) and dorsal hair (e-g) patterns ofHSGAL4, HSGAL4-UAShhACT andHSGAL4-UAShh embryos, respectively,following a single 20 min heat shock at8 h ( 0.5 h) of development. Note theelimination of the posterior-most row ofdenticles in (b) and (c), and the elimina-tion of type 3 dorsal hairs and theexpansion of region 2 in (f) and (g), bycontrast with the wild-type patternin (e). Panels (d) and (h) show the ven-tral and dorsal cuticle patterns, respec-tively, of an enGAL4-UAShh larva.Note the elimination of the posterior-most ventral denticles in each belt (d)and the wild-type dorsal hair pattern (h).

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fragment might spread from the expressing cells, at levelsbelow the limits of immunohistochemical detection, toexert a long-range effect across each segment. We there-fore asked whether increasing the levels of Hh proteinproduced at the parasegment boundary could lead tochanges in cellular identity across the parasegment. Todo this, we made use of another GAL4 line, in whichthe yeast protein is expressed under the control ofengrailed regulatory sequences (enGAL4; A. Brand andK. Yoffe, unpublished). This drives high-level expressionof the wild-type or modified Hh protein within thenormal hh transcriptional domain (Fig. 6, compare pan-els a and e). Despite the considerable increase in proteinlevels compared to wild type, the Hh-positive cellsremain demarcated by a sharp boundary, suggesting littlediffusion of protein away from the secreting cells.Embryos expressing such elevated levels of either thewild-type gene or the ACT construct do exhibit a slightexpansion of the wg expression domain, such that atstage 11, wg is transcribed in stripes up to two cellswide, compared to the single-cell-wide stripes typical ofwild-type embryos (Fig. 6f-i).

The effect of both the wild-type hh and the ACT con-struct on the transcription of ptc, another target of hhactivity, is rather more dramatic. Instead of resolving intotwo narrow stripes that flank the hh transcriptionaldomain (see Fig. 6b), expression of ptc remains wide-spread and accumulates at particularly high levels in abroad stripe posterior to each domain of hh expression(Fig. 6c,d). This suggests that when expressed at elevatedlevels, the amino-terminal portion of the Hh protein canspread beyond the cells adjacent to those from which it issecreted: that the ectopic activation of wg is restricted to asubset of cells that are responsive to Hh (compare Fig. 6panels g, h and i) suggests that this diffusion generatesa steep gradient of Hh, the transcription of ptc beingactivated by levels lower than those required to activatewg. Most embryos expressing either wild-type hh orACT construct under the control of enGAL4 completeembryogenesis and hatch. Strikingly, although the pat-terning of the ventral cuticle is slightly affected (Fig. 5d),the dorsal hair pattern appears essentially normal (Fig.5h). In the absence of the endogenous hh gene, expres-sion of ACT under enGAL4 control can partially rescue

Fig. 6. Effects of elevated levels of Hhprotein on wg and ptc transcription. (a)Distribution of the hhACT-encoded pro-tein driven by the enGAL4 enhancertrap line. Note the sharp demarcationbetween expressing and non-expressingcells and the elevated levels of Hh pro-tein compared to that produced by theendogenous gene shown in (e). Expres-sion of (b-d) ptc and (f-h) wg inembryos expressing hhASC, hhACT andhh, respectively, under the control ofenGAL4. In the case of hhASC, theexpression of both gene is identical towild-type. Note the limited expansion ofthe wg stripes (g,h) compared to thatseen when hh is expressed throughoutthe parasegment (i).

Signalling by the amino-terminal fragment of Hedgehog Fietz et al.

the effects of hh null mutations, although not as effectivelyas expression of the wild-type gene (data not shown).

Conclusions

Our findings provide conclusive evidence against thehypothesis that different forms of the Hh protein mediatedifferent aspects of its signalling activity. According to thismodel, the carboxy-terminal peptide is specifically res-ponsible for the patterning of the dorsal embryonic ecto-derm [13]. We find, however, that ectopic expression ofthe amino-terminal half of the protein results in effectssimilar to those induced by the wild-type protein, alter-ing the identity of cells of both the dorsal and ventralectoderm of the developing embryo and of cells of theanterior compartment of imaginal discs. Conversely, wedetect no effects on either larval or adult patterning fol-lowing ectopic expression of a form of the protein inwhich the signal cleavage sequence is mutated. Thus,although the amino-terminal peptide is normally foundin close association with the cell surface, its activitydepends critically upon its being secreted; moreover, asthis mutant form of Hh still undergoes autoproteolysis, itfollows that the carboxy-terminal peptide has no intrinsicsignalling activity of its own. We conclude that all sig-nalling activity of the Hh protein is most likely to residein the 19 kD amino-terminal fragment generated byautoproteolysis. The sole function of the carboxy-termi-nal portion of the protein seems to be to catalyse the mat-uration of the Hh protein into its active form, providing apotential control point for the regulation of Hh activity.

This conclusion is supported by experiments in whichwe raised the levels of hh expression without affecting itsspatial restriction. Although we have not directly. visual-ized the carboxy-terminal peptide in such embryos, wecan infer, on the basis of the elevated levels of the amino-terminal peptide that we observe, that the concentrationof the carboxy-terminal peptide across each segment issignificantly increased compared to wild-type. Despitethis, no effect on the patterning of the dorsal ectoderm isdetectable. Paradoxically, the expression domains of bothptc and wg, which are normally restricted to cells directlycontacting those expressing hh, are expanded by theoverexpression of either the full-length or carboxy-ter-minal-deleted form of the Hh protein. Thus, althoughthe amino-terminal peptide is closely associated with cellsfrom which it is secreted, its activity can spread beyondthese cells when it is expressed at sufficiently high levels.This property could provide the basis of the differentinducing activities of Hh-family proteins observed in ver-tebrate cells, the difference between contact-dependent[19] and diffusible [20] effects perhaps reflecting a differ-ence in concentration rather than form of the protein.

Whilst this paper was under review, a comparable analysisof the relative activities of the amino- and carboxy-ter-minal portions of Hh was published by Porter et al. [21],the results of which confirm those presented here.

Materials and methods

In vitro mutagenesis and germ line transformationIn order to generate hh ACT, the plasmid Bluhh, consisting of a1.9 kb cDNA fragment containing the complete coding regionof the D. melanogaster hh gene [22] cloned in Bluescript wasdigested with Xhol (nucleotide 1301) and filled-in with Kle-now polymerase. The fragment including the vector sequencesand the 5' end of the hh cDNA was isolated and ligated to a 3'fragment formed by cleaving Bluhh with FspI (nucleotide1524) and an enzyme in the polylinker. This resulted in thegeneration of a truncated form of the cDNA in which themethionine (encoded just after the XhoI site) is replaced by astop codon, effectively deleting the carboxy-terminal 19 kD ofthe protein.

ASC was generated by mutating the alanine and serine in thesignal cleavage sequence AHSC to arginine and asparagine,respectively. This was achieved using the polymerase chainreaction (PCR) with complementary oligonucleotides of thesequence: TTTAGCCCGCGGCACAACTGCGGTCCTG-GCCGAG (the mutated codons are underlined and the basechanges shown in bold face). Two fragments were amplified,one extending to the 5' end of the hh cDNA, the otherextending to the Sphl site (nucleotide 1041). These fragmentswere digested with SacII, which cuts at the unique site encod-ed in the oligonucleotide sequence, ligated and used to replacethe EcoR1-SphIl fragment from the wild-type cDNA clone.The entire amplified region was sequenced to ensure that noerrors were introduced by the PCR. Both constructs werecloned into the w+ P-element vector pUAST [15], and trans-formed into Drosophila embryos using standard microinjectionprocedures [23]. In each case, three independent lines (desig-nated UAShhASC20, 26 and 30 and UAShhACTMI, M5and M6) were established, and used in the described experi-ments to ensure that the observed effects are independent ofinsertion site.

Expression of transgenes in Drosophila embryos andimaginal discsSpatially restricted expression of UAS transgenes was achievedby crossing transgenic lines to ptcGAL4 [16], 30A [15] orenGAL4 (A. Brand and K. Yoffe, unpublished). Animals werecultured at 25 C on standard Drosophila medium unless other-wise stated. For transient ubiquitous expression of the trans-genes, flies carrying the UAS constructs were crossed to fliescarrying an HSGAL4 construct; embryos were collected fromover I h intervals, transferred to thin layers of I % agarose onglass microscope slides and incubated in a plastic Petri dishfloating in a water bath at 37 C for 20 min intervals. Followingheat treatment, embryos were returned to 25 C.

Western immunoblot analysisEmbryonic protein lysates were prepared by boiling embryosin protein sample buffer for 5 min. Following electrophoresis,the gel and nitrocellulose filter were preincubated in buffer(25 mM Tris, 192 mM glycine, 20 % v/v methanol, pH 8.3)prior to transfer for approximately I h at 100 V. Filters wereblocked in PAT (PBS, 1 % BSA, 0.1 % Triton and 0.2 % azide)for at least 1 h, before incubation overnight at 4 C with apolyclonal rabbit anti-Hedgehog antibody (1:5 000) raisedagainst a fusion protein containing the first 247 residues of theHh protein [14]. After washing, the filters were incubated withHRP-conjugated secondary antibody and the signal visualizedusing the ECL system (Amersham).

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In situ hybridizationWhole embryos were fixed and hybridized with deoxygenin-labelled single-stranded wg or ptc RNA probes, as described [10].

Larval and adult cuticle preparationsAdult flies and pharate larvae were dissected in 70 % ethanol,cleared by incubation in 10 % NaOH at 80 C for 5 min andmounted in Euparal for examination with the compoundmicroscope. Larvae were collected just prior to hatching,removed from their vitelline membranes manually and mountedin 1:1 lactic acid /Hoyer's medium.

ImmunohistochemistryFixed embryos and imaginal discs were incubated with poly-clonal rabbit anti-Hh antibody at 1:2 000 and visualized usingeither alkaline phosphatase- or fluorescein-coupled anti-rabbitIgG. Fluorescent specimens were imaged on a BioRad MRC1000 confocal microscope.

Acknowledgements: The first two authors contributed equally to thiswork. We are grateful to Uwe Hinz, Andrea Brand and NorbertPerrimon for supplying GAL4 enhancer trap lines and to RonBlackman for making the dpp-lacZ strain available. We are gratefulto Ian Goldsmith for synthesizing oligonucleotides, and to AndrewEdwards and Peter Jordan for help with the confocal microscopy.M.J.F. is a C.J. Martin Research Fellow of the Australian NH andMRC. A.J. is in receipt of a post-graduate bursary from ProgramaGulbenkian de Doutoramento em Biologia e Medicina and C.A isa European Community HCMP Fellow. This work was supportedby the ICRF.

References1. Mohler J: Requirements for hedgehog, a segment polarity gene, in

patterning larval and adult cuticle of Drosophila. Genetics 1988,120:1061-1072.

2. Basler K, Struhl : Compartment boundaries and the control ofDrosophila limb pattern by hedgehog protein. Nature 1994, 368:208-214.

3. Diaz-Benjumea FJ, Cohen B, Cohen SM: Cell-interaction betweencompartments establishes the proximal-distal axis of Drosophilalegs. Nature 1994, 372:1 75-1 79.

4. Capdevila J, Guerrero : Targetted expression of the signallingmolecule decapentaplegic induces pattern duplications and growthalterations in Drosophila wings. EMBO] 1994, 13:4459-4468.

5. Ingham PW, Fietz M: Quantitative effects of hedgehog anddecapentaplegic activity on the patterning of the Drosophila wing.Curr Biol 1995, 5:432-440.

6. Tabata T, Kornberg TB: Hedgehog is a signalling protein with a keyrole in patterning Drosophila imaginal discs. Cell 1994, 76:89-102.

7. Heemskerk , DiNardo S: Drosophila hedgehog acts as a morpho-gen in cellular patterning. Cell 1994, 76:449-460.

8. Hidalgo A, Ingham P: Cell patterning in the Drosophila segment:spatial regulation of the segment polarity gene patched. Develop-ment 1990, 110:291-302.

9. Hidalgo A: Interactions between segment polarity genes and thegeneration of the segmental pattern in Drosophila. Mech Dev1991, 35:77-87.

10. Ingham PW, Taylor AM, Nakano Y: Role of the Drosophila patchedgene in positional signalling. Nature 1991, 353:184-187.

11. Ingham PW, Hidalgo A: Regulation of wingless transcription in theDrosophila embryo. Development 1993, 117:283-291.

12. Lee JJ, von Kessler DP, Parks S, Beachy PA: Secretion and localizedtranscription suggests a role in positional signalling for products ofthe segmentation gene hedgehog. Cell 1992, 70:777-789.

13. Lee JJ, Ekker SC, von Kessler DP, Porter JA, Sun BI, Beachy PA: Auto-proteolysis in hedgehog protein biogenesis. Science 1994, 266:1528-1537.

14. Taylor AM, Nakano Y, Mohler J, Ingham PW: Contrasting distribu-tions of patched and hedgehog proteins in the Drosophila. MechDev 1993, 43:89-96.

15. Brand AH, Perrimon N: Targeted gene expression as a means ofaltering cell fates and generating dominant phenotypes. Develop-ment 1993, 118:401-415.

16. Speicher SA, Thomas U, Hinz U, Knust E: The Serrate locus ofDrosophila and its role in morphogenesis of imaginal discs: controlof cell proliferation. Development 1994, 120:535-544.

17. Ingham PW: Localised hedgehog activity controls spatially res-tricted transcription of wingless in the Drosophila embryo. Nature1993, 366:560-562.

18. Chang DT, Lopez A, von Kessler DP, Chiang C, Simandl BK, ZhaoRB, et al.: Products, genetic-linkage and limb patterning activity ofa murine hedgehog gene. Development 1994, 120:3339-3353.

19. Roelink H, Augsburger A, Heemskerk , Korzh V, Norlin S, Ruiz I, etal.: Floor plate and motor neuron induction by vhh-l, a vertebratehomolog of hedgehog expressed by the notochord. Cell 1994,76:761-775.

20. Fan C-M, Tessier-Lavigne M: Patterning of mammalian somites bythe surface ectoderm and the notochord: evidence for sclerotomeinduction by Sonic hedgehogVhh-1. Cell 1994, 79:1175-1186.

21. Porter JA, von Kessler DP, Ekker SC, Young KE, Lee JJ, Moses K,Beachy PA: The product of hedgehog autoproteolytic cleavageactive in local and long-range signalling. Nature 1995, 374:363-366.

22. Tashiro S, Michiue T, Higashijima S, Zenno S, Ishimaru S, TakahashiF, et al.: Structure and expression of hedgehog, a Drosophilasegment-polarity gene required for cell-cell communication. Gene1993, 124:183-189.

23. Ingham PW, Forbes Z: Microinjection into Drosophila Embryos.Essential Developmental Biology: A Practical Approach. Edited byStern CD, Holland PWH. Oxford: IRL Press; 1993.

Received: 15 March 1995; revised: 11 April 1995.Accepted: 11 April 1995.