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Malvolio, the Drosophila homologue of mouse NRAMP-1 (Bcg), is expressed in

macrophages and in the nervous system and is required for normal taste

behaviour

Article  in  The EMBO Journal · August 1995

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The EMBO Journal vol.14 no.13 pp.3007-3020, 1995

malvolio, the Drosophila homologue of mouseNRAMP-1 (Bcg), is expressed in macrophages and inthe nervous system and is required for normal tastebehaviour

Veronica Rodrigues1l2, Peh Yean Cheah,Krishanu Ray and William Chia

Institute for Molecular and Cell Biology, National University ofSingapore, 10, Kent Ridge Crescent, Singapore 0511'Present address: Molecular Biology Unit, Tata Institute ofFundamental Research, Homi Bhabha Rd, Bombay 400 005, India

2Corresponding author

We report the sequence, expression pattern and mutantphenotype of malvolio (mvl), the Drosophila homologueof mammalian natural resistance-associated macro-phage proteins (NRAMPs). In the mouse, this noveltransporter is encoded by Bcg, a dominant gene thatconfers natural resistance to intracellular parasites.mvl was identified in a screen for mutants that affecttaste behaviour. We show that loss-of-function as wellas insertional mutants in mvl display defects in tastebehaviour with no alterations in the physiology of thesensory neurons. Activity of the reporter enzymei-galactosidase, that reflects the expression patternof mvl, is seen in mature sensory neurons and inmacrophages. The conceptual translation of themvl cDNA shows a striking similarity (65% identity)with human NRAMP with almost complete identity ina conserved consensus motif found in a number ofATP-coupled transporters. Based on its phenotype andexpression pattern as well as its structural similaritiesto NRAMPs and a nitrate transporter in Aspergillusnidulans, we discuss a possible role for MVL in nitrite/nitrate transport and its implications.Key words: Drosophila/enhancer-traps/macrophages/taste/transporters

IntroductionAttempts to understand brain function are complicated bythe remarkable anatomical complexity of cell types andconnections even in relatively simple systems like thefruitfly Drosophila melanogaster. Over a million neuronsare present in the adult fly and they express severalthousand mRNA molecules, some at very low levels(Palazzolo et al., 1989; Jacobson, 1991; Truman et al.,1993). Mutagenesis screens for genes that are required forthe development of the nervous system have yieldedimportant results on the mechanisms that operate in thechoice of cell fate in the developing Drosophila eye(Greenwald and Rubin, 1992) and nervous system(Campos-Ortega, 1993; Ghysen et al., 1993; Jan and Jan,1994). However, screens that are based on visual inspectionof a region of the nervous system have several limitationsthat have been well recognized. First, null mutations ingenes that also have vital functions earlier than the stageof interest will be missed. Second, anatomical resolution

by visual inspection is limited by the tools available. Thus,many loss-of-function mutations result in lethality of theanimal but with no obviously identifiable phenotype exceptdeath (e.g. Bieber et al., 1989). Third, and in directcontrast to the above, several cases of loss-of-functionmutations are now known in which the animal is viable andwhere no phenotype is seen upon anatomical inspection(Elkins et al., 1990).

Behavioural genetic approaches to the study of thenervous system address some of the problems discussedabove and complement more common approaches to thestudy of development and function in the nervous system(Hall, 1985). Behaviour is a sensitive index of nervoussystem function (Benzer, 1973). The response of anorganism to its environment involves detection of stimuliby peripheral receptors, integration of the afferent informa-tion by the central nervous system and, finally, a motorresponse. Even relatively subtle defects in any of thesesteps are amplified and can be detected as an alteredbehavioural response. Behavioural defects in animals withnull or hypomorphic mutations in genes expressed in thenervous system thus allow the function and developmentof neural circuits to be addressed in complex situations(Tully et al., 1990; Ferrus and Canal, 1994; Hall,1994).We have used the gustatory pathway of the fly as a

system for the assay of gene function in the nervous system.Adult Drosophila demonstrate stereotyped responses toa number of chemical stimuli which can be assayed insimple gustatory paradigms (Rodrigues and Siddiqi, 1978;Tanimura et al., 1982). These assays, which measure theability of flies to detect and respond to sugars and salts,have been used to isolate mutations in more than a dozengenes affecting taste perception (Isono and Kikuchi, 1973;Falk and Atidia, 1975; Rodrigues and Siddiqi, 1978;Tompkins et al., 1979; Tanimura et al., 1982; Morea,1985; VijayRaghavan et al., 1992; Inamdar et al., 1993).It is clear that screens using behaviour as a phenotypewill yield hypomorphic alleles of genes playing moregeneral roles in development or function of the nervoussystem (Campbell et al., 1992; Murugasu-Oei et al., 1995)and other tissues. Potent strategies exist which allow theanalysis of the cellular function of genes in specificneuronal pathways using genetic mosaics (Xu andRubin, 1993).

In addition to the development of powerful behaviouralscreens, studies from several groups have resulted in amolecular, genetic, anatomical and physiological analysisof development and function of the taste hairs of the adultfly. The adult taste organs are bristles located on theproboscis, legs and wings (Stocker, 1994). The bristlestructure is made up of three support cells and eachorgan is innervated by one mechanosensory andfour chemosensory neurons (Nayak and Singh, 1983).

X Oxford University Press 3007

V.Rodrigues et al.

These bipolar neurons project in the labial nerve to thesub-oesophageal ganglion of the brain (Nayak and Singh,1985; Shanbhag and Singh, 1992). All the eight cellswhich comprise a single taste organ are derived from asingle sensory progenitor cell in the labial disc duringpupation (Ray et al., 1993). The four chemosensoryneurons perform distinct functional roles-one respondingto sugars, two to salts and one to water (Rodrigues andSiddiqi, 1978; Fujishiro et al., 1984). The mechanism bywhich individual cells acquire differential specificities isnot yet understood.

Studies on the nature of the molecular receptors involvedin detection of gustatory stimuli have lagged far behindthose on the multigene receptors families which have beenshown to be involved in olfaction (Buck and Axel,1991). Signal transduction in gustatory neurons, on theother hand, appears to occur by mechanisms comparableto those involved in visual system and in olfaction(Gilbertson, 1993). The functional complexity of theprocessing tasks of the central nervous system is muchmore difficult to decipher. Models which describe mechan-isms of integration in the nervous system have beeninferred by studying the behaviour and physiologicalproperties of mutants with lesions in different steps in thetaste circuits (Siddiqi et al., 1989; Balakrishnan andRodrigues, 1991).

Here we describe the isolation of a taste mutant which,like the character Malvolio in Shakespeare's 'TwelfthNight', 'taste(d) with distempered appetite'. The genehas been cloned and sequenced and found to encode atransmembrane molecule with a striking homology to aclass of transporters termed natural resistance-associatedmacrophage proteins (NRAMPs). The expression of mvlin Drosophila macrophages, as well as fully differentiatedcells of the nervous system, is discussed with referenceto its chemosensory phenotypes.

Resultsmvy adults are defective in perception of sugarsand other attractive stimuliThe Mv1971 mutation was isolated in a screen for dominantmutants defective in sugar perception. Heterozygotes aswell as homozygotes show a reduced preference for severalsugars and an increased acceptance of low concentrationsof sodium chloride (Figure 1). Chromosomal in situhybridization revealed the presence of a single P(w'-lacZ)insertion at 93B6 on the right arm of the third chromosome(data not shown). We verified that the behavioural defectsin mvl mutants were caused by the insertion of the Pelement, by selecting for transposase-mediated excisionof the P element based on a reversion of the w+ eyecolour marker. Homozygous excision lines were tested fortheir behavioural responses in the feeding preference assay.All 12 lines which behaved normally were shown to beprecise excisions within the limits of Southern analysis(data not shown).Homozygous Mv1971 flies (Figure 1 a-d), as well as six

new alleles generated by imprecise excision (see below),show a shift in the threshold of acceptance of sucrose,fructose and trehalose. These aberrations in behaviourwere observed using two different gustatory paradigms-the feeding preference test and the proboscis extension

test (Figure la, b; see Materials and methods). Higherconcentrations of stimulus are required to elicit a maximalbehavioural response. In addition, mutant animals showan increased preference for low concentrations of sodiumchloride compared to controls (Figure le).The behavioural tests employed measure the ability of

a stimulus to evoke an acceptance response from the fly.Defects in behaviour could, in principle, arise from alesion at any step in the gustatory pathway from stimulusdetection to execution of the motor response. Electro-physiological recordings from the labellar taste bristlesallow us to monitor the function of the sensory neuron.Stimulation of the bristle with sugars elicits neuronalactivity from the 'sugar neuron' which fires with a typicalspike in a concentration-dependent manner (Fujishiroet al., 1984). Data summarized in Figure If shows thatthe sugar neuron is physiologically unaltered in mvlmutants. The behavioural aberration could therefore beexplained by defects at a 'higher' level of the neuralpathway involving integration or processing of gustatoryinformation.The insertion mutation Mv197f shows a partial dominance

in taste behaviour; the homozygous strain shows a sig-nificantly stronger phenotype than the heterozygote(P < 0.001) (Table I). The phenotype of Mv197f in transwith Df(3R)eRl (93B3-5;93D2-4) which uncover the locusis not significantly stronger than that of the homozygote(P > 0.5) (Table I; Figure 2). If the insertion allele is anull as this data could suggest, than a single wild-typecopy of the locus as in Df(3R)eRl/+ would be expectedto show a mutant phenotype in gustatory tests.Df(3R)eRl/+ heterozygotes show normal responses tosugars (Table I) and to other taste stimuli (not shown).These data, together with the fact that Northern analysisrevealed the presence of the mvl transcript in Mv1971animals, rule out the possibility that the insertion leads toa null phenotype.

Since the nature of the Mv197f mutation is unclear, wegenerated additional alleles in order to ascertain thephenotype associated with mvl loss-of-function. Severalnew alleles were obtained by imprecise excision of the Pelement in Mv197f. These strains showed adult-specificgustatory defects and could be placed in an allelic serieswith respect to the severity of phenotype. In Mv15M andMvl12 there were deletions within the transposon leavingboth P element ends and the flanking genomic regionunaffected. These strains behaved like the original insertionstrain in their dominance as well as the expressivity ofthe defect (Table I). The second group of alleles, mvl8,mVdJ2, mvl23M and mvlA-46 are fully recessive. The pheno-types of mvl8 and mv1J2, as homozygotes, or in trans witha deficiency for the region, are not significantly differentfrom the insertion strain. In both these strains, most ofthe transposon has been excised leaving an -50 bp insertionin each case (not shown). Both mvl23M and mvlA-46 areweak hypomorphic alleles; the phenotype is significantlyenhanced when placed in trans with deficienciesuncovering the mvl locus. mvl23M did not reveal any changein the genomic region compared to the wild-type bySouthern analysis. In mvlA46 -800 bp was deleted on bothsides of the insertion site; the EcoRI site just upstream tothe translational start site is, however, not affected, leavingthe coding region intact (Figure 2). The deletion extends

3008

The mvy locus is required for normal taste behaviour

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Fig. 1. Behavioural and electrophysiological responses of Mv197f adults to gustatory stimuli. Wild-type (WT) in the figures refers to the response ofthe Canton-S strain (CS). The mvl mutant strains were isogenized to the CS genotype. Strains bearing a precise excision of the P element were

comparable to that of CS. (a) Proboscis extension test. Flies responding at least three times in five trials were taken as responders. At least 50 flieswere tested for each data point. (b-e) Feeding preference assay using sucrose (b), fructose (c) and trehalose (d) as stimuli in the uncoloured wells ofthe plate. The rest of the wells contained food dye but no stimulus. The number of uncoloured flies in the population gave a value of the percent

acceptance (e). Each data point gives the mean and standard deviation of at least 10 experiments. (f) Electrophysiological recordings of the response

of labellar taste hairs to sucrose. Individual taste bristles were stimulated and the number of spikes within a 450 ms interval starting 50 ms afterinitiation of the response were counted. Each point represents the mean and standard deviation from at least 200 bristles (20 flies). The student t test

was carried out between values from WT and Mvl97f recordings and the difference was found to be insignificant (P >0.1).

further 5' of the region to which the full-length cDNAhybridizes, but it is not clear whether it does delete thetranscriptional start site.

In mv1ml3, an imprecise excision of the P element, themvl transcription unit as well as -15 kb of 3' flankingsequence is deleted. mv1r'3 homozygotes as well as mvlml3/Df(3R)eRl embryos die late in embryogenesis. We didnot detect any obvious morphological changes in theseembryos using nervous system markers (mAb22C10) or

by examination using optical microscopy. While we cannotexclude the possibility that the lethality in mv1l13 homo-zygotes is due to a closely linked embryonic lethal, our

data does show that even total loss of mvl+ function doesnot result in any recognizable defects in development ofthe embryo.The behavioural and genetic data show that both

recessive loss-of-function as well as partial dominantmutations of mvl have defects in taste behaviour in adultflies. We have ruled out the possibility of effects due togenetic background by using isogenized strains; in thebehavioural analyses, revertant lines with 'precise'excisions were used as wild-type controls. The strongestmutant combinations that we have generated still leave a

residual (-30%) response to sugars.

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Table I. Behavioural responses of mvl alleles to 1 mM sucrose in the feeding preference test

Genotype Homozygote Strain/+ Strain/Df(3R)e-RI Strain/Insertion allele

Ex-l (WT) 78.5 ± 2.4 - 75.3 ± 5.5 54.9 + 11.4Mv197f 32.4 ± 5.8 55.9 + 11.4 27.2 ± 7.5 -

Mvl5M 31.0 ± 6.2 49.9 ± 8.4 30.0 ± 3.9 38.1 ± 6.3Mv112 29.4 ± 6.3 53.9 + 8.9 33.3 + 5.6 N.D.mvl8 20.9 ± 9.3 87.5 + 5.4 30.3 + 4.5 29.6 + 6.7mvlJ2 38.4 ± 9.7 86.5 ± 6.6 29.5 + 6.6 35.9 + 3.6mV123M 48.3 ± 10.6 89.4 ± 5.5 30.03 + 5.7 45.9 + 9.9mlVA-46 a 88.2 + 7.9 37.8 + 5.1 43.9 + 7.1mlvml3 lethal 86.9 + 7.1 lethal 33.3 + 9.1

Each value represents the mean and standard deviation of at least 10 independent experiments. Analysis on Southems showed that Ex- is a strain inwhich the transposon had excised precisely. In all behavioural assays carried out, Ex- 1 is comparable to Canton-S.aA closely linked lethal mutation was present on the mlvA-46 chromosome, hence the response of homozygotes could not be tested. The lethality wasnot uncovered by Df(3R)e-RI and therefore maps away from the mvl locus.N.D. not done.

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Fig. 2. Genomic organization of the mvl region. The proximal breakpoint of Df(3R)e (93B3-5) was located within the -30 kb genomic region aroundmvl and the deletion extends distally. The distal breakpoint of the mvlml3 lies within the P element and the deletion extends proximally. The mvjA-46deletion removes -800 bp around the insertion site. The genomic organization was deduced from hybridization analysis using fragments from threeoverlapping cDNA clones. (H, HindIlI; E, EcoRI; C, Sacl; S, Sall; B, BamHI; X, XhoI; R, EcoRV).

Df(3R)e-Rl¶breakpoint

Genomic organization of the mvy regionThe genomic DNA flanking 3' to the P element wasrescued and used as a probe to obtain genomic clonescovering -40 kb in the region of the insertion site (Figure2). The orientation of the clones on the chromosome wasassigned relative to the proximal breakpoint of Df(3R)e-RIat 93B3-5. The HindIII-SalI fragment spanning the inser-tion site recognized a single -3 kb transcript on Northernblots (Figure 3). Using this fragment as a probe, severalcDNA clones were obtained from two independentlygenerated libraries (Poole et al., 1985; Brown and Kafatos,1988). Analysis using several overlapping cDNA clonessuggested that the mvl mRNA is transcribed from withina 10 kb genomic region. The location of the transcriptionunit with respect to the genomic DNA and the mappedbreakpoints of some mutant alleles are shown in Figure2. The P element in the Mv1971 strain is inserted 313 bp

5' to the translation initiation site in the transcribed butuntranslated region of the transcription unit.

Expression of the mvl transcript was assessed byNorthern hybridization using poly(A)+ RNA from differentdevelopmental stages. mvl mRNA is detected duringembryonic stages and is down-regulated during the firstand second larval instars. Transcript levels rise againduring the third larval instar and are maintained duringpupation and adulthood (Figure 3). A maternal mvl tran-script of comparable size is also detected (not shown).

MVL is an integral membrane protein highlyhomologous to the natural resistance-associatedmacrophage protein (NRAMP)Sequence analysis of mvl cDNA clones revealed an openreading frame of 1457 bp. The first in-frame ATG waspreceded by stop codons in all three reading frames.

3010

The mvy locus is required for normal taste behaviour

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Fig. 3. Developmental profile of mvl transcripts. 3 jig of poly(A)+RNA from different developmental stages was loaded per lane. Arrowindicates the -3 kb mvl transcript. The bottom panel shows the filterstained with methylene blue showing approximately equal loading ineach lane.

to lie on the fifth cytoplasmic face (C5) of the membraneand mediates interaction between ATP binding subunitsof these transporters serving a role in energy coupling.The structure shown in Figure 5c is proposed based on

the consensus with NRAMP molecules and the hydropathyplot shown in Figure 4b (M.Cellier and P.Gros, personalcommunication). The 10 transmembrane (TM) domainswere chosen using a minimum of 16 hydrophobic residues;Arg and Lys residues were placed within the cytoplasm.The amino-terminal domain was placed within the cyto-plasm, based on its hydrophilic nature and by analogywith other members of this family (Vidal et al., 1993).The loop (P4) between TM7 and TM8 is highly hydrophilicand contains a single N-linked glycosylation site. Thesignature sequence discussed above is in the cytoplasmicloop C5. Three putative protein kinase C (PKC) phos-phorylation sites were located within the proposed cyto-plasmic domains of the MVL sequence using PROSITEanalysis (Bairoch, 1991); two of these sites lie within thefirst cytoplasmic domain (Cl) and the other lies withinC4 (Figure 5c). None of these sites are conserved withthe human or mouse homologue, although h-NRAMPdoes possess a PKC phosphorylation site in the firstcytoplasmic domain.

Several cDNA clones, as well as the correspondinggenomic region, have been sequenced to verify the pro-posed stop codon. Conceptual translation revealed a poly-peptide of 486 amino acids with an approximate molecularweight of 53 300 daltons (Figure 4). About 50% of theprotein is composed of the highly hydrophobic aminoacids leucine (10.3%), alanine (9.1%), valine (7.8%),isoleucine (7.2%), phenylalanine (6.0%) and the apolaramino acid glycine (9.1%). The charged amino acidslysine, aspartic and glutamic acids together comprise11.9% of the residues. Hydropathy analysis using Kyteand Doolittle algorithms (1982) suggests that MVL iscomposed of a minimum of eight (maximum 10) highlyhydrophobic stretches with mimimal charge density(Figure 4b). This implies that the protein is an integralmembrane-spanning molecule (see later). The amino-terminal domain does not possess any classical signalsequences.

Comparision of the MVL polypeptide with sequencesin the databases using BLAST programmes revealed astriking degree of homology with mouse NRAMP-1 (64%identity; 78% similarity L13732) and human NRAMP(65% identity, 79% similarity; L32185). The alignment ofMVL and the human NRAMP (h-NRAMP; Cellier et al.,1994) is shown in Figure 5a. A sequence motif known asthe 'binding protein-dependent transport system innermembrane component signature' (aa 396-415) in MVL isnearly identical to that of h-NRAMP with a single aminoacid difference. Figure Sb shows the alignment of themotif from some eukaryotic transporters. There are severalconserved residues between MVL and the Aspergillusnitrate transporter crnA (Figure Sb; Unkles et al., 1991).This motif was originally identified in the intracellularloops of the membrane anchors of bacterial periplasmicpermeases (Dassa and Hofnung, 1985; Kerppola andAmes, 1992) and has also been described in severaleukaryotic transporters (Vidal et al., 1993). In all thesemolecules, including MVL, this motif has been proposed

mvy is expressed in the nervous system and inmacrophagesThe P(w+-lacZ) transposon is inserted 313 bp upstreamof the translational start site within the transcribed, non-translated region of the mvl transcription unit (Figure 2).The tissue specificity of the reporter enzyme is thereforelikely to reflect the native expression of the mvl locus. Totest this assumption, we examined the tissue distributionof mvl transcripts using single-stranded cDNA probeslabelled with digoxygenin (Tautz and Pfeifle, 1989). Thepattern of mvl transcripts and P-galactosidase coincidewith each other in the stages that we have examined (datanot shown). The signals obtained in the RNA in situ weregenerally weaker and more diffuse than the f-galactosidasepattern observed in the Mv197f strain. Therefore, in thispaper, we describe the expression pattern of mvl duringdevelopment by following P-galactosidase localization inthe enhancer-trap allele (Figures 6 and 7). Our resultsshow that mvl is expressed in the peripheral and centralnervous system as well as in some of the cells of thehaematopoietic system, notably the macrophages.

In the adult brain, a large number of nuclei in thecellular cortex express the reporter enzyme ,B-galactosidase(Figure 6a). In addition to these neurons, several stainednuclei lying superficial to the ganglia are also stained(Figure 6b). When examined at higher resolution, thesecells were found to be large, multi-lobed, and looselyattached to the sheaths surrounding the ganglia (notshown). These cells, which by morphological criteriaappear to be cells of the blood or haematopoietic system,are associated with different tissue types throughout thefly (Figure 6c; Gateff, 1978).The sensory structures on the third segment of the

antenna (Figure 6d), the proboscis (Figure 6e) and themaxillary palps (Figure 6f, g) all express ,B-galactosidasein the enhancer-trap allele mv197f. Closer examination, inthick frozen sections, revealed that most of the stainednuclei correspond to neurons innervating these structures

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3012

The mvy locus is required for normal taste behaviour

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Fig. 5. (a) Alignment of MVL with h-NRAMP. The two proteins are 65% identical and 79% similar. Identical residues are connected by verticalsand similar residues are indicated by dots. (b) The binding protein-dependent inner membrane component signature motifs from the NRAMPs frommouse and humans and MVL are compared with CRNA-the nitrate transporter from Aspergillus. (c) The proposed structure of MVL. C(1-6) andP(1-5) indicate the putative cytoplasmic and extracellular domains respectively. The amino acid stretches comprising the transmembrane domains(TM) are indicated. The binding protein-dependent inner membrane component signature (arrowhead) in C5 is indicated. The 'Y-like' structureindicates a putative glycosylation site.

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The mvy locus is required for normal taste behaviour

of the developing antenna (arrow in Figure 6h), proboscis(not shown) and leg discs (Figure 6j). Observation usingNomarski optics revealed that these cells were associatedwith tissue debris within the disc lumen. Stained cells canalso be observed within the wing veins in the pupal wingat 40 h after pupation (Figure 6i).The identity of the large phagocytic cells observed

within the pupal imaginal discs and in the adult wasestablished by comparison with the macrophage-specificline P197 (Abrams et al., 1992). P197 contains a singleP(w+-lacZ) insertion on the second chromosome andhas been verified by histological analysis to expressspecifically in macrophages. We stained adults and pupaefrom this strain with the chromogenic substrate for P-galactosidase, X-gal. In the adult brain, superficial cellsassociated with the ganglia, very similar in morphologyand position to those shown in Figure 6b and c for mv197f,were observed. The large cells associated with the pupaldiscs, described above, were also stained in P197. Thewing disc from P197 is shown in Figure 6k for comparisonwith that from mvl97f in Figure 6i. In addition to the discs,stained macrophages were associated with developingmusculature (Figure 61) and attached to the basal surfaceof the epidermis in both Mv197f (Figure 6m and n) andP197 (Figure 6o).

These observations established that some of the stainedcells observed in the mvl enhancer-trap line were macro-phages. We further confirmed this by staining primarycultures of Mv1971 embryos with X-gal (Shields and Sang,1970). Macrophages can be easily recognized as largeflattened cells with a prominent nucleolus in 3-day-oldcultures. These cells were found to express ,-galactosidaseat a high level (data not shown).

In the embryo, expression of mvl, as detected bywholemount RNA in situ hybridization as well as reporterenzyme activity, occurs in several tissues although at verylow levels. Staining in the enhancer-trap line was firstdetected during germband extension in cells surroundingthe tracheal pits (arrowheads in Figure 7a) as well as inthe procephalic lobe (arrow, Figure 7b; Campos-Ortegaand Hartenstein, 1985). During germband retraction, nucleilying along the posterior border of the segment boundarieswere stained (thin arrows, Figure 7c). A large number ofcells within the amnioserosa are strongly stained through-out development and are visible in two rows during dorsalclosure (arrows, Figure 7d). The most prominent neuronalexpression is in the cells of the antenno-maxillary complex(Figure 7e, f). The cells shown in Figure 7f were estab-lished to be sensory neurons by comparison withmAb22C10-stained preparations at a similar develop-mental stage. mAb22C10 labels most neurons in the

peripheral as well as the central nervous system throughoutdevelopment (Fujita et al., 1982). In addition, a numberof non-neuronal cells in the vicinity of the antenno-maxillary complex express reporter activity (Figure ig)A few neurons in the cerebral hemisphere were alsostained (Figure 7e).The reporter gene in Mv197f provides evidence for the

expression of mvl+ in mature neurons in the central as wellas peripheral nervous system of the embryo and the adult.mvl is also expressed in macrophages which are associatedwith several structures throughout development.

DiscussionThe mvl locus was identified in a mutagenesis screendesigned to identify genes whose function is required fornormal taste behaviour. Behavioural analysis of mvl allelesshows that both loss-of-function and gain-of-functionmutations at the locus affect taste perception in adult flies.Electrophysiological responses of the peripheral neuronsto taste stimuli are apparently normal, suggesting that thelowered sensitivity of response is due to defects at thelevel of information processing rather than reception ofthe stimulus. The appearance of reporter enzyme activityin fully differentiated neurons argues for a direct role ofmvl+ in the functioning of gustatory circuits in Drosophila.On the other hand, mvl is also expressed in macrophagesthroughout development; defective macrophage functionin mutants could, in principle, lead to defects in thedevelopment of the taste circuitry (Truman, 1990; Abramset al., 1992; Steller and Grether, 1994; White et al., 1994).We did not notice any changes in the morphology of thegustatory centres of the brain of mvl mutants by examina-tion of stained sections at optical microscopic resolution(unpublished). Thus, we favour the view that the changesin behavioural acuity in mutant flies are attributable todefects in function of the gustatory pathway.

MVL belongs to a novel family of transporters thatmay transport nitrite/nitrate ionsmvl encodes a molecule with a striking similarity to afamily of novel transporters expressed in the mammalianimmune system, called NRAMP. NRAMP is a small familyof closely related genes with two members identified inmice, rats and humans and three members in swine andbirds (Gruenheid et al., 1995; P.Gros personal communica-tion). These molecules all possess a minimum of 10 andpossibly 12 transmembrane domains. We have proposeda structure for MVL with 10 transmembrane domains toaccommodate the homology with NRAMPs. This structurehas yet to be verified by biochemical analysis. The

Fig. 6. Reporter enzyme activity in Mv197f adults and pupae. (a) Adult brain wholemount showing staining in the cellular cortex (cc) in of the mid-brain and optic lobes. (b) Superficially located cells on the sheaths covering the optic ganglia. (c) Stained cells with large nuclei and severalprocesses are seen throughout the body. At higher resolution, the stained cells in (b) resemble those in c (see text). (d) Adult antenna from Mv197fshowing strong expression of ,-galactosidase in the olfactory organs in the third antennal segment (III). The second antennal segment (II) is notstained. (e) Thick section through the proboscis stained with the chromogenic reagent X-gal. The cell bodies of the neurons innervating the taste

organs (long arrow) as well some of the non-neuronal support cells (short arrow) are stained. Several cells within the pseudotrachea are also stained(arrowheads). (f) and (g) show staining in the cells below the bristles on the maxillary palp. Neurons below the sensory bristles (arrows in g) as wellas support cells can be distinguished. Scale bar in (a) and (b) = 100 ,um; (d) = 50 gm; (c), (e), (f) and (g) = 20 gm. Imaginal discs from animals42 h after pupation showing macrophages associated with developing structures; (h) antenna; (i) wing and (j) leg (long arrows). A wing disc fromthe macrophage-specific line P197 is shown for comparison (k). The sensory structures which are fully developed at this time are not stained eitherin Mv197f or in P197. (1) Developing musculature in Mv197f pupae showing aggregation of stained macrophages at the muscle attachment sites.(m) and (n) show individual macrophages attached to the basal side of the epidermis in the Mv197f adults and pupae. Stained cells of similarmorphology are seen in the macrophage-specific line P197. Scale bar (h), (i), (k), (1) = 25 ,um; (j) = 100 gm; (m), (n), (o) = 20 ,um.

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f

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Fig. 7. Expression pattern in Mv197f embryos visualized using an antibody against bacterial P-galactosidase. Staging is according to Campos-Ortegaand Hartenstein (1985). (a and b) Dorsal and lateral views respectively of a stage 10 embryo showing expression around the tracheal pits(arrowheads) and in the procephalic lobe (arrow). (c) Lateral views of stage 13 embryos showing staining in cells located on the posterior border ofeach segment. (d and e) Dorsal and ventral views of an early stage 16 embryo. Staining is seen in two rows of cells at the amnioserosa (arrow d),the cerebral hemispheres and in the antenno-maxillary complex (amx). This region is shown at higher magnification in (f). (g) is at a lower focalplane; several non-neuronal cells which are closely associated with the amx (arrow) are strongly stained. Scale bar (a-e) = 100 jum; (f, g) = 25 ,um.

NRAMP transporters are typified by the presence of aconserved 'binding protein-dependent inner membranecomponent signature' domain (Dassa and Hoffnung, 1985;Kerppola and Ames, 1992). MVL and the human andmouse NRAMPs differ in a single residue in this 20 aminoacid signature domain. The putative structure of NRAMPand MVL closely resembles that of the Aspergillus trans-porter CRNA which has been shown to play a role innitrate import (Unkles et al., 1991). Vidal et al. (1993)have proposed a role for NRAMP in nitrite and/or nitratetransport based on the close structural similarity withCRNA. Reactive nitrogen intermediates, in particular nitricoxide (NO) and its nitrite (NO2-) and nitrate (NO3-)derivatives play crucial roles in cytostatic and cytolyticactivity of macrophages (Lowenstein and Synder, 1992;Nathan and Xie, 1994). The radical NO is released fromthe guanidino group of arginine by the enzyme nitric oxidesynthetase. NO can diffuse freely through membranes buthas a short half-life and is readily converted to NO2- andNO3- ions in the presence of 02 and water; NO2- can beconverted back to NO by dismutation at low pH (Stuehrand Nathan, 1989). We speculate that while NO is freelydiffusible, the transport of NO2- and NO3- radicals acrosscell membranes may be facilitated by an energy-coupledtransporter. A transporter for NO2- or NO3- could haveimportant functional roles in modulation of NO levels and,in the case of macrophages, control of cytolytic action.

The expression of mvy in macrophagesdemonstrates conservation of molecules in similarcellular contextsGenetic analysis on the locus encoding the mouseNRAMP-1 (Bcg) is consistent with a role in the cytolysisof intracellular parasites (Vidal et al., 1993). Susceptibilityto mycobacterial infection in resistant and sensitive strainsof mice was associated with single amino acid changeswithin the NRAMP molecule (Malo et al., 1993). Further,mouse knock-outs null for the Bcg locus are hypersensitiveto parasitic infections (P.Gros, personal communication).NRAMP- 1 is an integral membrane protein expressedexclusively in the macrophage populations from thereticuloendothelial system. The finding that DrosophilaMVL, like its mammalian counterpart, is expressed inmacrophages suggests an amazing conservation of cellularfunction, in species separated by 540 million years ofevolution (Hultmark, 1993). Analysis of the Drosophiladorsal-related immunity factor and its relationship tothe NF-KB system suggests that mammalian and insectimmunity share a common ancestry (Ip et al., 1993).Insect macrophages perform tasks very similar to thoseof macrophages in the mammalian immune system, viz.scavenger receptor-mediated endocytosis, wound healing,pathogenesis and phagocytosis (Abrams et al., 1992). InDrosophila, as well as in the mouse, macrophages havebeen shown to play important roles in phagocytosis of

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The mvy locus is required for normal taste behaviour

apoptotic cells during embryonic and pupal development(Abrams et al., 1993; Lang and Bishop, 1993). Onepossibility is that the conservation of molecules usedin comparable cellular functions could imply a closerevolutionary conservation between these species. In thisscenario, these phagocytic cell types arise from a commonancestor with a strict conservation of important molecularentities. Alternatively, macrophages could have arisenseveral times during evolution but may have co-optedsimilar molecules for their function. A striking recentexample of a similar situation is seen from studies on thenatural history of the eye. The mammalian gene Pax-6encodes a transcription factor essential for the initial stagesof eye development, and mutants in mouse and humanslead to dramatic defects in which the eye fails to form(Hill and Hanson, 1992). Recently, Quiring et al. (1994)have shown that the Drosophila gene eyeless, encodes aprotein similar to Pax-6, and mutations in this gene too,lead to early and fundamental defects in compound eyedevelopment. Other examples of molecular conservationthat go together with roles in similar tissues include theDrosophila gene tinman and the mouse gene Csx, bothencoding homeodomain proteins which play roles in thedevelopment of the heart (Azpiazu and Frasch, 1993;Komuro and Izsumo, 1993). Such examples provideimportant insights into the development of organs and celltypes during evolution and the mechanisms that operateto allow the use of similar molecules in functionallysimilar cells or tissues in evolutionarily distant organisms.

The role of mvy in the nervous system raises thepossibility of its linkage to nitric oxide signallingThe second Nramp gene in mice, Nramp-2, is expressedubiquitously in all tissues including the nervous system(Gruenheid et al., 1995). In this respect, mvl resemblesNramp-2, although it has a somewhat more restrictedexpression mainly in the central and peripheral nervoussystem. mvl is expressed in the labellar chemosensoryneurons system, suggesting that its function in theseneurons is required for normal taste behaviour. However,electrophysiological studies have shown that the primaryevents in stimulus detection and signal transduction areunaffected and the behavioural defect is likely to occurbecause of a lesion at a more central level. One way inwhich these two results can be reconciled is if MVLfunctions at the integrative synapse between the sensoryneuron and its post-synaptic partner in the sub-oesophagealganglion. This is a readily testable prediction which awaitsfurther analysis.

There has been, to our knowledge, no demonstratedrole for nitrate or nitrite transport in neuronal physiology.NO, on the other hand, has been shown to play importanttransmitter and modulator roles in the peripheral andcentral nervous system (Kennedy, 1992). Since NO isfully diffusible, it can carry regulatory information frompost-synaptic sites to pre-synaptic terminals during induc-tion of long-term potentiation (Schuman and Madison,1991; Zhuo et al., 1993). Intake ofNO into target terminalsleads to increased cGMP levels through activation of asoluble guanylyl cyclase. Short- as well as long-termchanges of neuronal physiology can be attributed to theaction of cGMP-dependent protein kinases. In the retinaand the olfactory system of vertebrates, NO levels are

implicated as global neuromodulators (DeVries and Baylor,1993). It is not clear how the metabolites of NO turnover,NO2- and NO3-, are prevented from accumulating at siteswhere NO acts as a second messenger. One hypothesisis that these radicals are transported into subcellularcompartments for subsequent removal, or transported outof the cells in which they are produced. An energy-dependent transporter like MVL could play a role in thisclearing mechanism. In this model, mutations in mvlwould lead to accumulation of NO2- and NO3- radicals andconsequent defects in integration of sensory information atthe synapses.The amenable genetics and molecular biology of Droso-

phila should allow us to test our hypothesis on the roleof MVL in macrophage and neuronal function. Theavailability of dominant as well as recessive alleles of mvlwith easily assayable phenotypes allows the exploitationof powerful enhancer and suppressor screens to identifyother molecules participating with MVL in the transportprocess. These studies could shed light on a cellularprocess which has important implications in differentcellular contexts and in systems with less tractablegenetics.

Materials and methodsAll standard molecular biology procedures were performed according toSambrook et al. (1989). Protocols for Drosophila work were as describedin Ashburner (1989).

Drosophila stocks and librariesThe P element strain C(1)RM(w+)8 with eight immobile P elements onthe compound X chromosome was obtained from Bruce Hamilton(Palazollo et al., 1989). The strain Cy/Sp; (A2-3 ry+)ry Sb/TM6-Ubx contained a constitutively expressing transposase insertion at 99B(Robertson et al., 1988). The deletion strain Df(3R)e Rl (93B3-5;93D2-4)and balancer strains was obtained from the Bloomington Stock Center,Indiana. Details of markers and other strains are available in Lindsleyand Zimm (1992).

Stage-specific cDNA libraries in plasmid or in kgtlO were obtainedfrom Nick Brown (Brown and Kafatos, 1988) and Steve Poole (Pooleet al., 1985) respectively. Genomic libraries in EMBL3 were a kind giftfrom John Tamkun (Tamkun et al., 1992). The neuron-specific antibodymAb22C10 was kindly provided by the Benzer laboratory (Fujitaet al., 1982).

Isolation of P element insertion mutants defective in tastebehaviourJumpstarter males from the cross between C(l)RM(w+)8 and Cy/Sp;(A2-3ry+)ry Sb/TM6-Ubx were mass mated to wiw females. Maleswhich were w+ and therefore carried an insertion chromosome wereselected and screened in a feeding preference test (see below) using1 mM sucrose as a stimulus. At this concentration, -85% of flies eatfrom the stimulus-carrying wells; flies which failed in the test were re-screened. Isogenic lines were bred from those animals which failed intwo consecutive trials.

Excision of the P elementIn order to generate revertants as well as new alleles of mvl by excisionof the P element, we crossed the insertion strain to a line bearing thetransposase source. Excision of the P element was scored by followingthe loss of the eye colour marker w+. Lines were maintained by crossingto a balancer strain TM3Sb/TM6-Tb. Homozygous lines were tested inthe feeding preference assay and by Southern blotting. Lines in whichthe excision of the P element resulted in lethality were maintainedover balancers

Behavioural assaysFeeding preference test. The feeding preference test described byTanimura et al. (1982) was carried out with some modifications(Rodrigues et al., 1986). Alternate wells of a 6x 10 microtitre plate were

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filled with 1% agar containing the stimulus. The rest of the wellscontained 0.2% carmoisine red in agar. Control experiments establishedthat the food dye did not interfere with the test. Flies were starved inhumid conditions for 18 h prior to the test . Approximately 100 flieswere introduced into each test plate and left for I h in the dark. Followingthe test, flies were immobilized by cooling and scored by visualinspection for the colour in their abdomens. The acceptance response ofthe stimulus was calculated from the percentage of flies with uncolouredabdomens in the population. Means and standard deviations of each datapoint were obtained from at least 10 independent readings. The insertionstrain as well as the imprecise excision strains were isogenized beforebeing subjected to behaviour tests. When mutant strains were beingcharacterized, strains with precise excisions were taken as the wild-typecontrols in order to normalize for effects due to genetic background.Proboscis extension test. Two to 4-day-old flies were starved in moistchambers for 15 h prior to the test, immobilized by cooling, and fixedventral side up on a microscope slide using myristic acid wax (m.p.=58.5°C). After a recovery period of 3 h, flies were allowed to drinkdeionized water until satiated. The tarsus of the first leg was touchedwith a drop of the stimulus from a micro-syringe needle and the extensionof the proboscis scored. Each fly was given five trials and taken as aresponder if it extended its proboscis in at least three trials. When theresponse of mutants was being evaluated, the wild-type was tested inparallel under identical conditions.

Tip recording technique for electrophysiological analysisElectrophysiological recording from the labellar taste bristles was carriedout as described previously (Rodrigues and Siddiqi, 1978; Fujishiroet al., 1984). A 3-4-day-old fly was immobilized by cooling on ice and'threaded' through a tapered glass capillary. The protruding head wasimmobilized with myristic acid wax and the grounding Ag/AgCl electrodein a saline-filled microcapillary was impaled into the thorax of the fly.The recording electrode was connected to a high impedance pre-amplifierand signals were displayed on a storage oscilloscope. This electrode wasintroduced into a tapered glass capillary which also contained thestimulus. The tip of each taste hair on the proboscis was touched withthe recording electrode three times in succession. Spike trains generatedwere analysed in a 450 ms interval beginning 50 ms after the initiationof the response.

In situ hybridization to polytene chromosomesHybridization to salivary gland polytene chromosomes was carried outusing DNA probes labelled with biotinylated 16-dUTP. The procedurewas carried out as described in Ashburner (1989) and the signals werevisualized using horseradish peroxidase (HRP)-labelled antibodies.

Isolation of Mv! genomic and cDNA clonesDNA flanking the insertion site of the P element was rescued as describedin Ashburner (1989). The 'plasmid-rescued fragment' was used to screena wild-type library in EMBL3 (Tamkun et al., 1992). The genomicclones obtained were used as probes on Northern blots and to screenembryonic cDNA libraries (Poole et al., 1985; Brown and Kafatos, 1988).

Northern and Southern blot analysisGenomic DNA was prepared from adult flies and Southern hybridizationswere carried out as described by Sambrook et al. (1989). Total RNAswere prepared from different developmental stages and poly(A)+ RNAwas prepared using standard methods. Three gg of poly(A)+ fromeach developmental stage was separated on formaldehyde/agarose gels,transferred to nylon membranes and hybridized with labelled probes.Probes were labelled with [32P]dATP by random priming reactions.

DNA sequencingThe cDNA clones were subcloned into the single-stranded M13 vectorand sequenced using U.S. Biochemical Co. Sequenase kit (Version 2).Genomic cDNA was sequenced using oligonucleotide primers based onthe cDNA sequence. Protein homology searches were carried out usingthe data bases at the National Center of Biotechnology Information,National Institutes of Health (NIH) using BLASTp and BLASTnprogrammes.

Wholemount embryo RNA in situ hybridizationsLocalization of RNA in wholemount embryos was carried out essentiallyas described by Tautz and Pfeifle (1989) with modifications as describedby M.Mlodzik and N.Patel (unpublished). Single-stranded probes wereprepared by the polymerase chain reaction using digoxygenin-labelled

11-dUTP. The labelled plus strand was used as the control for background.Hybridized probe was detected using antibodies to digoxygenin coupledto alkaline phosphatase.

Visualization of P-galactosidase expression using activitystainingDissected preparations were fixed in 0.25% glutaraldehyde in phosphate-buffered saline (PBS) for 10-15 min at room temperature. They werewashed in PBS containing 0.3% Triton X-100 (PTX) and incubatedovernight at 37°C in X-gal staining solution (Simon et al., 1985). Stainedsamples were mounted in glycerol and examined in an Axiophotmicroscope with DIC optics.

Immunohistochemical staining of embryosEmbryos were fixed and immunostained according to Mitchinson andSedat (1983) with modifications by White and Gould (personal com-munication). Briefly, embryos were fixed in paraformaldehyde-heptaneand washed extensively in PBS containing 0.1% Triton X-100 (PBT).They were blocked for 1 h with 1% bovine serum albumin in PBT. Theywere incubated overnight at 4°C in 1:10 000 dilution of an antibodyagainst bacterial 3-galactosidase (Boehringer, Mannheim). Embryos werewashed in PBT and incubated with 1:200 dilution of biotinylatedsecondary antibody (Vector Labs). The reaction of the biotinylatedantibodies was detected using streptavidin-coupled HRP and then wasvisualized by staining for enzyme activity.

Stained embryos were mounted in glycerol and examined in a ZeissAxiophot using Normaski optics. Staging of the embryos was accordingto Campos-Ortega and Hartenstein (1985).

Accession numberThe sequence mvl has been deposited in the GenBank data library underaccession number U23948.

AcknowledgementsWe are indebted to Phillipe Gros and his colleagues for discussions andfor sharing their unpublished data with us. We thank Nick Brown, StevePoole and John Tamkun for Drosophila libraries; Chris Tate for help withthe structural predictions; Mani Ramaswami for advice on macrophageidentification and for valuable suggestions; K.VijayRaghavan for hisinterest and advice and K.S.Krishnan for comments on the manuscript.Marie Wong helped in several of the experiments and Oon Swee Huatand Bernie Murugasu-Oei helped us learn the intricacies of the computer.We gratefully acknowledge the members of the Chia laboratory for thecongenial scientific atmosphere and Jane Chia for christening the geneMalvolio. This work was supported by grants from the RockefellerFoundation (V.R. and B.C.) and the European Community (B.C.) as wellas core funding from the Institute of Molecular and Cell Biology.

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Received on February 6, 1995; revised on March 31, 1995

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