GENETIC INTERACTIONS PROVIDEEVIDENCE FOR THE ROLE OFINTEGRINS IN SPECIFYINGNORMAL OLFACTORY BEHAVIOR INDROSOPHILA MELANOGASTER

CHAMPAKALI AYYUBand JAYASHREE PARANJAPE#

Department of Biological Sciences,Tata Institute of Fundamental Research,Homi Bhabha Road, Mumbai 400 005, India

(Received 2 October 2001; accepted 15 April 2002).

In a previous paper, we showed that weak hypomorphic alleles at themyospheroid (mys) locus, which encodes the b-subunit of integrin, pos-sess defects in olfactory behavior in both adult and larva. In this paper,we show that another olfactory gene, olfE, exhibits haploinsufficientinteractions with recessive alleles at themys locus. olfE has recently beenshown to be an allele of swisscheese and is now designated as swsolfE. Ourfindings suggest an interaction between the sws protein and b-integrin inthe development and=or functioning of the olfactory system. Similarinteractions were also observed between sws and inflated, a gene en-coding the a2-subunit of integrin, as well asmys and multiple edematouswing (mew), a gene coding for a1 subunit of integrin. This study providesevidence for the roles of different integrin subunits and the sws productin regulating normal olfactory behavior in Drosophila.

The authors thank Prof. Veronica Rodrigues for her valuable suggestions. They also

thank Dr. K. Matthew of Bloomington Stock Centre, USA, for her help with the stocks.

Address correspondence to Champakali Ayyub, Department of Biological Sciences,

Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, India.

E-mail: kali@mailhost.tifr.res.in#Present address: Department of Cancer Biology, Lerner Research Institute, Cleveland

Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.

J. Neurogenetics, 16: 165–174, 2002

Copyright # 2002 Taylor & Francis

0167-7063/02 $12.00 þ .00

DOI: 10.1080/01677060290024637

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Keywords: olfE; swisscheese; inflated; myspheroid; multiple edematouswing mutants

INTRODUCTION

Adults of Drosophila melanogaster show very sensitive and stereotypedresponses to a wide range of chemical stimuli. There are approximately1200 olfactory neurons innervating the sense organs on the antenna and120 on the maxillary palp (Venkatesh & Singh, 1984; Stocker, 1994).These neurons send projections to 43 glomeruli of the antennal lobe(Stocker, 1994; Laissue et al., 1999). Recently, about 60 putative odorantreceptors have been identified, although the function of only one of thesehas been established (Clyne et al., 1999; Vosshall et al., 1999; Wetzel et al.,2001; Storkuhl et al., 2001). Each sensory neuron expresses either one ora small number of putative olfactory receptors; neurons expressing asingle olfactory receptor project to a single glomerulus within the olfac-tory lobe (Vosshall et al., 2000). Functional experiments by 2-deoxy-glucose mapping in the fruit fly (Rodriguer & Buchner, 1984; Rodrigues,1988) and by calcium imaging in the honey bee (Joerges et al., 1997;Faber et al., 1998) have demonstrated that the olfactory glomeruli serveas functional units of odor coding. Information about the quality of achemical stimulus is processed as a spatial pattern of neural activityacross the olfactory lobes.

The neurogenetic approach aims to identify all the genes that con-tribute to normal olfactory behavior in Drosophila (Rodrigues & Siddiqi,1978). Since the olfactory behavioral assays are sensitive, they are likelyto identify genes whose hypomorphic alleles lead to subtle defects in brainmorphology or function. The behavioral screens that we employ requirethat the fly is normal in all other modalities viz, locomotion, vision,courtship, and mating. The mutations thus selected affect chemosensorybehavior somewhat selectively, although null alleles could well affectother functions.

The olfC mutations have recently been shown to be allelic to the pre-viously characterised myospheroid (mys, 7D5–6) mutants. Hence,hypomorphic alleles of mys result in defects in olfactory behavior. Thesemutants are designated mysolfC (Ayyub et al., 1990; 2000).

olfE, an olfactory gene closely linked to mysolfC, was isolated for itsreduced sensitivity to benzaldehyde at both larval and adult stages. Bygenetic and molecular mapping, it was placed between 7C9 and 7D1(Ayyub et al., 1990; Hasan, 1990). Kretzschmar et al. (1997) showed thatthe olfE þ transgene can rescue a neurodegenerative mutant swisscheese(sws), which maps at the same cytological position. The gene encodes aprotein of 1425 amino acids. This conceptual protein shows two domainswith homology to the regulatory subunits of protein kinase A and to a

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conceptual protein of yet unknown function in yeast, worm, and human.Mutants of sws show age-dependent neurodegeneration in pupa andadult. Their early death is caused by hyperwrapping of neurons by glialsheath causing neural death (Kretzschmar et al., 1997). We believe thatolfE is a weak allele of sws and henceforth refer to it as swsolfE.

It is clear that molecules such as integrins would have general roles indevelopment and function. The availability of viable alleles showingspecific behavioral phenotypes provides us with a means to identifymolecules that interact with integrins in the olfactory pathway. We ex-amined the haplo-insufficient interaction between integrin mutations andother olfactory mutants in behavioral assays. The demonstration of a‘synthetic’ phenotype created by two recessive hypomorphic allelesin trans suggests a direct interaction between the molecules in generatingnormal olfactory behavior.

MATERIALS AND METHODS

Drosophila cultures were grown on standard cornmeal-agar medium at25� 1�C, unless otherwise stated. For bahavioral tests, freshly eclosedflies were transferred into fresh culture bottles and left for 2–4 daysbefore testing. An isogenised derivative of the Canton-Special strain(Canton-S, or CS) was used as the wild-type in these studies. Flies car-rying viable mutations at mys (namely olfCX3, olfCX5, olfCX10, and

olfCX17) and at the swsolfE locus (called olfEX26) were obtained from theDrosophila Stock collection at Tata Institute of Fundamental Research,Mumbai. mys10, an allele of myospheroid (mys) and hs-mysþ transfor-mant, were obtained from D. Brower, University of Arizona at Tucson,USA. The hs-mysþ transformant strain contains two copies of a P(wþ

hsmysþ ) insertion; mys cDNA was cloned in frame with an hsp-70promoter (Bunch et al., 1992). Two alleles of inflated (if ), g2 if k27e f 36a,and g2if B2 f 36a, and an allele of mew ( yl mewM6 f 36a) were obtained fromtheDrosophila Stock Centre at Indiana University (Bloomington, Indiana,USA). All three lethal strains were maintained over a balancer, FM7.To make a heterozygote of any one of these lethal strains and an ol-factory mutant, balanced females of the former were mated with malesof the latter. From the progeny, nonbalanced females were picked forthe olfactory assay. Details of markers, balancers, and rearrangementsare available in Lindsley and Zimm (1992).

Measurement of Olfactory Behavior

The response of flies to an odor was tested using the odor-inducedjump test. This paradigm depends on the ability of flies to jump whenexposed to a strong puff of an odor. A modification of the test described

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by McKenna et al. (1989) was used (Ayyub et al., 1990). Flies wereimmobilised by cooling and a batch of five flies were placed into eachtube. When they had climbed two-thirds of the height of the tube, air wasblown through the odor into the jump tube. Normal flies jump inresponse to the stimulus and fall to the bottom of the tube; the number offlies jumping within 5 s of the onset of the stimulus was counted. Eachscore was calculated based on the response of twenty flies. In allexperiments mutants and wild-type controls were run in parallel. Eachdata point represents the mean (� SEM) of at least 10 independentexperiments.

RESULTS AND DISCUSSION

Trans-heterozygotes of mysolfC and swsolfE Are Defective inResponse to Both Benzaldehyde and Iso-amyl Acetate

Previous work had shown that the olfactory mutants at the myslocus can be divided into 2 classes—class I mutants (mysolfCX2,mysolfCX3, and mysolfCX10) are defective to both ethyl acetate andiso-amyl acetate whereas class II mutants (mysolfCX5, mysolfCX14, andmysolfCX17) show a defect to iso-amyl acetate alone. This classificationwas based on results obtained in the Y-maze test as well as the odor-induced jump assay. Similarly, larval responses measured in the larvalplate assay also allowed us to divide the alleles into two functionalclasses. Figure 1A depicts responses of four of the alleles to ethylacetate (open bar) and iso-amyl acetate (filled bar). All the mutantsshow normal responses to benzaldehyde (data not shown) and are fullyrecessive (Figure 1B).

swsolfE is an extant olfactory mutant at the sws locus. It shows normalresponses to both these acetate esters (open and filled bars), but a reducedresponse to benzaldehyde (stippled bar). This defect is also fully recessive(Figure 1C).

In order to test for genetic interactions between mys and sws,we constructed pairwise heteroallelic combinations of mys alleles andswsolfE. Adults were tested in the odor-induced jump paradigm. We haveestablished that all mys alleles, as well as swsolfE, are fully recessive; that is,they do not show any behavioral defect to acetate esters or benzaldehydeas heterozygotes with a wild-type strain. Trans-allelic combinations ofmysolfCX3 or mysolfCX10 with swsolfE show normal responses to both iso-amyl acetate and benzaldehyde. However,mysolfCX5 ormysolfCX17, in transwith swsolfE, show reduced responses to both iso-amyl acetate and ben-zaldehyde (Figure 2A). We ascertained that this defect was due to a failure

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FIGURE 1 A–C. Behavioral responses to olfactory stimuli measured in the odor-

induced jump-test. Each bar represents mean of at least 10 experiments and theerror bar indicates SEM. (A) Responses of wild-type (þ ) and mysolfC flies to iso-amyl acetate (IAA) and ethyl acetate (EA). (B) Responses to iso-amyl acetate

and ethyl acetate of all four alleles of mysolfC as heterozygotes with wild-type. (C)Responses of wild-type (þ ) and swsolfE flies to iso-amyl acetate, ethyl acetate, andbenzaldehyde (BENZ) and that of heterozygotes of wild-type and swsolfE to

benzaldehyde.

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FIGURE 2 Phenotypes of different trans-heterozygotes in the odor-induced

jump-test using iso-amyl acetate (IAA), ethyl acetate (EA) and benzaldehyde(BENZ). Each bar represents mean of at least 10 experiments and the error barindicates SEM. Responses of trans-heterozygotes of swsolfE and alleles of mysolfC

to iso-amyl acetate and benzaldehyde (A) and to ethyl acetate (B). With onlymysolfCX5 and mysolfCX17, swsolfE showed mutant responses. (C) Phenotypes oftrans-heterozygotes carrying swsolfE and mys10 to all three odourants. Theresponses were normal in all cases. (D) Responses of trans-heterozygotes of

Df(1)snc128 with mutants of swsolfE and mysolfC. The trans-heterozygotes havereduced response for only the odor the mutant is defective.

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in olfaction rather than amotor deficit since these genotypes showanormalresponse to ethyl acetate in the odor-induced jump test (Figure 2B).

It is interesting to note that only alleles belonging to class II (i.e.,mysolfCX5 and mysolfCX17) showed negative interaction with swsolfE.Members of class I (mysolfCX3 or mysolfCX10) showed normal olfactory re-sponse in transwith swsolfE. The basis of this specific interaction is not clear.

Class II alleles show recessive defects to iso-amyl acetate but not toethyl acetate. However, when placed over a deficiency (Df(1)snc128) un-covering both sws and mys loci, these alleles showed defects to both esters(Ayyub et al., 1990). mysolfCX17=Df(1)snc128 heterozygotes, on the otherhand, did not have any behavioral defect to benzaldehyde unlike my-solfCX17=swsolfE (Figure 2A). Similarly, sws and mys, in trans with

Df(1)snc128, did not show an additional defect other than those to whichthe homozygote was defective (Figure 2D).

The null allele of mys, mys10, did not show any behavioral deficitin trans with swsolfE (Figure 2C). The failure of trans-heterozygotic in-

teraction between swsolfE and null alleles of mys suggests an antimorphiccombination between mutated products of the two genes (Figure 2A).

Transgenes of sws and mys can Rescue the Interaction BetweenMutants

The olfactory defect in swsolfE can be rescued by a transgene,P(olfE þ), that carries a 14 kb genomic fragment from the 7C9; 7D1 region(Hasan, 1990). P(hs-mysþ) with a cDNA corresponding to the translatedsequence of the mys gene rescues the abnormalities in the mutants of themys locus, including the olfactory defect (Bunch et al., 1992; Ayyub et al.,

TABLE I Transgenes of swsolfE and mys in Rescue of Olfactory Phenotype

Iso-amyl acetate Benzaldehyde

swsolfE=mysolfCX17; P(olfE þ )=þ 83.0 � 0.8 86.5 � 0.8mysolfCX17=Y; P(olfE þ )=þ 45.0 � 1.2 85.0 � 1.2swsolfE=mysolfCX17; P(hsmys þ )=þ 60.4 � 4.2 47.3 � 4.9swsolfE=Y; P(hs-mys þ )=þ 60.0 � 4.3 40.4 � 4.0

Rescue of the olfactory phenotype of the trans-heterozygote carrying swsolfE

and mysolfCX17 by the P(hs-mysþ) and P(olfEþ) transgenes in the odor-inducedjump-test using iso-amyl acetate and benzaldehyde. Each data point represents

mean � SEM of at least 10 experiments. In the trans-heterozygote, the P(olfEþ)transgene restored a totally normal response whereas the P(hs-mysþ) transgenerescued the phenotype partially.

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FIGURE 3 Interaction between (A) swsolfE and two alleles of if and (B) mew anddifferent alleles of mysolfC. Responses of the trans-heterozygotes were measured inthe odor-induced jump-test using iso-amyl acetate (IAA), ehtyl acetate (EA), and

benzaldehyde (BENZ). Each bar represents mean of at least 10 experiments andthe error bar indicates SEM. swsolfE showed interaction with if only when testedagainst benzaldehyde whereas mysolfC and mew interacted for both ethyl acetate

and iso-amyl acetate.

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2000). We tested whether these transformants were equally competent torescue the interaction described above.

Results summarized in Table I indicate that P(olfE þ) is capableof ameliorating this interaction between swsolfE and mysolfC. On the otherhand, P(hs-mysþ) only partially rescues this same interaction. Over-expression of sws or mys in a mysolfC or swsolfE background did not resultin any change in behavior.

swsolfE Interacts also with Mutants of a2 Integrin, Inflated

Integrins exist as a heterodimer of a and b components. Datasummarised above suggest that swsolfE interacts with the b integrinsubunit. We tested whether this haplo-insufficient interaction could beextended to alleles of the a2 integrin product as well. a2 integrin isencoded by the gene inflated (if ); most alleles of if, including if k27e andif B2 used in our study, are lethal. Heterozygotes of swsolfE and each ofthese two alleles of if were tested for their responses to the odours iso-amyl acetate, benzaldehyde, and ethyl acetate in the odour-inducedjump assay.

Figure 3A shows that both the alleles of if are recessive for theirolfactory phenotype. However, as a heterozygote with swsolfE, each ofthem exhibits a reduced response only to benzaldehyde. A similarfinding was obtained when these flies were tested in Y-maze (data notshown). Hence, we suggest that a complex of inetgrins b, a2, and swsolfE

plays an important role in the development and=or function of theolfactory system in Drosophila.

a1 Integrin Encoded by mew also Participates in Olfaction

Figure 3B summarises the results on interaction between mew andmysolfC. mewM6 is a recessive mutant. A heterozygote of mewM6 andeither of the alleles of mysolfC showed a reduced response to both ethylacetate and iso-amyl acetate. This confirms our expectation that the a1integrin too is involved in olfaction of Drosophila.

In summary, by a combined genetic and behavioral study, we haveshown that three different integrin genes are involved in the olfactionof Drosophila. The neighbouring genes swsolfE and mysolfC interactwith each other, as well as with inflated. Moreover, mysolfC interactswith mew. We conclude that the olfactory system of Drosophila needsall three integrins as well as swsolfE to elicit a normal olfactory re-sponse.

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REFERENCES

Ayyub, C., Paranjape, J., Rodrigues, V., & Siddiqi, O. (1990). Genetics of olfactory beha-

vior in Drosophila melanogaster. J. Neurogenet. 6, 243–262.

Ayyub, C., Rodrigues, V., Hasan, G., & Siddiqi, O. (2001). Genetic analysis of olfC

demonstrates a role for the position-specific integrins in the olfactory system of Dro-

sophila melanogaster. Mol. Gen. Genet. 263, 498–504.

Bunch, T.A., Salatino, R., Engelsgjerd, M.C., Mukai, L., West, F., & Brower, D.L. (1992).

Genetic characterization of mutant alleles of myospheroid, the gene encoding b subunit of

the Drosophila PS integrins. Genetics 132, 519–528.

Clyne, P.J., Certel, S.J., de Bruyne, M., Zaslavsky, L., Johnson, W.A., & Carlson, J. (1999).

A novel family of divergent seven-transmembrane proteins: Candidate odorant receptors

in Drosophila. Neuron 22, 327–338.

Faber, T., Joerges, J., & Menzel, R. (1998). Associative learning modifies neural repre-

sentations of odours in the insect brain. Nat. Neurosci. 2, 74–78.

Hasan, G. (1990). Molecular cloning of an olfactory gene from Drosophila melanogaster.

Proc. Natl. Acad. Sci. USA 87, 9037–9041.

Joerges, J., Kuttner, A., Galizia, C.G., & Menzel, R. (1997). Representation of odours and

odour mixtures visualised in the honeybee brain. Nature 387, 285–288.

Kretzschmar, D., Hasan, G., Sharma, S., Heisenberg, M., & Benzer, S. (1997). The swiss

cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila.

J. Neurosci. 17, 7425–7432.

Laissue, P.P., Reiter, C., Hiesinger, P.R., Halter, S., Fischbach, K.F., & Stocker, R.F.

(1999). Three-dimensional reconstruction of the antennal lobe in Drosophila melanoga-

ster. J. Comp. Neurol. 405, 543–552.

Lindsley, D.L., & Zimm, G.C. (1992). The genome of Drosophila melanogaster. Academic

Press, San Diego.

McKenna, M., Monte, P., Helfand, S., Woodard, C., & Carlson, J. (1989). A simple che-

mosensory response and the isolation of acj mutants in which it is affected. Proc. Natl.

Acad. Sci. USA 86, 8118–8122.

Rodrigues, V., & Siddiqi, O. (1978). Genetic analysis of chemosensory pathway. Proc.

Indian. Acad. Sc. (B) 87B, 147–160.

Rodrigues, V., & Buchner, E. (1984). [3H]2-deoxyglucose mapping of odor-induced neuronal

activity in the antennal lobes of Drosophila melanogaster. Brain Res. 324, 374–378.

Rodrigues, V. (1988). Spatial coding of olfactory information in the antennal lobe of

Drosophila melanogaster. Brain Res. 453, 299–307.

Stocker, R.F. (1994). The organisation of the chemosensory system in Drosophila melano-

gaster: a review. Cell Tissue Res. 275, 3–26.

Storkuhl, K.F., & Kettler, R. (2001). Functional analysis of an olfactory receptor in Dro-

sophila melanogaster. Proc. Natl. Acad. Sci. 98, 9381–9385.

Venkatesh, S., & Singh, R.N. (1984). Sensilla on the third antennal segment of Drosophila

melanogaster meigen. J. Insect Morphol. Embryol. 13, 51–63.

Vosshall, L.B., Amrein, H., Morozov, P.S., Rzhetsky, A., & Axel, R. (1999). A spatial map

of olfactory receptor in the Drosophila antenna. Cell 96, 729–736.

Vosshall, L.B., Wong, A.M., & Axel, R. (2000). An olfactory receptor map in the fly brain.

Cell 102, 147–159.

Wetzel, C.H., Behrendt, H., Gisselmann, G., Storkuhl, K.F., Hovermann, B., & Hatt, H.

(2001). Functional expression and characterization of a Drosophila odorant receptor in

a heterologous cell system. Proc. Natl. Acad. Sci. 98, 9377–9385.

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