Chem. Senses 26: 215-221,
2001
© Oxford University Press 2001
SYMPOSIUM: AChemS XXII Symposium |
Functional Genomics of Odor-guided Behavior in Drosophila melanogaster
Departments of 1 Zoology and 2 Genetics, W.M. Keck Center for Behavioral Biology, Box 7617, North Carolina State University, Raleigh, NC 27695-7617, USA *Authors are listed alphabetically
Correspondence to be sent to: R.R.H. Anholt, Department of Zoology, W.M. Keck Center for Behavioral Biology, Box 7617, North Carolina State University, Raleigh, NC 27695-7617, USA
Abstract
The avoidance response to repellent odorants in Drosophila melanogaster, a response essential for survival, provides an advantageous model for studies on the genetic architecture of olfactory behavior. Transposon tagging in a highly inbred strain of flies in combination with a rapid and simple statistical behavioral assay enables the identification of not only large phenotypic effects, but also small aberrations from wild-type avoidance behavior. The recent completion of the sequence of the Drosophila genome facilitates the molecular characterization of transposon-tagged genes and correlation between gene expression and behavior in smell-impaired (smi) mutant lines. Quantitative genetic analyses of a collection of smi lines in a co-isogenic background revealed an extensive network of epistatic interactions among genes that shape the olfactory avoidance response. Candidate genes for several of these transposon-tagged smi loci implicate genes that mediate odorant recognition, including a novel odorant binding protein; signal propagation, including a voltage-gated sodium channel; and a protein containing multiple leucine rich repeats and PDZ domains likely to be involved in postsynaptic organization in the olfactory pathway. Several novel genes of unknown function have also been implicated, including a novel tyrosine-regulated protein kinase. The discovery and characterization of novel gene products that have major, hitherto unappreciated effects on olfactory behavior will provide new insights in the generation and regulation of odor-guided behavior. The identification and functional characterization of proteins encoded by smi genes that form part of the olfactory subgenome and correlation of polymorphisms in these genes with variation in odor-guided behavior in natural populations will advance our understanding of the genetic architecture of chemosensory behavior.
Introduction
Odor-guided behavior is essential for the survival and reproduction of most animals. In recent years considerable progress has been made in elucidating the molecular mechanisms that underlie odor recognition and odor-guided behavior. In vertebrates, nematodes and insects large gene families encoding G protein-coupled odorant receptors have been identified (Buck and Axel, 1991
; Ngai et al., 1993; Troemel et al., 1995; Sengupta et al., 1996; Clyne et al., 1999; Gao and Chess, 1999; Vosshall et al., 1999). A surprisingly large percentage of the genome comprises genes that mediate olfaction. In vertebrates, odorant receptor genes alone have been estimated to comprise up to 1% of the genome (Buck and Axel, 1991). In Drosophila melanogaster it has been estimated that 
4% of the genome contributes directly to the avoidance response of the repellent odorant, benzaldehyde (Anholt et al., 1996). Our studies are aimed at understanding the genetic architecture of odor-guided behavior using Drosophila as a model system.
Drosophila melanogaster as a model system
Odor-guided behavior is a complex trait that is shaped through the coordinated expression of ensembles of genes. Relatively little is known, however, about which genes are members of these ensembles and to what extent individual genes contribute to phenotypic variation in behavior. Drosophila melanogaster is an ideal model system for studying the genetic basis of complex traits. Because the generation time is short, highly inbred lines can be established readily. Thus, flies with a common genetic background can be generated allowing us to study the effects of a single locus in the absence of other segregating loci that could be contributing to the trait of interest. Furthermore, ‘balancer’ chromosomes, containing multiple overlapping inversions and a dominant visible marker, have been synthesized for the three major chromosomes. These chromosomes suppress recombinations in their homologues, enabling the cloning of entire chromosomes, construction of chromosome substitution lines and maintenance of mutant stocks that are not viable as homozygotes. Moreover, the entire Drosophila genome has recently been sequenced, which will greatly facilitate the identification of novel genes important for olfaction (Adams et al., 2000; Rubin, 2000).
In addition, the olfactory system of D. melanogaster, which consists of the third antennal segments and the maxillary palps, is quantitatively less complex than its vertebrate counterpart. Each third antennal segment contains only 1000 olfactory receptor cells and each maxillary palp carries 120 chemosensory neurons [reviewed by Stocker (Stocker, 1994)]. These neurons project to 43 morphologically identified glomeruli in the antennal lobe (Laissue et al., 1999; Gao et al., 2000; Vosshall et al., 2000). This represents an 40-fold reduction in complexity compared with the 1800 glomeruli in the mouse olfactory bulb (Pomeroy et al., 1990). Similarly, whereas the mouse odorant receptor repertoire has been estimated at 1000 genes (Buck and Axel, 1991), 60 putative odorant receptors have been identified in the Drosophila genome (Rubin, 2000). Its suitability for genetic studies and the relative simplicity of its olfactory system make D. melanogaster an ideal model organism for studies on the functional genomics of odor-guided behavior.
Quantifying odor-guided behavior
Early studies on olfaction in Drosophila identified mutants using a variety of behavioral assays, such as the chemosensory jump assay (Helfand et al., 1989; McKenna et al., 1989), Y- or T-maze assays (Rodrigues and Siddiqi, 1978; Ayyub et al., 1990) and the olfactory trap assay (Woodard et al., 1989). We developed a simple, rapid and highly reproducible ‘dipstick’ assay to quantify avoidance responses to the repellent odorant, benzaldehyde (Anholt et al., 1996). Single sex groups of five individuals are placed in test vials without food for 2 h. The test vials are divided into three compartments by placing marks on the wall 3 and 6 cm from the bottom of the vial. The odorant is introduced into the top compartment of the vial (mostly occupied by the plug) as an aqueous solution on a cotton wool swab, the tip of which is aligned with the 6 cm mark (Figure 1). The number of flies migrating to the compartment remote from the odor source is measured at 5 s intervals, from 15 to 60 s after introduction of the odor source (Figure 1). The ‘avoidance score’ of the replicate is the average of these 10 counts, giving a possible range of avoidance scores between 0 (maximal attraction to the odor source) and 5 (all flies are in the compartment away from the odor source for the entire assay period, i.e. a maximal repellent response). The response to distilled water is used as a control. Many replicate assays can be done for each line to obtain average values with reduced standard deviations. The elimination of genetic variation through the use of an isogenic genetic background together with our ability to rapidly accumulate large data sets for each line through repeated measurements provides us with the statistical power to reproducibly resolve not only large phenotypic effects, but also small smell impairments.
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The genetic architecture of olfactory behavior in D. melanogaster
To assess variation in olfactory behavior in a natural population, 43 X chromosomes and 35 third chromosomes were extracted from a natural population and substituted into a common inbred background (Mackay et al., 1996) (Figure 2). Measurements of avoidance responses to benzaldehyde demonstrated significant genetic variation in avoidance scores. Surprisingly, the genetic correlations between the sexes for olfactory avoidance behavior were extremely low, suggesting that different genes contribute to variation in avoidance scores in males and females (Mackay et al., 1996) (Figure 2). This may reflect the fact that odor-guided behavior serves different functions in males and females, e.g. only females select or reject oviposition sites. The lack of genetic correlation of avoidance scores between the sexes has profound evolutionary implications. Because the genetic architecture for odor-guided behavior appears sexually dimorphic, it is clear that the olfactory subgenome in males and females evolves along different evolutionary trajectories. This sex x genotype interaction facilitates the maintenance of genetic variation for olfactory behavior, since no single genotype can satisfy optimal fitness requirements for both sexes. Maintaining variation in the trait could ensure survival of the species under the diverse environmental conditions encountered over evolutionary time. Transposon tagging olfactory genes in a controlled genetic background represents one strategy that can be used to systematically dissect the complex genomic architecture that underlies odor-guided behavior.
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P-element insertional mutagenesis and epistasis
A comprehensive understanding of the genetic architecture of odor-guided behavior will ultimately require the identification of all the genes that contribute to this trait and characterization of their interactions. One strategy that can, in principle, accomplish this daunting task is the use of P-transposable-element insertional mutagenesis, which enables phenotypic effects to be linked directly to gene expression (Cooley et al., 1988; Bellen et al., 1989). The introduction of a transposon in the genome of D. melanogaster can result in gene disruption at or near the site of insertion of the transposable element. Introduction of a reporter gene in genetically engineered transposable element constructs (e.g. P[lArB]), which can be driven by promoter/ enhancer elements near the insertion site, can reveal expression patterns of the affected gene (‘enhancer trap’). Furthermore, the use of a cloning vector, such as pBluescript, in the construct can facilitate cloning of flanking sequences adjacent to the site of P-element insertion.
We generated 379 isogenic P[lArB]-element insert lines (188 are on the second chromosome and 191 on the third chromosome) and screened them for aberrant olfactory responsiveness to benzaldehyde (Anholt et al., 1996). Fourteen P-element insert lines were identified that showed a statistically significant reduction in avoidance response to benzaldehyde. Of those lines, four were sexually dimorphic in their responses to benzaldehyde, with females being more severely affected than males (Anholt et al., 1996). P[lArB] insertion sites were mapped to specific cytological band positions by in situ hybridizations to polytene chromosomes of larval salivary glands. These P[lArB]-tagged mutant lines were designated ‘smell-impaired’ (smi) and each line was named according to its cytological band location (e.g. smi21F). Of the 14 smi lines originally identified, 12 are amenable to further characterization.
Having available a collection of smi lines in a common isogenic background enabled us to investigate whether these genes interact in shaping odor-guided behavior. One method for identifying and ordering genes in functionally interacting groups is to study the enhancer/suppressor effects of one gene on the function or expression of another (García-Bellido, 1981; Botas et al., 1982; Kennison and Russell, 1987; Dambly-Chaudière et al., 1988). We crossed all 12 homozygous smi lines in a half-diallel design to generate all 66 possible double heterozygotes and quantified their behavioral responses. The common genetic background of the smi lines enabled us to separate average heterozygous effects from epistatic interactions. Significant epistatic interactions were found for nine of the transheterozygous lines, involving 10 of the 12 smi loci (Fedorowicz et al., 1998). Interactions among eight of these loci can be represented as an interaction diagram (Figure 3). Thus, these experiments demonstrate that the olfactory subgenome is characterized by a network of genes that display extensive epistasis.
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Molecular characterization of smi genes
After identifying this set of smi lines and demonstrating epistatic interactions among them, the next challenge is to characterize each smi locus indicated in Figure 3 at the molecular level. Since the P[lArB] construct contains the pBluescript cloning vector, DNA sequences adjacent to the P-element insertion site can be rapidly cloned and sequenced. Such sequence information can then be used to identify genes in the vicinity of the P[lArB] insertion site that may be responsible for the smell impaired phenotype (Table 1). To be able to conclude that altered expression of a candidate gene is indeed responsible for the observed smell impairment requires extensive further experimentation. First, the P-element can be excised and phenotypic revertants can be generated to demonstrate that the P[lArB] insertion, rather than an unrelated mutation, is responsible for the smell-impaired phenotype. Secondly, avoidance responses in flies carrying different alleles in the candidate gene must be evaluated, and complementation tests between the original P[lArB] insertion line and flies that contain deficiencies or other P-element insertions in the region of interest must be performed to provide additional genetic evidence that the candidate gene is indeed associated with the smell-impaired phenotype. Thirdly, it is necessary to demonstrate that expression of the candidate gene is reduced in mutant flies as compared with wild-type or phenotypic revertants. Fourthly, in situ hybridization can be used to show that expression patterns in wild-type flies resemble lacZ reporter gene expression patterns in the smi mutant. Finally, definitive proof that disruption of the candidate smi gene is indeed responsible for the smell impaired phenotype can be obtained by demonstrating that introduction of the wild-type gene into the mutant background rescues the mutant phenotype.
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Candidate smi genes include a wide spectrum of structural and regulatory gene products that may contribute both to the development of the olfactory system and to processes that mediate recognition, processing and integration of olfactory information. Deficits in odorant receptors are likely to generate olfactory impairments specific for certain odorants and the magnitude of such impairment will depend on the redundancy of recognition of the odorant by other receptors. The diversity and restricted size of the Drosophila odorant receptor family suggests that this system has less redundancy in odorant recognition than its mammalian counterpart. The best documented example of a specific olfactory deficit due to absence of an odorant receptor has been described for Caenorhabditis elegans, where a null mutation in the odr-10 gene, which encodes an odorant receptor for diacetyl, results in impaired chemotaxis of mutant nematodes to diacetyl (Sengupta et al., 1996
).
Whereas disruption of genes encoding odorant binding proteins and odorant receptors will result specifically in chemosensory impairments, gene products that mediate signal transduction, transfer of the signal from the periphery to the central nervous system, signal integration and the generation of a behavioral response are likely to be pleiotropic. For example, mutations in the retinal degeneration B (rdgB) gene, which encodes a phosphatidyl inositol transfer protein, and in the norpA gene, which encodes a phospholipase C, result in both visual and olfactory impairments (Smith et al., 1991; Woodard et al., 1992; Vihtelic et al., 1993; Riesgo-Escovar et al., 1994, 1995).
Candidate genes for previously identified smi lines are listed in Table 1. Preliminary characterization of several of these genes implicate a novel putative odorant binding protein (smi21F; note its central position in the epistasis diagram in Figure 3); a voltage-gated sodium channel (smi60E); Scribble, a gene that encodes a protein containing multiple leucine rich repeats and PDZ domains (smi97B; Bilder and Perrimon, 2000); and a novel dual-specificity tyrosine-regulated protein kinase, dyrk2 (smi35A). Ultimately, a complete understanding of the genetic architecture of odor-guided behavior requires characterization of all the genes involved and epistatic interactions among them, as well as understanding to what extent polymorphisms in each gene contribute to variation in olfactory behavior in nature. Rapid technological advances in functional genomics are bringing the realization of this ambitious goal within reach.
Acknowledgments
Work on odor-guided behavior in our laboratories is supported by NIH grants GM59469, GM45344 and GM45146 and by the W.M. Keck Foundation. J.J.F. and S.M.R. are recipients of W.M. Keck postdoctoral fellowships. I.G. is a W.M. Keck predoctoral fellow. J.J.F. is the recipient of a CONICET fellowship from Argentina. S.M.R. is the recipient of an NRSA award (GM/DC20897) from the National Institutes of Health. This is a publication from the W.M. Keck Center for Behavioral Biology at North Carolina State University.
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Accepted October 31, 2000
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