Chem. Senses 26: 195-199,
2001
© Oxford University Press 2001
SYMPOSIUM: AChemS XXII Symposium |
The Invertebrate Odorant-binding Protein LUSH is required for Normal Olfactory Behavior in Drosophila
Department of Pharmacology and Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA
Correspondence to be sent to: Dean P. Smith, Department of Pharmacology and Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-9111, USA
Abstract
The invertebrate odorant-binding proteins consist of a large family of low-molecular-weight, highly divergent proteins expressed exclusively in the chemosensory sensilla of insects. Each member of this family studied to date is secreted into the sensillum lymph of a small subset of sensilla by non-neuronal support cells. These expression patterns suggests an odor-specific function for these proteins as opposed to a general role in sensillum biology. Consistent with this notion, mutants defective for LUSH, a Drosophila member of this family, have odor-specific defects in olfactory behavior. The Drosophila genome contains at least 32 members of this gene family, rivaling the number of odorant receptors in this species. The relationship between these two protein families and how they act to determine odor specificity of olfactory neurons will be the topic of future studies.
The molecular basis of olfactory detection and information processing is beginning to be understood. In vertebrate animals, millions of olfactory neurons are located in the nasal epithelium, overlaid by a thin layer of mucus. Odor specificity of vertebrate olfactory neurons appears to be determined by the odorant receptor expressed in the neuron (Buck and Axel, 1991
; Zhou et al., 1998; Malnic et al., 1999). Each olfactory neuron expresses a single one of the 1000 or so odorant receptor genes and all neurons expressing the same receptor gene project their axons to the same glomerulus in the olfactory bulb (Vassar et al., 1994; Sullivan et al., 1995). The sensation of ‘odor’ is thought to result from the brain interpreting the pattern of glomerular activity in the bulb, which corresponds to the array of receptors activated at the periphery.
Non-neuronal support cells secrete a small number of vertebrate odorant-binding proteins (v-OBPs) into the mucus. The function of these proteins in olfaction is unknown. However, many odorants are hydrophobic molecules that have low aqueous solubility. Binding experiments show that v-OBPs bind odorants with a diverse array of molecular structures (Pelosi et al., 1982; Pevsner et al., 1985, 1990). These proteins are thought to increase the ability of hydrophobic molecules to enter the mucus layer [reviewed by Pelosi (Pelosi, 1995)]. Consistent with this notion, v-OBPs are members of the lipocalin family of hydrophobic molecule transporters that include transport molecules like the retinol-binding protein (Flower, 1996).
By contrast, the invertebrate odorant-binding proteins (i-OBPs) in insects are an independent gene family. Indeed, X-ray crystal structure data from the two types of OBPs reveal no structural relationship. v-OBPs bind odorants at the interface of a dimer (Bianchet et al., 1996) while the i-OBPs bind ligand as monomers (Sandler et al., 2000). Like the v-OBPs, the function of the i-OBPs are not known, but we have identified a mutant defective for a Drosophila i-OBP member. This mutant has odor-specific defects in olfactory behavior, implying a role for these proteins in olfactory discrimination and behavior.
The anatomy of the insect peripheral olfactory system is distinct from vertebrate and nematode olfactory model systems. In Drosophila, the 2000 or so olfactory neurons reside within segregated compartments called sensilla (Figure 1). Three morphologic classes of sensillum are present on the Drosophila antenna, including basiconic, trichoid and coeloconic sensilla [reviewed by Stocker (Stocker, 1994)]. All three classes detect odorants (Siddiqi, 1987; Clyne et al., 1997), but the significance of the morphological differences is not known. Each sensillum is a hollow, hair-like structure filled with fluid called sensillum lymph that bathes the dendrites of the olfactory neurons contained within it. Each sensillum contains the dendrites of between one and four olfactory neurons. Different sensilla have different odor specificities (Siddiqi, 1987; Clyne et al., 1997). While the recently discovered odor receptor gene products expressed in the olfactory neurons are likely to be major determinants of odor specificity for olfactory neurons in Drosophila (Clyne et al., 1999; Gao and Chess, 1999; Vosshall et al., 1999), there may be additional factors that modulate chemical specificity. Specifically, the segregation of neurons into sensilla provides insects with the opportunity to independently regulate components of the sensillar lymph which, in turn, regulate the concentration of odorants in the lymph, ultimately influencing the odor specificity of the neurons. The invertebrate OBPs are specifically secreted directly into the sensillum lymph. Some members are conserved across different Drosophila species (Hekmat-Scafe et al., 2000), suggesting an essential role in olfactory function. Members of this family have been shown to directly bind odorant molecules (Vogt and Riddiford, 1981; Du and Prestwich, 1995; Sandler et al., 2000). Furthermore, each member of the family is expressed in a small subset of sensilla, implying these molecules perform an odor-specific function (McKenna et al., 1994; Pikielny et al., 1994; Kim et al., 1998). What is the function of the i-OBP family?
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In Drosophila seven members of the i-OBP family have been reported, none of which are expressed in a sex-specific manner (McKenna et al., 1994
; Pikielny et al., 1994; Kim et al., 1998). Each Drosophila member is expressed in a subset of sensilla. We identified LUSH as a new Drosophila member of the i-OBP family using an enhancer trapping screen designed to identify genes expressed exclusively in the Drosophila antenna (Kim et al., 1998). Briefly, a transposable genetic element containing the LacZ gene was randomly integrated into the Drosophila genome. Several thousand lines were generated, each containing a single transposon. Expression of the LacZ gene depends on acquiring local enhancer elements at the integration site to activate transcription of LacZ. Thus, the LacZ expression should mimic the expression pattern of endogenous genes located at the transposon integration site. Each transposon line was screened for ß-galactosidase activity restricted to the antenna. One line integrated within 300 nucleotides of lush, the gene coding for LUSH.
LUSH has the hallmark features of a member of the i-OBP family, including olfactory-specific expression, a signal sequence for secretion from the non-neuronal support cells that secrete it into the lymph, and a series of six cysteines with conserved spacing (Raming et al., 1990; Vogt et al., 1991). Antiserum raised against LUSH protein confirmed that LUSH is secreted into the sensillum lymph of trichoid sensilla (Figure 2A).
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While the existence of i-OBPs has been appreciated for >20 years, we still have little insight into the role of these proteins. Mutants defective for an i-OBP were not available. Having a transposon close to the lush gene provided an opportunity to generate the first OBP mutant. To create loss-of-function mutations in lush, we generated small deletions at the transposon integration site by ‘jumping’ the transposon out of the chromosome and screening for small local deletions. One deletion we recovered eliminated 3 kb of genomic DNA flanking the P element. This lesion completely removed the lush transcription unit, but did not appear to affect any other genes. Flies homozygous for this deletion are viable and fertile, and, as expected from the deletion, make no LUSH protein (Figure 2B).
To determine if the loss of a single i-OBP results in olfactory defects, we compared olfactory discrimination between lush mutants and wild-type adults using the olfactory trap assay (Woodard et al., 1989). Briefly, 10 wild-type or mutant flies were placed in a Petri plate with a single odorant trap, and the number of flies within the trap was determined after a set time period. We screened a panel of 60 simple volatile organic compounds at different concentrations to test for differences in distribution between control and lush flies. Odorants were tested at 1:1000 and 1:4 dilutions in agarose. Comparing the responses of lush mutants with the strain carrying the transposon from which the mutants were derived minimized genetic background differences. These flies are expected to be genetically identical except for the presence of the transposon in the controls (which does not disrupt lush expression) and the lack of the lush gene in the experimental group. (Different strains of flies have dramatically different olfactory behavioral responses, making comparisons between hybrid strains uninterpretable.) Table 1 shows some of these data. As expected from the restricted expression pattern of LUSH in a subset of sensilla, the majority of the compounds attract similar proportions of wild-type and lush mutant flies, indicating that there is no global olfactory defect associated with the loss of the lush gene. However, odor-specific defects in chemosensory behavior are observed in lush mutant flies when challenged with three chemically related odors. We observed a significant increase in the number of mutant flies in traps containing high concentrations of ethanol, propanol and butanol compared with control flies. Responses to a variety of other alcohols are not different to the wild type. Interestingly, the apparent increased attraction of lush flies for ethanol, propanol and butanol is specific to high odorant concentrations (Figure 3A). Figure 3B reveals the dose-dependent, abnormal attraction of lush mutants for ethanol. lush flies are attracted to yeast extract, ethyl acetate and low concentrations of ethanol to a similar extent as the wild type. However, the mutant flies display an abnormal attraction to traps containing high concentrations of ethanol (Figure 3B, 1:100, 1:4). We named this deletion mutant ‘lush’ to reflect their increased affinity for ethanol-rich environments. We conclude that lush mutants have odor-specific defects in chemosensory behavior and are abnormally attracted to high concentrations of a subset of odorants, including ethanol, propanol and butanol.
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The increased likelihood of lush mutant flies to enter traps containing high concentrations of these alcohols could result either from increased attraction to these odorants or from a defect in avoidance of high concentrations of these compounds. If there is a defect in chemoavoidance to ethanol in lush mutants, we should be able to demonstrate this behavioral response in wild-type flies. To determine this, we tested the effects of mixing ethanol with yeast extract, a strong chemoattractant. Figure 3C shows that wild-type flies are attracted to dilute yeast extract (left panel, open bars). However, when the same amount of yeast is mixed with 25% ethanol, wild-type flies are significantly less likely to enter these traps (compare open bars). Therefore, the presence of high levels of ethanol reduces the attractiveness of the yeast for wild-type flies. This demonstrates that there is an active avoidance mechanism in wild-type flies that is stimulated by high concentrations of ethanol. lush mutants are equally attracted to yeast as wild-type flies (filled bar, left graph) but are defective for the avoidance behavioral response (Figure 3C, filled bars). In fact the lush mutants are significantly more attracted to the mixture of yeast and concentrated ethanol than to yeast alone. These results are consistent with the notion that neurons within the sensilla expressing LUSH mediate avoidance to concentrated alcohols and that LUSH is important for the activity of these neurons. However, alternative models are also consistent with these data. For example, the LUSH-positive sensilla may contain olfactory neurons that mediate attraction to alcohol, and LUSH normally functions to remove alcohol from the lymph when the levels get very high.
To prove that the chemosensory defects we observed in the lush mutants are due entirely and specifically to loss of LUSH protein in the trichoid sensilla, we introduced a cloned wild-type copy of this gene under control of its own promoter into lush mutant flies by germline transformation. Expression of a lush transgene under control of its own promoter in the mutant background restores LUSH expression to normal levels (Figure 2B, Rescue). Furthermore, the transgene completely restores wild-type olfactory behavioral responses to the lush mutants (Figures 3B, striped bars). Therefore, the abnormal chemoattraction of lush mutants to high levels of alcohol results specifically from loss of LUSH protein in a small subset of trichoid sensilla. We conclude that lush mutants have defective chemosensory responses to a subset of odorants resulting from loss of a single odorant-binding protein in the sensillum lymph of a small subset of trichoid chemosensory sensilla.
Future prospects
There are 
60 odorant receptors in the Drosophila genome (Vosshall et al., 2000). We have scanned the Drosophila genome for candidate odorant-binding proteins and have identified 25 new potential members of this family, making the total number of potential Drosophila odorant-binding proteins to 32 (D.P. Smith, unpublished data). The challenge for the future will be to determine how these molecules function at the biochemical level to influence olfactory behavior.
Acknowledgments
This work was supported by a grant from the National Institute of Health (DC02539) to D.P.S.
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Accepted October 31, 2000
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