Characterization of Drosophila odorant receptors

Animals can detect a vast array of odors with remarkable sensitivity and discrimination. Olfactory information is first received by olfactory receptor neurons (ORNs), which transmit signals into the CNS where they are processed; this ultimately leads to behavioral responses. Electrophysiological and anatomical studies suggest that there are on the order of 36 classes of ORNs in the adult fly (30 on the antenna and 6 on the maxillary palp), each class with a distinct odor sensitivity. Classes of ORNs found in the antenna are arrayed in zones, while the classes of ORNs found in the maxillary palp are distributed in a less ordered fashion. ORNs in both the maxillary palp and the antenna extend their axons to the antennal lobe of the brain, where first-order processing of olfactory information occurs. The lobe contains 40 olfactory glomeruli, spheroidal modules where ORN axons converge and where their terminal branches form synapses with the dendrites of their target interneurons (Clyne, 1999a and references).

What is the molecular basis for the distinct odor sensitivities of the different classes of ORNs? One possibility is that each class of ORN expresses a unique odorant receptor, as has been proposed for vertebrate olfactory systems (Buck, 1996). In mammals a combinatorial code is used to discriminate between odors. In mammals, any one odorant receptor recognizes multiple odorants and one odorant is recognized by multiple ORs, but different odorants are recognized by different combinations of ORs (Malnic, 1999). Another set of receptors in mammals, associated with ORNs of the vomeronasal organ, distinguishes pheromones (Matsunami, 1997). A second possibility is that each class of ORN might express a unique combination of a large set of receptors, as found in chemosensory cells of the nematode, C. elegans (Troemel, 1995). ORNs have been found to express up to four ORs each in C. elegans. Both models call for a family of receptor genes, and several lines of evidence suggest that for insects such a family would belong to the superfamily of seven-transmembrane G protein-coupled receptors (GPCRs). A genomics approach was taken to identify the odorant receptors in Drosophila. This approach, involving a search through known genome sequences for GPCRs, is arguably the best example of the power of genomics when applied to the discovery of a family of proteins. Sixteen genes have been discovered using computer programs that identify diagnostic features of the protein structure of the seven-transmembrane GPCR superfamily (Clyne, 1999a). A second studied discovered eleven GPCR proteins coding for ORNs (Vosshall, 1999). Members of this new family are highly divergent from previously defined genes. Nearly all of the genes are found to be expressed in one or both of the olfactory organs, and for a number of genes this expression is restricted to a subset of ORNs. Expression of different genes is initiated at different times during the development of the adult antenna (Clyne, 1999a), and expression of a subset of these candidate receptor genes depends on the POU-domain transcription factor, Acj6, also known as Ipou (Clyne, 1999b).

A two-part strategy was adopted to identify odorant receptor genes from the genomic database. Initially, a computer algorithm was designed to search the Drosophila genomic sequence for open reading frames (ORFs) from candidate odorant receptor genes. Reverse transcription polymerase chain reaction (RT-PCR) was then used to see if transcripts from any of these ORFs were expressed in olfactory organs. For the computational screens, the genomic sequence data obtained by FTP from the Berkeley Drosophila Genome Project was used. ORFs of 300 bases or longer in all six frames were identified. Next, a program written to identify GPCRs statistically by their physicochemical profile was used to screen for candidate ORFs. The number of possible candidates was reduced by comparing them to Drosophila codon usage tables. Further analysis revealed that 8 of the 34 candidate ORFs correspond to genes of known function, as for example, a cyclic nucleotide-gated channel. RT-PCR with primers designed from two of the final candidates yielded amplification products from antennal cDNA. These two genes are located within 500 bp of each other at cytological position 22A (see Odorant receptor 22a and Odorant receptor 22b), and their predicted proteins are 75% identical at the amino acid level (Clyne, 1999a).

To determine if these two candidates were part of a larger family of genes encoding seven transmembrane domain proteins, their sequences were used in BLAST searches of the Drosophila genome database to identify related genes. Homologs of the two candidates were found, and in turn, their sequences were used for further database searches. In total, 16 genes have been identified from the 16% genomic sequence currently available. This family of genes has been named DOR (for Drosophila olfactory receptor), and each individual gene is named based on its cytogenetic location in the genome. Thus, the two genes identified initially are Odorant receptor 22a and Odorant receptor 22b, which are abbreviated here as 22A.1 and 22A.2 (The final digit in this nomenclature is used to distinguish the genes at a site and does not refer to the cytogenetic band number). Of the 16 family members, 13 have been found to be expressed in either the antenna or the maxillary palp, or in both, based upon RT-PCR analysis and in situ hybridizations to RNA in tissue sections (Clyne, 1999a).

The DOR genes have no significant similarities to any known genes, and do not appear in any of the Drosophila EST databases. However, hydropathy plots of the predicted proteins show that each has approximately seven peaks that could represent transmembrane domains. The lengths of the 16 proteins are between 369 and 403 amino acids, similar to the lengths of most previously described families of GPCRs. In addition, the spacing of the putative transmembrane domains gives rise to predicted intracellular and extracellular loops similar in size to those in many families of GPCRs. Amino acid sequence identity among the DOR genes ranges from 10%-75%, with many genes showing a relatively low level of identity to each other (20%). Two pairs of clustered genes, 22A.1/22A.2 and 33B.1/33B.2 show the highest identity, with 75% and 57% identities, respectively. However, not all clustered genes show high degrees of similarity. 33B.3, for example, is only 28% identical to both 33B.1 and 33B.2, and 46F.1 and 46F.2 are only 29% identical. In addition to exhibiting sequence identity, many of the genes contain introns in corresponding locations, consistent with their constituting a family derived from a common ancestral gene. There are 67 residues that are conserved among at least 50% of the genes, and most of these are in the C-terminal halves of the proteins. Among the conserved residues are a serine and a threonine in the intracellular C-terminal tail, residues frequently conserved in this region of GPCRs. The most divergent region in the sequences is a stretch of 30 amino acids representing part of the first extracellular loop and nearly all of transmembrane domain 3. The divergence in this region also occurs in the most conserved pairs of genes: 22A.1 and 22A.2 are 75% identical overall but only 50% identical in this region, and 33B.1 and 33B.2 are 57% identical overall but only 33% identical in this region. Transmembrane domains 3, 4, and 5 are exceptionally divergent in rat odorant receptors (Clyne, 1999a) and have been proposed to play a role in odorant binding (Buck, 1991). Some of the genes are clustered in the genome, while others are apparently isolated. Within a cluster, the average intergenic distance is on the order of 500 bp. Clustered DOR genes do not necessarily have introns in corresponding locations (e.g., 46F.1 and 46F.2), but all clustered genes have their transcriptional orientations in the same direction (Clyne, 1999a).

To determine whether any of the DOR genes have closely related homologs, coding regions from nine of the genes were used to probe Southern blots of Drosophila genomic DNA at high or reduced stringency. Each probe appeared to detect only its own sequence at high stringency, while at lower stringency most genes detected one or two novel bands. As expected, because of the overall low level of similarity, none of these extra bands corresponded to any of the other known DOR genes. These data indicate that some of these genes have one or two closely related homologs but that none belongs to a large subfamily of highly related genes (Clyne, 1999a).

Although, at present, the clusters of DOR genes identified in Drosophila contain smaller numbers of genes (three or fewer) than in other organisms (Troemel, 1995; Sullivan, 1996; Barth, 1996), a number of interesting features of the clustered genes are already apparent. As found in other organisms (Barth, 1996), Drosophila odorant receptor genes within a cluster are not necessarily coordinately regulated, such that genes within a cluster are expressed in different subsets of cells, and even in different olfactory organs (e.g., 46F.1 is expressed in the maxillary palp, whereas 46F.2 is expressed in the antenna). So far, all genes identified within a cluster, however, are transcribed in the same orientation. Genes within a cluster sometimes do, but sometimes do not, share intron positions, suggesting that introns may have become lost following gene duplication; a phylogenetic study revealed extensive gene duplication and intron loss among the chemoreceptor genes of C. elegans (Robertson, 1998).

Functional analysis of odorant receptors

A functional analysis was undertaken of the odorant receptor repertoire in the Drosophila antenna. Each receptor was expressed in a mutant olfactory receptor neuron (ORN) used as a 'decoder,' and the odor response spectrum conferred by the receptor was determined in vivo by electrophysiological recordings. The spectra of these receptors were then matched to those of defined ORNs to establish a receptor-to-neuron map. In addition to the odor response spectrum, the receptors dictate the signaling mode, i.e., excitation or inhibition, and the response dynamics of the neuron. An individual receptor can mediate both excitatory and inhibitory responses to different odorants in the same cell, suggesting a model of odorant receptor transduction. Receptors vary widely in their breadth of tuning, and odorants vary widely in the number of receptors they activate. Together, these properties provide a molecular basis for odor coding by the receptor repertoire of an olfactory organ (Hallem, 2004).

The odorant receptor (Or) genes in Drosophila are a highly diverse family of 60 genes. Two of them, Or22a and Or22b, have been characterized in detail and were shown to be coexpressed specifically in the ab3A antennal neuron. A deletion mutant called Δhalo that lacks these receptor genes suffers loss of odorant response in the ab3A neuron. This mutant ab3A neuron has been used to characterize other odorant receptors introducing these receptors into the mutant neuron and recording the electrophysiological response to odorants (Hallem, 2004).

For the ORNs to which a receptor has been mapped, a single odorant receptor appears sufficient to account for the complete odor response spectrum of the ORN. The simplest interpretation of these data is that these Drosophila ORNs express a single functional odorant receptor. Thus, this study provides functional data consistent with the 'one receptor-one ORN' model proposed for mammalian ORNs on the basis of molecular expression studies. Moreover, the ability of these odorant receptors to confer the odor response spectrum of a 'donor' ORN upon the recipient Δab3A neuron indicates that the receptors are the primary determinants of the odor response spectrum. These receptors do not appear to require neuron-specific or sensillum-specific perireceptor molecules in order to confer the odor response spectrum (Hallem, 2004).

Eleven of the receptors expressed conferred odor response spectra that did not match those of identified ORNs. Not all antennal ORNs have been characterized, e.g., only a limited survey of ORNs in trichoid sensilla is currently available. It seems likely that most of these 11 unmapped receptors derive from ORNs that have not yet been defined. At the same time, a number of defined ORN classes, such as ab1A, have not been matched to a receptor. One possibility is that these ORNs express Or genes whose expression was not initially detected in the antenna and that have not been tested in this study. Alternatively, some could express gustatory receptor (Gr) genes, many of which are expressed in taste organs, where some have been functionally implicated in taste or pheromone perception but at least three of which are expressed in the antenna. Another formal possibility is that some of the unmapped receptors in fact derive from unmatched ORNs but act in pairs in these ORNs or in conjunction with perireceptor molecules that are not available to receptors expressed in ab3A. Several Or genes did not confer detectable odorant sensitivity upon the Δab3A neuron. These receptors could be nonfunctional in vivo, or they could respond specifically to a ligand not present in the odorant panel, such as a pheromone. Finally, two of the receptors analyzed, Or43a and Or43b, have also been functionally characterized by others using different approaches. These results are similar to those reported previously, with only a few exceptions (Hallem, 2004).

The receptor-to-neuron map provided in this study does not reveal a simple logic relating the ORN and the receptor that it expresses. For example, adjacent ORNs do not consistently express receptors encoded by adjacent genes. Although some pairs of neurons (such as ab5A, ab5B and ab2A, ab2B) express receptors that are relatively closely related by sequence similarity, this relationship does not hold universally: the receptors of ab3A and ab3B are distantly related. Thus, these studies define a problem whose solution is likely to be complex: the evolution of the receptor-to-neuron map (Hallem, 2004).

The odorant receptor dictates the odor response spectrum of the ORN in which it is expressed in many and perhaps all cases. The results of this study also indicate that the receptor is the primary determinant of three other ORN response properties: spontaneous firing rate, signaling mode, and response dynamics. All four of these properties are likely to play critical roles in odor coding, and some are closely related. For example, the level of spontaneous activity affects the capacity of inhibitory signaling as a mode of information transmission: a high level of spontaneous activity provides a wide operating range in which inhibition can act. A high spontaneous firing level could also affect the sensitivity of the ORN or could have effects on the state of postsynaptic neurons in the antennal lobe. Thus, these results demonstrate a critical role for the odorant receptor in multiple aspects of odor coding. The complexity of the odor code transmitted from the peripheral olfactory organs to the brain depends primarily on the functional properties of odorant receptors (Hallem, 2004).

The signaling mode of an ORN was found to be determined by its odorant receptor. Different receptors, when expressed in the same ORN and given the same odorant stimulus, can confer responses that differ in signaling mode. A second finding is that a single receptor can mediate both excitatory and inhibitory responses (Hallem, 2004).

A simple model could explain how the receptor determines both the signaling mode and spontaneous firing rate of the ORN. According to this model, in the absence of odorants, receptors exist in an equilibrium between an 'active' conformation that leads to activation of the G protein-mediated signal transduction cascade and an 'inactive' conformation that does not. The equilibrum constant differs for different receptors, thus explaining differences in spontaneous firing rate among ORNs. The binding of an excitatory odorant stabilizes the active conformation of the receptor, leading to an increase in firing rate. The binding of an inhibitory odorant stabilizes the inactive conformation, leading to a decrease in firing rate. A particular odorant, such as 1-hexanol, might stabilize the active conformation of some receptors, such as Or67a, but stabilize the inactive conformation of other receptors, such as Or47b. Similar models have also been proposed for other GPCRs (Hallem, 2004).

Response termination kinetics, like spontaneous firing rate, signaling mode, and odor response spectrum, is determined by the odorant receptor. By what mechanism does termination kinetics depend on the receptor but not on the cellular context in which the receptor is expressed? One possibility is that termination kinetics depends primarily on the dissociation constant of the receptor for its odorant ligand. This possibility could explain why an individual receptor can show differences in the termination rate for two odorants, as has been observed with Or7a for E2-hexenal and benzaldehyde. It could also explain why two receptors can show differences in the termination rate for the same odorant, as observed with Or47a and Or98a for pentyl acetate (Hallem, 2004).

Drosophila ORNs operate in different environments. They reside in different olfactory organs, in sensilla of radically different morphology, and in different molecular contexts, e.g., in proximity to different OBPs. Moreover, the receptors are themselves remarkably divergent in sequence. Given this heterogeneity, one might have expected severe limitations on the ability of receptors to function normally when expressed ectopically in different ORNs (Hallem, 2004).

Many odorant receptors were found to function normally with respect to a variety of parameters when expressed in the ab3A neuron, and at least some receptors were found to function in a number of diverse neurons. Receptors normally expressed in ORNs of trichoid and coeloconic sensilla (atXA and acXB, respectively) can function in a basiconic sensillum (ab3), despite differences in morphology and OBP content, and antennal receptors can function in the maxillary palp, a developmentally and morphologically distinct organ. Odorant receptors from the malaria vector mosquito Anopheles gambiae have been shown to function in a Drosophila ORN. While it is certainly possible that some receptors, such as those specialized for pheromone detection, might function normally only in their native contexts, the results suggest a broad compatibility between most receptors and ORNs (Hallem, 2004).

Nearly the entire repertoire of Or receptors has been examined in a highly sensitive olfactory organ, the Drosophila antenna. This analysis has allowed a consideration of the molecular basis of odor coding across an entire olfactory organ, with respect both to the mechanisms of coding and to the functional organization of the system (Hallem, 2004).

The odor response spectra of these receptors was examined with an odorant panel that is both chemically diverse and ecologically relevant. The odorants include acetate esters, organic acids, alcohols, an aldehyde, ketones, and a monoterpene ester. All of these odorants can be found in either bananas, apples, oranges, pineapples, or black currants. Ethyl acetate, for example, constitutes 33% of the volatiles in pineapple (Hallem, 2004).

In their natural environment, flies encounter not only a vast array of odorants but also a vast range of odorant concentrations, ranging from low concentrations for a fly in flight to high concentrations for a fly immersed in rotting fruit. In this study, 500 ms pulses of air were directed over odorant solutions that varied in dilution from 10^-8 to 10^-2. Although these doses are referred to in terms of the dilutions of odorant in the solvent, i.e., '10^-2,' these air pulses then undergo a large dilution in another air stream before reaching the fly. It is not known how many molecules of odorant are thereby carried from their hydrophobic solvent to the antenna at room temperature or how this exposure compares to that of a fly standing on a fermenting fruit at higher temperatures. However, virtually all of the firing rates measured in this study are below the maximum firing rates observed for Drosophila ORNs and are thus within the dynamic ranges of ORNs. Moreover, the responses observed are comparable in magnitude to those produced by exposure to natural food sources such as banana, orange, pineapple, mango, and grape, all of which was found to yield responses of ~80-270 spikes/s from ab2A and ab3A neurons. It will be of interest to extend the sampling of odor space to include not only a broader panel of odorants at a wide range of concentrations but, perhaps most important, mixtures of odorants, as flies in the wild rarely encounter a pure odorant (Hallem, 2004).

All receptors characterized are distinct. Odor response spectra differ between receptors that are encoded by tightly linked genes, receptors that map to neighboring neurons in the same sensillum, and receptors that are more closely related in sequence. At the same time, there is overlap among response spectra. Some odorants elicited strong responses from ~1/3 of the tested receptors. Different receptors vary in their breadth of tuning with respect to the odorant panel: some respond strongly to a single odorant and others to as many as ~70% of the volatile odorants selected for inclusion in the panel (Hallem, 2004).

The functional overlap among receptors expands the coding capacity of the system by allowing for combinatorial coding, which has been documented previously in other systems. Coding capacity is further expanded, however, by additional diversity in receptor function: receptors confer not only the odor response spectrum but also the response mode and the response dynamics upon the ORNs that express them, as well as the level of spontaneous activity. Thus, there are several degrees of freedom available to each receptor, and the response of the system is multidimensional not only by virtue of its multiplicity of receptors but also by virtue of the multiplicity of response characteristics exhibited by each receptor (Hallem, 2004).

The olfactory system encodes not only odorant quality, i.e., the identity of an odorant stimulus, but also its intensity. Analysis of a large population of receptors revealed that different odorants are encoded differently across different concentrations. Some odorants elicit strong responses from multiple receptors even at low concentrations, whereas others do not. These results show that differential receptor activation provides a rich coding space in which to register odor intensity (Hallem, 2004).

These results provide an underlying molecular basis for odor coding, whose cellular basis has been the focus of several recent studies. Optical imaging and electrophysiological studies showed that different odorants activate distinct but overlapping subsets of glomeruli in the antennal lobe of Drosophila and that higher odorant concentrations elicit stronger responses and activate larger numbers of glomeruli. Extensive electrophysiological recordings from projection neurons reveal that they differ in breadth of tuning, signaling mode, and response dynamics, and it will be of interest to determine how the diverse odorant receptor responses described in this study are ultimately transformed into those of the projection neurons. Of particular interest in the representation of odors in the antennal lobe is the role of local interneurons, which form widespread connections among glomeruli and which could register the simultaneous activation of receptors that recognize different features of an odor stimulus (Hallem, 2004).

The patterns of receptor activation described in this study may not provide all the information necessary for odorant discrimination. For example, the temporal structure of olfactory information has been shown to be critical for odor coding in several systems, and there are other ways of analyzing the temporal dynamics of neuronal activity. However, all of the parameters measured in this study are likely to be essential to odor coding. Olfactory responses are based on the activities of the first-order neurons of the system, the ORNs, and the activities of these neurons are in turn based on the activities of the receptors that have been characterized in this study (Hallem, 2004).

Odor coding depends on the existence of multiple ORN classes, each with different response characteristics. This organization depends ultimately on the regulated expression of individual receptors in defined subsets of ORNs. Little is known about the mechanisms by which ORNs select, from among a large repertoire, which genes to express. The receptor-to-neuron map established in this study provides a foundation for exploring the developmental mechanisms by which the molecular basis of odor coding in this system is established (Hallem, 2004).

Insect olfactory receptors are heteromeric ligand-gated ion channels

In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. This study provides evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes show extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment (Sato, 2008).

Taken together, these data provide compelling evidence that a heteromeric complex of a conventional insect OR and the highly conserved Or83b family co-receptor has the characteristics of a cation non-selective ion channel directly gated by odour or pheromone ligands. It is concluded that G-protein-mediated signalling is negligible in producing the current elicited by odour activation of insect OR heteromultimers. These findings provide insight into long-argued insect olfactory transduction mechanisms and may explain the lack of clear consensus on the role of second messengers in this process. The insect ORs share no homology with any previously described ion channel and do not contain any known ion selectivity filter motifs. Insect OR activity is not inhibited by Gd³⁺, a lanthanide that is a broad-spectrum ion channel inhibitor. Therefore, although the ionic permeability reported in this study for Na⁺, K⁺ and Ca²⁺ would be consistent with the properties of non-selective cation channels, a molecular basis for this novel ionotropic activity remains to be elucidated. The spontaneous activity of the OR complex found in this study seems to account for previous observations that olfactory sensory neurons exhibit bipolar electrical activity and become electrically negative on the deletion of Or83b in vivo. Given that there are 62 and 79 potential ligand-binding OR subunits in Drosophila and Anopheles, respectively, the insect ORs may represent the largest single family of ion-channel-like proteins in any organism. This work also raises the intriguing possibility that the insect gustatory system, which senses bitter and sweet tastants as well as carbon dioxide, shares this ionotropic coupling mechanism with the insect ORs. In fact, an ionotropic sugar-gated channel in fleshfly taste cells has previously been reported. This finding offers the caveat that other orphan receptors classified as G-protein-coupled receptors merely because of their putative seven-transmembrane topology may instead possess ligand-gated channel activities, as has been shown previously for light-activated channelrhodopsin. This work has important implications for worldwide efforts to identify specific inhibitors for the insect ORs, which may prove useful in controlling host-seeking behaviours of disease-vector insects such as mosquitoes (Sato, 2008).

From worm to man, many odorant signals are perceived by the binding of volatile ligands to odorant receptors1 that belong to the G-protein-coupled receptor (GPCR) family. They couple to heterotrimeric G-proteins, most of which induce cAMP production. This second messenger then activates cyclic-nucleotide-gated ion channels to depolarize the olfactory receptor neuron, thus providing a signal for further neuronal processing. Recent findings, however, have challenged this concept of odorant signal transduction in insects, because their odorant receptors, which lack any sequence similarity to other GPCRs, are composed of conventional odorant receptors (for example, Or22a), dimerized with a ubiquitously expressed chaperone protein, such as Or83b in Drosophila6. Or83b has a structure akin to GPCRs, but has an inverted orientation in the plasma membrane. However, G proteins are expressed in insect olfactory receptor neurons, and olfactory perception is modified by mutations affecting the cAMP transduction pathway. This study shows that application of odorants to mammalian cells co-expressing Or22a and Or83b results in non-selective cation currents activated by means of an ionotropic and a metabotropic pathway, and a subsequent increase in the intracellular Ca²⁺ concentration. Expression of Or83b alone leads to functional ion channels not directly responding to odorants, but being directly activated by intracellular cAMP or cGMP. Insect odorant receptors thus form ligand-gated channels as well as complexes of odorant-sensing units and cyclic-nucleotide-activated non-selective cation channels. Thereby, they provide rapid and transient as well as sensitive and prolonged odorant signalling (Wicher, 2008).

Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila

Ionotropic glutamate receptors (iGluRs; see Ionotropic receptor 8a) mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. This paper characterizes a family of iGluR-related genes in Drosophila, which have been named ionotropic receptors (IRs). These receptors do not belong to the well-described kainate, AMPA, or NMDA classes of iGluRs, and they have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Misexpression of IRs in different olfactory neurons is sufficient to confer ectopic odor responsiveness. Together, these results lead to the proposal that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues (Benton, 2009).

Species as diverse as bacteria, plants, and humans have the capacity to sense small molecules in the environment. Chemical cues can transmit the presence of food, alarm signals, and messages from conspecifics that signify mating compatibility. Peripheral chemical recognition largely relies on membrane receptor proteins that interact with external ligands and convert this binding into intracellular responses. The vast majority of identified chemosensory receptors in multicellular organisms belong to the seven transmembrane domain G protein-coupled receptor (GPCR) superfamily, including odorant, gustatory and pheromone receptors in mammals, birds, reptiles, amphibians, fish, and nematodes. Unicellular organisms also use GPCRs for chemoreception, such as the pheromone receptors in budding yeast (Benton, 2009).

Insects can detect a wide range of environmental chemicals: bitter, sweet, and salty tastants, odors, pheromones, humidity, carbon dioxide, and carbonated water. Most of these chemosensory stimuli are recognized by members of two evolutionarily related insect-specific chemosensory receptor families, the Odorant Receptors (ORs) and Gustatory Receptors (GRs). These proteins contain seven predicted transmembrane domains but are evolutionarily unrelated to GPCRs and adopt a distinct membrane topology. Recent analysis has indicated that insect ORs function as odor-gated ion channels (Sato, 2008; Wicher, 2008), setting them mechanistically apart from metabotropic vertebrate ORs (Benton, 2009).

Comprehensive analysis of the expression of these receptors in Drosophila, has hinted at the existence of other types of insect chemosensory receptors (Couto, 2005; Yao, 2005). This is particularly apparent in the major olfactory organ, the third segment of the antenna, which bears three types of olfactory sensory hairs (sensilla): basiconic, trichoid, and coeloconic. All olfactory sensory neurons (OSNs) innervating basiconic and trichoid sensilla generally express one OR, along with the OR83b co-receptor. However, with the exception of OR35a/OR83b-expressing neurons (Yao, 2005), OSNs housed in coeloconic sensilla do not express OR83b or members of the OR or GR gene families. Nevertheless, electrophysiological analysis has revealed the existence of multiple types of coeloconic OSNs tuned to acids, ammonia and humidity (Yao, 2005), suggesting that other types of insect chemosensory receptors exist (Benton, 2009).

A bioinformatic screen has been carried out for insect-specific genes enriched in OSNs (Benton, 2007). Among these, a large expansion was found of the ionotropic glutamate receptor (iGluR) gene family of unknown biological function (Littleton, 2000). This study provides evidence that these variant iGluRs represent a novel class of chemosensory receptor (Benton, 2009).

The screen identified 6 antennal-expressed genes encoding proteins annotated as ionotropic glutamate receptors (iGluRs) (Littleton, 2000). Using these novel receptor sequences as queries, exhaustive BLAST searches of the Drosophila genome identified a family of 61 predicted genes and 1 pseudogene. These genes are distributed throughout the genome, both as individual sequences and in tandem arrays of up to four genes. This family was named the Ionotropic Receptors (IRs), and individual gene names were assigned to the IRs using nomenclature conventions of Drosophila ORs (Benton, 2009).

Phylogenetic analysis of predicted IR protein sequences revealed that they are not closely related to members of the canonical families of iGluRs (AMPA, kainate, NMDA, or delta). However, they appear to have a similar modular organization to iGluRs, comprising an extracellular N-terminus, a bipartite ligand-binding domain, whose two lobes (S1 and S2) are separated by an ion channel domain, and a short cytoplasmic C-terminus. It is noted that the gene structure and protein sequence of most receptors are presently only computational predictions. Nevertheless, the family is extremely divergent, exhibiting overall amino acid sequence identity of 10-70%. The most conserved region between IRs and iGluRs spans the ion channel pore, suggesting that IRs retain ion-conducting properties (Benton, 2009).

The ligand-binding domains are considerably more variable, although alignment of small regions of the S1 and S2 lobes of IRs and iGluRs allowed examination of conservation in amino acid positions that make direct contact with glutamate or artificial agonists in iGluRs. While all iGluRs have an arginine (R) residue in S1 that binds the glutamate α-carboxyl group, only 19/61 (31%) IRs retain this residue. In the first half of the S2 domain, 9/61 (15%) of IRs retain a threonine (T), which contacts the glutamate γ-carboxyl group in all AMPA and kainate receptors. Interestingly, the iGluRs that lack this T residue (NR1, NR3A, delta) have glycine or serine and not glutamate as a preferred ligand (Mayer, 2006; Naur, 2007). Finally, in the second half of the S2 domain, 100% of the iGluRs have a conserved aspartate (D) or glutamate (E) that interacts with the α-amino group of the glutamate ligand, compared with 10/61 (16%) IRs. Of 61 IRs, only three (IR8a, IR75a, IR75c) retain the R, D/E, and T residues characteristic of iGluRs, although these residues lie within a divergent structural backbone. Other IRs have a diversity of different amino acids at one or more of these positions. Thus, the ligand-binding specificity of most or all IRs is likely to be both distinct from that of iGluRs and varied within the IR family (Benton, 2009).

The expression of the IR family was determined by both tissue-specific RT-PCR and RNA in situ hybridization. Fifteen IR genes are expressed in the antenna. Transcripts of these genes were not detected elsewhere in the adult head, body or appendages, except for IR25a and IR76b, which are also expressed in the proboscis. Expression of the remaining 46 IR genes was not reproducibly detected in any adult tissue. It is unclear whether these genes are not expressed, expressed at different life stages, or expressed in at levels below the detection threshold of these assays (Benton, 2009).

Analysis of where in the antenna IR genes are expressed compared to ORs was performed by double RNA in situ hybridization with probes for the OR co-receptor OR83b and one of several IR genes, including IR64a, IR76b, IR31a, and IR40a. IRs are not expressed in basiconic and trichoid sensilla, as they are not co-expressed with OR83b, and IR expression persists in mutants for the proneural gene absent md neurons and olfactory sensilla (amos), which completely lack these sensilla types. However, expression of these IRs is dependent upon the proneural gene atonal, which specifies the coeloconic sensilla as well as a feather-like projection called the arista, and a three-chambered pocket called the sacculus. Thus, ORs and IRs are expressed in developmentally distinct sensory lineages in the antenna. One exception is the subpopulation of coeloconic OSNs that expresses both IR76b and OR35a and OR83b. It was confirmed that IR-expressing cells in the antenna are neurons by demonstrating that they co-express the neuronal marker elav (Benton, 2009).

A comprehensive map was generated of IR expression. Each IR was observed to have a topologically-defined expression pattern that is conserved across individuals of both sexes. IR8a and IR25a, which encode closely related receptors, are broadly expressed, detected in overlapping populations of neurons around the sacculus and in the main portion of the antenna. IR25a but not IR8a is also detected in the arista. IR21a is expressed in approximately 6 neurons in the arista, as well as 5-10 neurons near the third chamber of the sacculus. Three IRs display specific expression in neurons surrounding the sacculus: IR40a and IR93a are co-expressed in 10-15 neurons adjacent to the first and second sacculus chambers, while IR64a is found in 10-15 neurons surrounding the third chamber (Benton, 2009).

The remaining 9 IRs are expressed in coeloconic OSNs distributed across the antenna. Double and triple RNA in situ hybridization revealed that individual neurons express between 1 and 3 different IR genes and are organized into specific clusters of two or three neurons. Four distinct clusters (cluster A-cluster D), containing two (cluster C) or three (cluster A, B, and D) neurons, could be defined by their expression of stereotyped combinations of IR genes. Cluster C includes a coeloconic neuron that expresses OR35a and OR83b in addition to IR76b. Although each cluster is distinct, there is overlap between the IRs they express. IR76b is expressed in one neuron in all four clusters, IR75d in three clusters and IR75a in two clusters. In additional to these selectively-expressed receptors, individual neurons are likely to express one or both of the broadly-expressed IR8a and IR25a. The combinatorial expression patterns of the IRs raise the possibility that these genes define specific functional properties of these neurons (Benton, 2009).

Definition of four distinct clusters of IR-expressing neurons in the antenna is consistent with the identification of four types of coeloconic sensilla, named ac1-ac4, which have distinct yet partially overlapping sensory specificities (Yao, 2005). To examine whether IR expression correlates with the chemosensory properties of these OSNs, the spatial organization of IR-expressing neurons was compared using probes for unique IR markers for each cluster type to these functionally distinct sensilla types. As a unique molecular marker for Cluster B is lacking, this cluster was defined as those containing IR75a-expressing OSNs (present in Cluster B and Cluster C) that are not paired with OR35a-expressing cluster C neurons. It was found that each cluster has a different, though overlapping, spatial distribution in the antenna. For example, Cluster A neurons (marked by IR31a) are restricted to a zone at the anterior of the antenna, just below the arista, while cluster C neurons (marked by IR75b) are found exclusively in the posterior of the antenna. These stereotyped IR neuron distributions were observed in antennae from over 20 animals (Benton, 2009).

The initial description of the coeloconic sensilla classes did not describe their spatial distribution (Yao, 2005). This study therefore recorded odor-evoked responses in >100 coeloconic sensilla in several dozen animals across most of the accessible antennal surface, using a panel of odorants that allowed identification unambiguously of each sensilla type (ammonia for ac1, 1,4-diaminobutane for ac2, propanal and hexanol for ac3, and phenylacetaldehyde for ac4) (Yao, 2005). After electrophysiological identification, the location of the sensilla on the antennal surface was noted (Benton, 2009).

This mapping process allowed a correlation of the electrophysiological and molecular properties of the coeloconic sensilla. For example, ac1 sensilla were detected only in a region on the anterior antennal surface just ventral to the arista, and therefore are most likely correspond to cluster A, containing IR31a-IR75d-IR76b/IR92a-expressing neurons. The data fit well with the previous assignment of the OR35a-expressing neuron to the ac3 sensillum (Yao, 2005), which is found on the posterior of the antenna and is the only coeloconic sensillum class that unambiguously houses two neurons (Yao, 2005). While these results allow initial assignment of IRs to different coeloconic sensilla classes, it is noted that assignment of specific odor responses to individual IR-expressing OSNs is not possible from these data alone (Benton, 2009).

All neurons expressing a given OR extend axons that converge upon a single antennal lobe glomerulus, resulting in the representation of a cognate odor ligand as a spatially-defined pattern of neural activity within the brain. To ask whether IR-expressing neurons have the same wiring logic, the targeting of OSNs expressing IR76a was investigated by constructing an IR76a-promoter GAL4 driver that recapitulates the endogenous expression pattern. Labeling of these neurons with mCD8:GFP revealed convergence of their axons on to a single glomerulus, ventral medial 4 (VM4), in the antennal lobe. This glomerulus is one of approximately eight that was previously unaccounted for by maps of axonal projections of OR-expressing OSNs (Benton, 2009).

To determine where IRs localize in sensory neurons, antibodies were generated against IR25a. Broad expression of IR25a protein was detected in sensory neurons of the arista, sacculus, and coeloconic sensilla. All anti-IR25a immunoreactivity was abolished in an IR25a null mutant. Low levels of IR25a could be detected in the axon segment adjacent to the cell body in some neurons but no staining was observed along the axons as they entered the brain, or at synapses within antennal lobe glomeruli. In coeloconic neurons, prominent anti-IR25a staining was detected both in the cell body and in the distal tip of the dendrite, which corresponds to the ciliated outer dendritic segment innervating the sensory hair. Relatively low levels were detected in the inner dendrites, suggesting the existence of a transport mechanism to concentrate receptor protein in cilia. A similar subcellular localization was observed in sacculus and aristal sensory neurons. The specific targeting of an IR to sensory cilia suggests a role for these proteins in sensory detection (Benton, 2009).

To test the hypothesis that IR genes encode chemosensory receptors, whether ectopic IR expression could induce novel olfactory specificities was investigated. Three IRs expressed in ac4 sensilla (IR84a, IR76a and IR75d) were individually mis-expressed in ac3 sensilla using the OR35a-GAL4 driver. Single sensillum recordings were used to examine which, if any, of these three IRs, could confer sensitivity to phenylacetaldehyde, the only known robust ligand for ac4 but not ac3 sensilla (Yao, 2005). Mis-expression of IR84a conferred a strong response to phenylacetaldehyde that was not observed in control strains or in animals mis-expressing either IR76a or IR75d. Ectopically-expressed IR84a did not confer sensitivity to the structurally related odor, phenylacetonitrile, which does not activate either ac3 or ac4 neurons (Yao, 2005). This indicates that mis-expressed IR84a does not simply generate non-specific ligand sensitivity in these neurons (Benton, 2009).

Next the novel odor responses conferred by IR84a mis-expression were compared to the endogenous phenylacetaldehyde responses of ac4 sensilla by generating dose-response curves. Stimulus evoked spike frequencies of ac3 sensilla ectopically expressing IR84a are quantitatively very similar to those in ac4 sensilla, even exceeding the endogenous ac4 responses at higher odor concentrations. These elevated responses are likely to be due to the contribution of weak endogenous phenylacetaldehyde responses that were observed in ac3 sensilla at high stimulus concentrations, as subtraction of these values produces an IR84a-dependent phenylacetaldehyde dose-response curve that is statistically the same as that of ac4 sensilla. Thus, ectopic expression of a single IR in ac3 is sufficient to confer a novel ligand- and receptor-specific odor sensitivity that is physiologically indistinguishable from endogenous responses (Benton, 2009).

To extend this analysis to a second IR, whether mis-expression of one of the IR genes uniquely expressed in ammonia-sensitive ac1 neurons (IR31a and IR92a) was sufficient to confer ectopic responsiveness to this odor was examined. Because ac3 sensilla neurons display endogenous ammonia-evoked responses at modest stimulus concentrations, these experiments used the IR76a-promoter GAL4 transgene to mis-express these receptors in ammonia-insensitive ac4 sensilla (Yao, 2005). ac4 sensilla mis-expressing IR92a, but not IR31a, displayed responses to ammonia. 1,4-diaminobutane, a control stimulus that does not activate either ac1 or ac4 neurons (Yao, 2005), did not stimulate ac4 sensilla mis-expressing IR92a. It was noted that the magnitude of the ectopic IR92a ammonia response is lower than native ammonia-evoked responses of ac1 sensilla (Yao, 2005). This may be due to the lack of co-factors present in ac1 sensilla but not in ac4 sensilla. Nevertheless, these results suggest that IR92a comprises at least part of an ammonia-specific chemosensory receptor (Benton, 2009).

The specific combinatorial expression patterns of IRs in sensory neurons and the diversity in their ligand-binding domains is difficult to rationalize with a general role in signal transduction, independent of ligand recognition. More importantly, the novel olfactory sensitivity induced by ectopic expression of IR84a and IR92a provides evidence that IR proteins function directly as ligand-specific, chemosensory receptors. While these experiments demonstrate a sufficiency of IRs for conferring odor-responsiveness, definitive proof of their necessity will require analysis of loss-of-function mutations (Benton, 2009).

In animal nervous systems, iGluRs mediate neuronal communication by forming glutamate-gated ion channels, and it is speculated that IRs also form ion channels, gated by odors and other chemosensory stimuli. A growing number of ionotropic mechanisms in chemoreception are known. For example, members of the transient receptor potential (TRP) family of ion channels are the primary receptors for nociceptive compounds including capsaicin and menthol and have also been implicated in gustatory detection of acids. Insect ORs also display functional properties of ion channels. Proof that IRs function as ion channels will necessitate electrophysiological characterization of these receptors in heterologous expression systems, and evidence for direct binding of chemosensory ligands to IRs will require biochemical assays in vitro (Benton, 2009).

iGluRs normally function as heterotetrameric assemblies of variable subunit composition that exhibit differing functional properties such as ligand sensitivity and ion permeability. The current analysis indicates that up to five different IRs may be co-expressed in a single sensory neuron, raising the possibility that these receptors also form multimeric protein assemblies with subunit-dependent characteristics. Of particular interest are the two broadly-expressed members of the family, IR8a and IR25a, which may represent common subunits in many different types of IR complexes. Their function is unclear, but it is possible that they have a co-receptor function with other IRs, analogous to that of OR83b. Preliminary analysis of IR25a mutants revealed no obvious defects in odor-evoked responses in coeloconic sensilla, but this may be due to redundancy of IR25a with IR8a or the existence of homomeric IR receptors without IR8a or IR25a. Other IRs, such as IR75a and IR76b, are expressed in two or more types of coeloconic sensory neurons. In these cases, the response properties may be defined by the combination of IRs expressed in these distinct neuronal populations. However, the present lack of knowledge of relevant ligands for several coeloconic OSNs makes it difficult to match specific ligands to individual IR neurons based on the expression map alone (Benton, 2009).

The IR repertoire is remarkably similar in size, overall genomic organization and sequence divergence to Drosophila ORs. Like the ORs, individual IRs are specifically expressed in small subpopulations of chemosensory neurons, and this expression is regulated by relatively short (< 1-2 kb) upstream regulatory regions. Furthermore, at least one population of IR-expressing neurons converges on to a single glomerulus in the antennal lobe, similar to the wiring logic established for OR-expressing neurons both in invertebrate and vertebrate olfactory systems. Some differences are observed, however, in the organizational logic of IR and OR expression. Most OR-expressing neurons express a single OR gene, along with OR83b, in distinct clusters that innervate specific olfactory hairs. In contrast, many IR-expressing neurons identified in the antenna express 2 or 3 IR genes, in addition to one or both of the broadly-expressed IR8a and IR25a genes. Moreover, overlap is observed both between the molecular composition of different IR neurons and the combination of neurons that innervate a given sensillum. For example, IR76b is co-expressed with at least two other different IR genes in at least two different sensilla - with IR92a in ac1 and with IR76a in ac4 - as well as being co-expressed with OR35a and OR83b in ac3. While the precise biological logic of IR co-expression awaits the matching of specific chemosensory ligands to IR-expressing neurons, combinatorial expression of IRs may contribute more significantly to their role in sensory detection than for ORs (Benton, 2009).

Why does Drosophila possess two types of antennal chemosensory receptors? Although both may be ionotropic, IRs and ORs are not simply slight evolutionary variants. The receptor families are molecularly unrelated, are under the control of distinct developmental programs, and housed within sensory structures of radically different morphology. Thus, it seems likely that these chemosensory receptors fulfill distinct functions in chemosensation. Analysis of the chemosensory behaviors mediated by IR sensory circuits (now possible with the identification of specific molecular markers for these pathways) may provide insights into the contributions of these different olfactory subsystems. IRs may also have functions in other chemosensory modalities, as two antennal IRs are also detected in the proboscis, and the expression of 46 members of the repertoire remains unknown (Benton, 2009).

Chemosensation is an ancient sensory modality that predates the evolution of the eukaryotes. Are there traces of conservation in the molecular mechanism by which prokaryotes and eukaryotes sense external chemicals? iGluRs have long been recognized to have prokaryotic origins. Their ion channel domain is homologous to bacterial potassium channels, and the ligand binding domain is structurally related to bacterial periplasmic binding proteins (PBPs), extracellular proteins that scavenge or sense amino acids, carbohydrates and metal ions by coupling to transporters or chemotaxis receptors. Evolutionary connections between iGluRs and PBP function have not often been considered, perhaps in part due to their very weak primary sequence similarity, the widespread occurrence of the PBP fold -also present, for example, in bacterial transcription regulators - and the dedicated role for iGluRs in mediating or regulating synaptic transmission, a process seemingly distant from bacterial solute uptake and chemotaxis (Benton, 2009).

This discovery of a family of divergent iGluR-like proteins that may act as peripheral chemosensors provides a link between the disparate functions of these protein modules. While a role for IRs in detecting diverse external ligands is analogous to the function of bacterial PBPs, the primary sequence and neuronal expression of IRs is clearly closer to the properties of iGluRs. Intriguingly, a large family of iGluR-related proteins, the GLRs has also been identified in the plant Arabidopsis thaliana (Lam, 1998; Chiu, 1999). Almost nothing is known about their physiological functions, but bioinformatic analysis of GLRs suggests that glutamate is unlikely to be their natural ligand (Dubos, 2003; Qi, 2006). It is possible that GLRs may have roles as chemosensors, for example in detection of soil nutrients or airborne volatiles. Thus, while iGluRs have been intensely studied for their roles in synaptic communication, this characterization of the IRs leads to the suggestion that the ancestral function of this protein family may have been in detecting diverse chemical ligands to mediate both intercellular communication and environmental chemical sensing (Benton, 2009).

A role for a phospholipid intermediate in insect olfactory transduction

Mechanisms by which G-protein-coupled odorant receptors transduce information in insects still need elucidation. This study shows that mutations in the Drosophila gene for G_qα (dgq) significantly reduce both the amplitude of the field potentials recorded from the whole antenna in responses to odorants as well as the frequency of evoked responses of individual sensory neurons. This requirement for G_qα is for adult function and not during antennal development. Conversely, brief expression of a dominant-active form of G_qα in adults leads to enhanced odor responses. To understand signaling downstream of G_qα in olfactory sensory neurons, genetic interactions of dgq were tested with mutants in genes known to affect phospholipid signaling. dgq mutant phenotypes were further enhanced by mutants in a PLCβ (phospholipase Cβ) gene, plc21C. Interestingly although, the olfactory phenotype of mutant alleles of diacylglycerol kinase (rdgA) was rescued by dgq mutant alleles. These results suggest that G_qα-mediated olfactory transduction in Drosophila requires a phospholipid second messenger the levels of which are regulated by a cycle of phosphatidylinositol 1,4-bisphosphate breakdown and regeneration (Kain, 2008).

The dgq gene is located at 49B on the second chromosome and encodes the only known functional Gq-like α subunit of heterotrimeric G-proteins in Drosophila. The gene encodes at least two functional isoforms, which arise by alternative splicing. Among these, dgqα1 is expressed primarily in the adult eye (Lee, 1990), whereas dgqα3 mRNA is expressed widely through development including the adult head and appendages (Talluri, 1995; Ratnaparkhi, 2002). Based on RNA expression analysis and behavioral studies with an RNAi construct (Kalidas, 2002), it has been suggested that a G_qα isoform might function downstream of olfactory G-protein-coupled receptors in antennal sensory neurons. To test this idea directly, mutations were generated in the dgq gene by excision of a P-element in the 5'-UTR of dgq, and by ethyl methane sulfonate mutagenesis. Molecular analysis demonstrated that the P-excision allele, dgq^221c, carries a lesion in the 5' end of dgq spanning the translation start site in exon 3, thus rendering it null. Exons located 3' to exon 3 are intact (Banerjee, 2006). Both dgq^221c and dgq¹³⁷⁰ (induced by chemical mutagenesis) are lethal as homozygotes and as heterozygotes with Df(2R)vg-C, which uncovers the dgq locus. The lethality in dgq¹³⁷⁰ fails to complement that of dgq^221c. Homozygous dgq¹³⁷⁰ mutants produce a transcript with a G->A mutation at base pair 1933. This would result in a change from arginine (AGA) to lysine (AAA) at residue 207, which lies in the switch II helix region that is highly conserved among α subunits of all heterotrimeric G-proteins. Structural studies show that this region undergoes a conformational switch on GTP binding, which is considered essential for effector function. Specifically, the arginines at positions 204 and 207 of Dgqα3 correspond to Arg205 and Arg208 of Gαi. Both these residues have been shown to provide important stabilizing contacts after GTP binding with glutamates located further downstream. Presumably, the mutation in dgq¹³⁷⁰ destabilizes the GTP-bound state of G_q and renders it effectively dead by preventing activation of the downstream effector. Normal levels of the mutant protein are present in dgq¹³⁷⁰ homozygous larvae (Kain, 2008).

Animals homozygous for the null alleles, dgq^221c and dgq¹³⁷⁰, die as either first- or early second-instar larvae. To study the odorant response from adult antennas, animals were generated with homozygous mutant clones on the antenna using the MARCM method. Mitotic recombination at the FRT^42B target site was induced using Flipase (Flp) driven by the eyeless (ey) promoter, which has been previously demonstrated to generate clones mostly in visual and olfactory sensory neurons (OSNs) and only minimally in the central brain. Mutant clones identified by GFP driven by the OSN-specific 'driver' Or83bGAL4 were obtained in >80% of progeny of the appropriate genotypes (FRT^42B, dgq^null/FRT^42B, tubPGAL80; ey-Flp/OR83bGAL4, UAS2XEGFP). Animals with strong GFP fluorescence in their antennas were chosen for the experiment, whereas animals with no visible GFP expression (i.e., no mutant clones) were control for effects attributable to background genotypes. Confocal sectioning of antenna bearing large clones, revealed significant mixing of marked and nonmarked cells suggesting that ey-FLP induces FRT-mediated recombination at multiple times during antennal development. The olfactory response was measured after pulsed odor delivery. EAGs were obtained after each successive pulse of a concentration of each of five different odorants. Odorant concentrations used were chosen such that they lay within the range of the log linear response to that specific odorant, when tested on wild-type flies. The response of wild-type (CS) and control flies bearing no detectable GFP-positive cells gave a comparable range of amplitudes for each of the odors tested. Antennas bearing clones of homozygous dgq^221c or dgq¹³⁷⁰ tissue showed a response reduced to 3-10 mV compared with 8-16 mV in the controls. The mutation did not affect the morphology of the antenna. Antennal sensory neurons and their projections also appear normal as judged by immunohistochemical staining with mAb22C10 and anti-GFP (Kain, 2008).

It is known that a mutation in dgq affecting the visual splice variant (dgqα1) compromises the light response as measured by electroretinograms (ERGs) (Scott, 1995). As expected, ey-Flp also generated clones of dgq nulls in the retina. As an independent confirmation of the clonal analysis, ERGs were measured from flies with visible antennal clones and it was found that responses were reduced to the same level as seen with the dgq¹ allele. Moreover, dgq^221c/dgq¹ and dgq¹³⁷⁰/dgq¹ flies are viable and show compromised phototransduction, although not to the same extent as shown previously by dgq¹/Df flies (Scott, 1995). The residual visual response observed in dgq^221c/dgq¹ and dgq¹³⁷⁰/dgq¹ animals may arise from different genetic backgrounds of the three dgq alleles tested. Because the dgq¹ allele affects only the visual splice variant of dgq (Scott, 1995; Ratnaparkhi, 2002), neither dgq^221c/dgq¹ nor dgq¹³⁷⁰/dgq¹ flies exhibited any defects in EAG measurements. These data together with EAG recordings suggest that light and odor transduction in Drosophila exploit common mechanisms albeit using distinct transcripts of the dgq gene (Kain, 2008).

In the canonical receptor and G-protein-coupled signaling pathway, the enzyme phospholipase Cβ (PLCβ) is activated on GTP binding to G_qα, which cleaves the membrane bound phospholipid, PIP₂, to generate soluble InsP₃ and membrane-bound diacylglycerol (DAG). The Drosophila genome has two genes encoding for PLCβ -norpA and plc21C. A previous study found that dgq^221c and plc21C^P319 mutant alleles have synergistic effects on larval viability and adult flight (Banerjee, 2006), indicating that PLCβ21C can function together with Drosophila G_qα. The plc21C^P319 mutant allele is a hypomorph, with a P-insert in the first intron, and is homozygous viable, whereas plc21C^p60a is a small deficiency that removes the 5' end of plc21C plus a neighboring essential gene p60. A reduction of plc21C RNA levels in plc21C^P319 homozygotes and plc21C^p60a heterozygotes was confirmed by RT-PCR. UASplc21C⁵⁵⁷ is a recently generated UASRNAi strain reported as specific for plc21C (Kain, 2008).

EAG responses of plc21C^P319 homozygotes and plc21C^P319/p60A combinations were reduced to <6 mV for all chemicals tested. The EAG responses to ethyl acetate (from basiconic sensilla) and propionic acid (from coeloconic sensilla) were found to be reduced at several concentrations. plc21C/+ and dgq/+ heterozygotes resulted in a small but significant decrease in EAG responses compared with wild-type (CS) controls (Kain, 2008).

A combination of plc21C/dgq resulted in a 7-9 mV reduction in EAG responses compared with wild-type controls. This reduction is significantly greater than the value expected to arise from a mere additive effect of plc21C/+ and dgq/+ heterozygotes. Although other explanations for this haploinsufficent interaction are formally possible, the explanation is favored that PLCβ21C interacts with G_qα in normal olfactory transduction. This observation was supported by the significantly reduced EAG response to all five odorants obtained in an RNAi-driven OSN-specific downregulation of plc21C, and by downregulation of plc21c in the dgq expression domain (GqGAL4/RNAiplc21c⁵⁵⁷). EAG responses of the plc21C RNAi strain were reduced further on introduction of UASGq^1F1 (RNAiGq). In the double mutant dgq-plc21C RNAi strain, EAG responses were reduced to ~2 mV but are still not abolished completely. Moreover, expression of plc21C RNAi in the OSNs resulted in a significant reduction of the spike frequency of ab2a neurons on stimulation by ethyl acetate. Thus, odor transduction in ab2a neurons requires both G_qα and Plc21C (Kain, 2008).

Because small, but significant, odor responses remain in all dgq and plc21C mutant genotypes tested, these data further highlight the likelihood of other signaling mechanisms that collaborate with G_qα during odor detection. As reported previously, EAG responses recorded from a null allele for the second PLCβ, norpA^P24, appear close to normal for the five odorants tested. The introduction of norpA^P24 in plc21C/dgq animals had no additional effect in reducing EAG amplitudes. norpA thus has no significant effect on antennal physiology in contrast to its proposed effect on signaling in OSNs located on the maxillary palp and the major role played by this gene in visual transduction (Kain, 2008).

The results so far show that signaling downstream of olfactory receptors in the Drosophila antennas requires Dgqα3 and PLCβ21C. To identify which of the two second messengers generated by G_qα activation of PLCβ21C, DAG or InsP₃, are required for the electrical response to odorants, mutants were tested that function in the two arms of the pathway. The rdgA gene codes for an ATP-dependant DAG kinase that converts DAG to phosphatidic acid, which in turn is a precursor for PIP₂ formation. Homozygotes for two rdgA mutant alleles rdgA¹ and rdgA³ show reduced EAG responses to all five odorants tested, whereas heterozygotes appear close to normal. rdgA¹ homozygotes also show reduced EAG responses to multiple concentrations of two odorants tested, ethyl acetate and propionic acid. Interestingly, unlike previously observed effect of rdgA mutants on ERG termination kinetics, slower termination was not observed of the EAG response in rdgA mutants. Moreover, unlike eyes of 1-d-old rdgA¹/rdgA¹ adults, which exhibit strong retinal degeneration, both rdgA alleles have normal antennal morphology even 6 d after eclosion. Thus, the reduced EAGs observed are not a consequence of antennal neuron degeneration (Kain, 2008).

Next, the EAG response was tested after introducing a single mutant copy of dgq in rdgA homozygotes (rdgA/rdgA;dgq/+). Interestingly, it was found that reduction of Dgq levels to 50% by introduction of a single copy of the dgq^null allele rescued the EAG responses of rdgA homozygotes bringing them back to near normal. The extent of rescue varies with specific dgq and rdgA mutant allele combinations. Strongest effects were seen in rdgA¹/rdgA¹; dgq¹³⁷⁰/+ animals. Reducing dgq function by expression of the RNAi construct UASGq^1F1 also rescued the EAG defects produced by both rdgA mutant homozygous alleles. The rescue of EAG responses in rdgA mutants by dgq mutants was further verified by testing RNAi lines specific for rdgA and dgq in appropriate combinations. EAG responses were significantly higher in the presence of RNAi constructs for both rdgA and dgq compared with the response from individual RNAi lines. A possible explanation for these data is that failure of conversion of DAG back to phosphatidic acid and PIP₂ through rdgA encoded DAG kinase compromised the generation of an odor-evoked response but does not cause degeneration of OSNs. Reduction in the signaling flux through G_qα (by lower Dgq levels in dgq^null/+ genotypes or by G_qα RNAi expression) compensates for this defect and rescues the olfactory defect of rdgA (Kain, 2008).

Because there is a significant body of data from studies with other organisms suggesting that InsP₃ is a second messenger in invertebrate olfactory transduction (Ache, 2005), EAG responses were measured from single copies of the two dgq alleles with two different mutations of the InsP₃ receptor as heterozygotes (dgq^null/+; itpr^EMS/+). itpr^sv35 is a null, whereas itpr^ka901 can act as a gain-of-function allele. The effect of these alleles on antennal olfactory responses has not been investigated before. No significant change in the response to any chemical was observed in the eight strains tested. These data are in agreement with a previous study from Drosophila, in which EAGs measured from the antennas of viable alleles for the InsP₃ receptor were normal. Thus, primary olfactory responses from the Drosophila antennas do not require the InsP₃ receptor but are dependent on a DAG kinase (rdgA) (Kain, 2008).

This study has shown that mutations in the G-protein G_qα result in reduced sensitivity of Drosophila antennal sensory neurons to several different chemical stimuli. Genetic analysis suggested that its downstream effector is phospholipase Cβ encoded by the plc21C gene. PLCβ enzymes catalyze breakdown of the membrane-bound lipid PIP₂ to generate DAG and InsP₃. Reduced EAG amplitudes in DAG kinase mutants (rdgA) and their rescue by lowering G_qα levels, argue that the extent of PIP₂ depletion after G_qα and PLCβ activation is responsible for this arm of olfactory transduction. Altered PIP₂ levels have been shown to gate or modulate membrane conductances in other contexts. The presence of a residual olfactory response in antennal neurons that are null for dgq supports existence of an additional transduction mechanism (Kain, 2008).

Odorant detection in animals occurs through a diverse class of G-protein-coupled receptors. In mammalian olfactory sensory neurons, it appears that all odorant receptors activate a single G-protein G_olf and generate one second messenger, cAMP. In Drosophila dgq mutants, a reduced response was found to all five odorants tested, indicating that multiple odorant receptors activate G_qα. Ethyl acetate, isoamyl acetate, and benzaldehyde stimulate receptors located in the basiconic sensilla, whereas butanol and propionic acid activate neurons innervating the coeloconic sensilla. It has been proposed that the basiconic sensilla primarily detect food odors and the coeloconics express receptors that respond to certain specialized odors. The results suggest that these functional distinctions are not based on a change in signal transduction machinery (Kain, 2008).

The residual odor-induced response observed in G_qα mutant antennas and in single ab2a neurons argues for the presence of an alternate transduction mechanism(s) in OSNs. Odorants could stimulate either other G-proteins, which alter cyclic nucleotide levels, or a direct odor-gated ion channel, as in heteromers of Manduca OR/OR83b or both. Precisely how the alternate mechanism of odor transduction interacts with G_qα-mediated transduction described here requires additional study. The strong genetic evidence presented here, supporting a role for a G_qα-mediated signaling cascade during olfactory transduction, needs to be reconciled with recent data suggesting that Drosophila odorant receptors do not conform to the structure or topology typical of other G-protein-coupled receptors (Kain, 2008).

Based on the quality and strength of the odor stimulus it receives, each odorant receptor could stimulate different transduction pathways to varying extents. It is proposed that firing frequency of the OSN is an integration of this information, perhaps by cross-sensitization or desensitization of membrane conductances gated by the individual pathways. Interestingly, it has been demonstrated that a single odorant receptor (e.g., Or59b), present within the same neuron can mediate both excitatory and inhibitory responses as a consequence of stimulation by two different odorants. Precedents for multiple signaling pathways in odor detection within a single organism are present in invertebrates (Ache, 2005), although their existence within the same olfactory neuron has not been demonstrated so far (Kain, 2008).

The requirement of phospholipase C for odor-induced activity of OSNs in the maxillary palp highlights an overlap in signaling mechanisms during olfaction and vision. The data further highlight parallels in these two sensory systems. In Drosophila photoreceptors, signal amplification begins with activation of a number of G-protein molecules by a single photoisomerised rhodopsin molecule, followed by activation of one PLC molecule for each G-protein . Although the current methods of measuring olfactory responses in Drosophila do not allow direct measurement of signal amplification, the enhanced EAG amplitudes observed in AcGq3-expressing animals and synergism between dgq and plc21C mutant alleles observed in this study supports the idea of signal amplification by G_qα (Kain, 2008).

The rdgA gene has a role in both amplification and termination of the visual response. The observation that rdgA¹ and rdgA³ homozygotes exhibit reduced EAG responses, in the absence of antennal neuron degeneration, suggest that the rate of DAG turnover to generate other intermediates leading to PIP₂ is important for membrane depolarization of OSNs. The absence of antennal degeneration predicts a low level of basal PLCβ activity in OSNs, and perhaps strong adaptive mechanisms that prevent activation of membrane channels in normal odor-rich environments. This needs to be investigated further. The fact that reduced EAGs can be rescued by a single copy of dgq mutant alleles, supports the existence of a cycle of phosphoinositide turnover in OSNs, in which reduced levels of G_qα help to slow down this cycle and thus balance the rate of PIP₂ regeneration in rdgA mutants. The observed suppression appears qualitatively similar to the interaction of rdgA and dgq during the amplification phase of Drosophila visual transduction in which rdgA mutants enhance the sensitivity of dgqα¹ mutants as measured by the amplitude of quantum bumps. Finally, either PIP₂ depletion, or a lipid intermediate presumably gates a depolarizing membrane channel. The identity of this channel can only be conjectured on at this stage. However, based on studies from other G-protein-coupled lipid signaling pathways, a transient receptor potential (TRP) family channel might be a possible candidate. Among this family, the two most well studied members in Drosophila are TRP and TRPL (TRP-like), both of which are enriched in photoreceptors. A previous study on the olfactory responses of trp mutants ruled out a role for this gene in primary sensory transduction of odor stimuli. Several members of this family exist in Drosophila and need to be investigated for their role in olfactory transduction (Kain, 2008).

The structural biology of olfactory organs

(1) Organization and function of maxillary palp olfactory receptor neurons and brain olfactory glomeruli

There are two olfactory organs on the adult fly: the third segment of the antenna and the maxillary palp. In both organs, ORNs are housed in sensory hairs called sensilla. In contrast to the antenna, the organization of the 120 ORNs of the maxillary palp is simpler. There are 60 sensilla basiconica on the maxillary palp, each housing two ORNs. The 120 ORNs fall into six different classes based upon their odorant response profiles. Neurons of the six ORN classes are always found in characteristic pairs in three functional types of sensilla basiconica, with the total number of neurons in each class being equal. Each class is distributed broadly over all, or almost all, of the olfactory surface of the maxillary palp.

Olfactory receptor neurons of the adult fly are located in both the antenna and the maxillary palp. To ask whether any of the DOR genes are expressed in these neurons, in situ hybridization to RNA was carried out in adult tissue sections. Of 11 genes examined, 7 show detectable expression, which in every case is observed only in the olfactory organs. The 46F.1 probe hybridizes to a subset of ORNs in the maxillary palp. A count of labeled ORNs in serial sections reveals that the total number of 46F.1-staining ORNs per maxillary palp is 18, or 15% of the 120 olfactory neurons in the maxillary palp. A similar number of neurons, 17, was labeled by another probe, 33B.3. The neuronal identity of the labeled cells is apparent from the presence in many cases of a well-defined axon projecting from the labeled cell body and joining the maxillary nerve. For both probes, the labeled neurons are distributed broadly over the olfactory surface of the organ, and are interspersed among unlabeled neurons. Staining in many cells appears annular, which is interpreted to reflect a perinuclear distribution of mRNA, as expected of an mRNA present at highest concentrations in the cell bodies of these ORNs. The 33B.3 and 46F.1 genes are evidently expressed in different subsets of ORNs, because the number of neurons hybridizing with a mixed probe is greater than the number of neurons that hybridized when either probe is used individually. For neither probe was hybridization detected in the antenna, head, or thorax (Clyne, 1999a)

The number and broad distribution of maxillary palp neurons expressing 46F.1 and 33B.3 are intriguing in light of electrophysiological studies. There are about 120 ORNs on the palp, which fall into six different classes based on their odorant response profiles. Each class contains roughly equal numbers of neurons, distributed broadly over the olfactory surface of the palp. Thus, if an individual receptor gene is expressed in all ORNs of a functional class, one might expect a gene to be expressed in a broad distribution, in about 20 neurons, in good agreement with the distribution and numbers observed for both 46F.1 and 33B.3 (Clyne, 1999).

Three DOR genes that are expressed in the maxillary palp come from the 16% of the genome analyzed. Since these three genes, like most DOR genes, are not clustered in the genome, linear extrapolation suggests that the entire genome contains on the order of 18 DOR genes expressed in the maxillary palp, an organ that has six functional classes of neurons. If all neurons within a functional class, i.e., with the same odor specificity, are identical in terms of their receptor expression, then the ratio of expressed genes to neuronal classes (three genes per neuronal class) in this organ would be consistent with a model in which an individual ORN expresses a small number of odorant receptors (Clyne, 1999a and Vosshall, 1999).

ORNs in Drosophila and other insects project to an olfactory processing center, the antennal lobe, which is much like the olfactory bulb of vertebrates. Like its vertebrate counterpart, the antennal lobe contains olfactory glomeruli, of which the antennal lobe of Drosophila has a total of 40 olfactory glomeruli. In vertebrates, there is an approximate equivalence between the estimated number of odorant receptor genes and the number of glomeruli. If, in fact, the number of Drosophila odorant receptor genes is on the order of 100, then the ratio of odorant receptor genes to glomeruli would exceed two to one, and would rise if additional families of odorant receptor genes were discovered. It is noted that the number of glomeruli receiving input from the maxillary palp has been variously estimated as three and five; if the estimate of 18 genes expressed in the maxillary palp is correct, then the ratio of these receptor genes to their corresponding glomeruli would fall in the range of three to six (Clyne, 1999a).

2) Organization of antennal olfactory receptor neurons

Whereas the maxillary palp has approximately 120 ORNs, the antenna has 1200. The organization of the 1200 ORNs of the antenna is complex but ordered. On the antenna, there are different morphological categories of sensilla: sensilla trichodea, sensilla coeloconica, large sensilla basiconica, and small sensilla basiconica. The different morphological categories of sensilla are distributed in overlapping patterns across the surface of the antenna. Electrophysiological studies show that each morphological category of sensilla can be divided into different functional types, defined by the characteristic response profiles of their ORNs. For sensilla trichodea, the different functional types are segregated into zones on the surface of the antenna; segregation is also observed for the different functional types of sensilla coeloconica. This zonal organization is less conspicuous for large and small sensilla basiconica, of which different functional types are intermingled. Electrophysiological data suggest that there are on the order of 30 different classes of ORNs in the antenna, a rough estimate based on the odor response profiles of individual ORNs (Clyne, 1999a and references).

Many of the DOR genes are expressed in the antenna and not in the maxillary palp. The 47E.1 probe hybridizes to 40 cells in a broad area across the antenna, including both anterior and posterior faces, similar to the distribution pattern of small sensilla basiconica. A probe from the 25A.1 gene hybridizes to fewer cells (16) but in a region of the antenna similar to that of 47E.1 staining, as judged by reconstruction of serial sections. The 22A.2 probe hybridizes to 22 cells in a different distribution, clustered in the dorsomedial region of the antenna. This pattern matches the distribution of the large sensilla basiconica (Clyne, 1999).

The Vosshall (1999) study reveals topographically defined subpopulations of olfactory sensory neurons in either the antenna or the maxillary palp. In situ hybridization experiments reveal that each receptor is expressed in a spatially restricted subpopulation of neurons in the antenna or maxillary palp. The total number of cells expressing each receptor per antenna was obtained by counting the positive cells in serial sections of antennae from multiple flies. DOR67 and DOR53, for example, are expressed by about 20 neurons on the medial-proximal edge of the antenna, whereas DOR62 and DOR87 are expressed by subpopulations of 20 cells at the distal edge of the antenna. Approximately ten cells in the distal domain express DOR64. Each of three linked genes, dor71, dor72, and dor73, is expressed in different neurons. dor72 is expressed in approximately 15 antennal cells, while dor73 is expressed in 1 to 2 cells at the distal edge of the antenna. In contrast, dor71 is expressed in approximately ten maxillary palp neurons but is not detected in the antenna. The three sensillar types are represented in a coarse topographic map across the third antennal segment. The proximal-medial region, for example, contains largely basiconic sensilla. Receptors expressed in this region (dor53 and dor67) are therefore likely to be restricted to the large basiconic sensilla. More distal regions contain a mixture of all three sensilla types, and it is therefore not possible from these data to assign specific receptors to specific sensillar types. The spatial pattern of neurons expressing a given receptor is conserved between individuals. In situ hybridization with two receptor probes to three individual flies reveals that both the frequency and spatial distributions of the hybridizing neurons are conserved in different individuals. At present, the precision of this topographic map cannot be determined and it can only argued that given receptors are expressed in localized domains.

Receptors for mate recognition in Drosophila

Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila. The trichoid sensilla, one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, were systematically examined by electrophysiological analysis. None respond strongly to food odors but all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone transferred from males to females during mating. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin-female extract as well. Thus, males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. All members of the Odor receptor (Or) gene family that are expressed in trichoid sensilla were then systematically tested by using an in vivo expression system. Four receptors respond to fly odors in this system: Two respond to extracts of both males and virgin females, and two respond to cVA. A model is proposed describing how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic (van der Goes van Naters, 2007).

The responses of ORNs in trichoid sensilla of the antenna were measured by single-unit electrophysiology. All three trichoid-sensilla subtypes, T1, T2, and T3, which contain one, two, and three ORNs, respectively, were tested. These three subtypes occupy distinct but overlapping regions of the antennal surface and together comprise more than 20% of the sensilla in the antennae. Initially, 86 compounds were tested, most of which are found in fruits or are fermentation products. These compounds were tested on 60 trichoid sensilla, 30 from males and 30 from females. The compounds were tested in mixtures, and no mixture elicited a response greater than 20 impulses/s, which represents less than 10% of the maximal response of these ORNs. Some mixtures inhibited the spontaneous activity of T2 and T3 sensilla and produced decreases of 10–20 impulses/s in the action-potential rate. The three most inhibitory odors were subsequently determined to be 1-hexanol, hexyl acetate, and butyl acetate. The paucity of strong excitatory responses to food odors is consistent with the results of an earlier screen with a limited number of chemicals; in this earlier screen, no strong responses were found, although modest responses were elicited by trans-2-hexenal and cis-vaccenyl acetate (cVA) (van der Goes van Naters, 2007).

The odor of live flies was tested. 50 flies were placed in a glass tube that was closed at both ends with a cotton mesh. Air was puffed through the tube toward the antenna of a fly mounted for electrophysiological recording. 75 individual trichoid sensilla, of all three subtypes, were tested for responses to the odors of both males and virgin females. Air passing over male flies elicited a strong response from ORNs in a large group of trichoid sensilla. These ORNs did not respond to the odor of virgin females. These sensilla correspond to the T1 subtype, each of which houses a single ORN. T1 sensilla are found on both male and female antennae; in both cases they respond to the odor of males but not of virgin females. The T2 and T3 sensilla did not produce responses to fly odors when they were tested in this paradigm (van der Goes van Naters, 2007).

These experiments showed that at least some trichoid sensilla respond to fly odors. However, whether other trichoid sensilla might show responses to fly odors was tested in a more sensitive assay. A new paradigm was developed. Because flies approach each other closely during courtship, it was reasoned that some pheromone-sensitive sensilla might be adapted for short-range information reception. Some of the chemical cues that influence courtship behavior in Drosophila are present in the cuticle, i.e., on the surface of the fly, and are long-chain unsaturated hydrocarbons of very limited volatility. Although some of these cues are believed to be detected via the taste system, it seemed possible that the olfactory system might also contribute to the reception of cuticular components at very close range during courtship (van der Goes van Naters, 2007).

Accordingly, rather than adding odor stimuli to an air stream directed at the fly from a distance, stimuli were presented by approaching the antenna with the tip of a glass capillary carrying the odor. This procedure was designed to simulate the proximity of two interacting flies. As an initial test of the feasibility of this paradigm, 500 pl of a solution of cVA was draw into the capillary. cVA has been shown to act as an anti-aphrodisiac pheromone in Drosophila; there is also evidence for its playing a role as an aggregation pheromone. As the capillary tip approached certain trichoid sensilla, the impulse rates of certain ORNs increased and reached a maximum of >200 impulses/s upon physical contact of the capillary tip with the sensillum shaft. Control stimuli prepared with the hexane solvent alone gave no response (van der Goes van Naters, 2007).

Having established a short-range delivery paradigm, the responses, initially to cVA, of trichoid sensilla were systematically examined across the entire antennal surface. Mature male flies contain approximately 1 μg of cVA, primarily in the ejaculatory bulb. A capillary tip was loaded with 5 ng of cVA (0.005 fly equivalent) and 189 trichoid sensilla were approached individually. Strong responses of >100 impulses/s in were observed 169 of the 189 sensilla. Previous reports had shown that the ORN in T1 sensilla responds to cVA, and this study confirmed this finding. Responses to 5 ng of cVA exceeded 200 impulses/s in T1 sensilla. Also in agreement with the previous reports, some sensilla immediately adjacent to the zone containing T1 did not respond to cVA. However, it was determined that, in addition to the T1 subtype, a large number of sensilla more distolateral on the antennal surface also contained ORNs that are sensitive to cVA in this paradigm. Neurons in the distolateral sensilla responded to the cVA stimulus with a rate increase of more than 100 impulses/s. Thus, there appear to be at least two populations of sensilla with ORNs that respond to this pheromone (van der Goes van Naters, 2007).

To expand the scope of this analysis from a single defined pheromone, cVA, to a broad representation of the cuticular pheromone profile, hexane extracts of males and virgin females were prepared. Approximately 500 pl of extract was drawn into the capillary tip; this amount is equal to 0.25% of the material extracted from a single fly (van der Goes van Naters, 2007).

When a male extract was used as the odor source, all 147 trichoid sensilla tested, from all regions of the antennal surface, yielded responses. Different ORNs began to respond to the approaching odor source at different distances. The T1 sensilla, which house a single ORN, appeared to be particularly sensitive; they showed responses greater than 20 impulses/s when the odor source came within a 1 cm radius. As the odor source became still closer, the impulse rates increased rapidly. ORNs in T2 and T3 sensilla appeared to be less sensitive and had impulse rates increasing only after the odor source approached a distance of 200 μm, as determined with an ocular micrometer. The responses were dose dependent; when the dose was increased from 0.25% fly equivalent to 5% fly equivalent, the response radius increased from 200 μm to 500 μm (van der Goes van Naters, 2007).

When an extract from virgin females was used as the stimulus, strong responses were observed in ORNs of all trichoid sensilla except T1. Thus, T1 sensilla appear to be tuned to male odor, whereas T2 and T3 sensilla yield strong responses to both males and virgin females. Sensitivity to male and virgin-female extracts was comparable in T2 and T3 sensilla. These in vivo recordings, taken together, demonstrate that trichoid sensilla respond to fly odors and that the odors of males and virgin females are registered differently across the ensemble of trichoid sensilla. A limitation of the analysis is that it is difficult to ascribe responses to individual ORNs within trichoid sensilla. With the exception of T1, trichoid sensilla contain multiple ORNs. In recordings, this is evident from summation and cancellation events between impulses in the traces. In most cases it was not possible to discriminate the activities of the individual ORNs because the action potentials, as recorded extracellularly, did not differ significantly in size or shape. Because of the inability to classify action potentials with confidence, it was not possible to determine whether there is a functional subdivision among the ORNs sharing a sensillum. To address this limitation, advantage was taken of another experimental system, the “empty neuron” system, in an effort to analyze the responses of trichoid sensilla at a higher resolution (van der Goes van Naters, 2007).

Drosophila contains a family of 60 Or (Odor receptor) genes, and the following 12 family members have been reported to map to individual ORNs of trichoid sensilla: Or2a, Or19a, Or19b, Or23a, Or43a, Or47b, Or65a, Or65b, Or65c, Or67d, Or83c, and Or88a. Each of these 12 Or genes were expressed in the “empty neuron” system, an in vivo expression system based on a mutant ORN, ab3A, that resides in a basiconic sensillum. The endogenous receptor genes of this ORN, Or22a and Or22b, are deleted, and the promoter of Or22a drives ectopic expression of another odor receptor in ab3A via the UAS-GAL4 system. The odor responses conferred upon ab3A by the ectopically expressed receptor are then measured by single-unit electrophysiology (van der Goes van Naters, 2007).

The 12 trichoid receptors were systematically tested in the empty-neuron system with a panel of fly-derived chemicals: hexane extracts of males and virgin females, material from the genital regions of flies (males, virgin females, and mated females), and cVA. The genital odors were obtained by drawing a glass capillary, with a tip pulled to a diameter of 3 μm, across the genital region of a fly such that material visibly coated the tip. Preliminary experiments showed that the responses could be quantified most reproducibly not during the approach of a stimulus to the antenna but after the capillary tip contacted the sensillum. Responses mediated by the trichoid receptors were were therefore quantified by determining impulse rates of the ORN after contact. The 12 receptors were expressed and tested in both male and female recipients with all six stimuli, and no differences between the responses of male and female flies were identified (van der Goes van Naters, 2007).

Of the 12 receptors, four mediated responses to fly odors in this system. All four, Or47b, Or65a, Or67d, and Or88a, responded to male extract, and their action-potential frequencies increased by 50–200 impulses/s. Two of these receptors, Or65a and Or67d, did not respond to extract from virgin females. The sex specificity of Or65a and Or67d is consistent with a role for these receptors in the detection of male-specific pheromones. The other two receptors, Or47b and Or88a, responded to extract from virgin females; these responses were comparable to those they gave to male extracts. It was noted that both Or47b and Or88a were previously tested in the empty-neuron system with a panel of 110 odors, most of which were present in fruits and were of widely varying chemical structures, and no excitatory responses were recorded. These results are consistent with the hypothesis that Or47b and Or88a detect a pheromone secreted by both males and females (van der Goes van Naters, 2007).

Male genital material elicited strong responses from Or65a, Or67d, and Or88a. Genital material from virgin females did not elicit a strong response from any of the 12 receptors. However, material from the genital region of females that were mated 1–4 hr previously produced responses from these three receptors, which, yielded firing rates comparable to those observed with male genital material. These results suggest that during copulation the male transfers compounds that activate these receptors (van der Goes van Naters, 2007).

One compound that the male transfers to the female during copulation is cVA. The sensitivity of Or67d to cVA is consistent with previous observations; expression studies have shown that Or67d is expressed in T1 sensilla, which are sensitive to cVA, and ectopic expression of Or67d in other trichoid sensilla conferred sensitivity to cVA. However, the results indicate that there are multiple receptors for cVA. Both Or67d and Or65a responded most strongly to cVA among a panel of six related compounds. The two receptors differed in their specificities, however; Or67d gave a relatively stronger response than did Or65a to cis-vaccenyl alcohol, for example. It is noted that the detection of a second cVA receptor, which has not been reported previously, may reflect the sensitivity of the short-range delivery paradigm that was designed (van der Goes van Naters, 2007).

The response specificity of Or67d, as measured in the empty-neuron system, is nearly identical to that of the ORN in the T1 sensillum. However, it is noted that the magnitude of the response to cVA in the expression system is approximately half that in T1. Dose-response curves show that the response threshold is also lower in the native T1 sensillum; it appears as though the T1 neuron can detect a dose of approximately 10⁻⁴ ng, whereas the expressed Or67d receptor may require a dose of approximately 10⁻² ng for detection. Slower rise and decay rates were also found, along with higher levels of spontaneous firing in the expression system. These results suggest that the expression system may lack a component that is present in the endogenous context; for example, the odorant-binding protein LUSH was found to be required for normal response to cVA in T1 sensilla (van der Goes van Naters, 2007).

Whereas Or67d mediates responses to cVA in T1 sensilla, Or65a is expressed in the ORNs of trichoid sensilla that are more distolateral on the antenna and that also respond to cVA. It is noted that the Or65a gene is in close proximity to Or65b and Or65c and that the three genes are coexpressed in a single ORN. Although neither Or65b nor Or65c mediated responses to any of the fly odors tested in the empty-neuron system, the possibility was considered that they might contribute to the response of the ORN if they were coexpressed with Or65a, perhaps via heterodimer formation. Accordingly, all pairwise combinations of the three receptor genes were co-expressed. It was found that coexpression of Or65b or Or65c with Or65a did not increase the response mediated by Or65a to any stimulus or change the level of spontaneous activity. Coexpression of Or65b and Or65c yielded little, if any, response to any stimulus (van der Goes van Naters, 2007).

Finally, it is noted with interest that although Or88a conferred responses to male genital material, it did not mediate responses to cVA, suggesting that it detects an additional pheromone that is also transferred from males to females upon mating (van der Goes van Naters, 2007).

This study has identified four receptors that mediate responses to fly odors. Or47b and Or88a mediate responses to the odors of both males and virgin females. Or65a and Or67d mediate responses to cVA, a male-specific lipid that is present in male genital material, is presumably extracted in hexane extracts, and is transferred to females upon mating. Or88a also responds to a compound in male genitalia, but this compound is distinct from cVA (van der Goes van Naters, 2007).

The responses of these receptors suggest a working model of the olfactory basis of mate recognition by males. In this model, neural activity mediated by Or47b and Or88a reports the proximity of a fly, either male or female. This olfactory recognition may contribute to the recognition mediated by other sensory modalities; recognition of conspecifics is a prerequisite to successful courtship. The activity of Or65a, Or67d, or both would indicate that the partner is a male or a recently mated female; thus, when the antenna of a male is in close proximity to another fly, the activation of Or65a and/or Or67d would report that the other fly is unsuitable as a mate. The lack of a signal from these receptors would permit continued courtship activity by the male (van der Goes van Naters, 2007).

A well-documented phenomenon can be interpreted in terms of this model. Mature males not only court virgin females but also vigorously court newly eclosed males. Young males, like virgin females, lack cVA and would not be expected to activate Or65a and Or67d, allowing courtship to proceed (van der Goes van Naters, 2007).

Why would Or65a and Or67d not be activated in the antenna of a male by material in its own genital region? Perhaps very little of the internal genital material is released to the air unless the region is manipulated by a capillary tip or washed in hexane, and perhaps what little is released under natural conditions can normally be detected only at very close range; if cVA were released in large amounts and inhibited mating over a long range, then mating might be inhibited at sites where flies congregate and often mate, such as rich food sources. It is also possible that the fly adapts to the ambient level of cVA, produced by its own genital region, and is sensitive to increases above that level (van der Goes van Naters, 2007).

Why are there two cVA receptors, expressed in two distinct ORNs, in different subtypes of trichoid sensilla? There is evidence that cVA serves two functions as a pheromone in Drosophila. (1) cVA has been shown to act as an anti-aphrodisiac, detering males from courting with a recently mated female. (2) cVA is deposited by females during egg laying, and there is evidence that it enhances the attractiveness of the oviposition substrate to other flies. Perhaps Or65a and Or67d activate two distinct behavioral circuits and thereby separately mediate two functions of cVA in conjunction with other cues (van der Goes van Naters, 2007).

Interestingly, no receptor for female-specific odors was identifed, although there is evidence that 7,11-heptacosadiene and 7,11-nonacosadiene, two female-specific hydrocarbons, act as aphrodisiacs. It is possible that some of the trichoid receptors respond to these compounds, which were not tested individually, or other female-specific compounds but do not function efficiently in the expression system. It is also possible that these compounds are detected by gustatory receptors, perhaps members of the Gr family. One class of gustatory neuron, which expresses Gr68a, has been shown to be required for normal courtship. Finally, the possibility is noted that some of the receptors that did not respond to the tested stimuli might detect pheromones of other Drosophila species (van der Goes van Naters, 2007).

It is striking that no differences were observed between males' and females' antennal responses to any of the fly odors tested. This similarity is in stark contrast to the extreme sexual dimorphism in antennal responses to pheromones in moths, such as Bombyx mori and Manduca sexta. The similarity in Drosophila peripheral olfactory responses suggests that in the fly, differences in male and female behavioral responses may be determined by differences in reception of other classes of sensory input, such as taste information, or by differences in the transmission or processing of olfactory information. It is possible that cVA, for example, is sensed through the same peripheral mechanisms in males and females but that only in males is the primary representation transformed in a way that accords it a negative valence (van der Goes van Naters, 2007).

In summary, a systematic analysis was carried out of the trichoid sensilla, one of the three major types of sensilla on the Drosophila antenna. These sensilla appear to be specialized for sensing fly odors, as opposed to food odors. The differential activity of ORNs in trichoid sensilla provides an olfactory basis for a male's ability to discriminate suitable from unsuitable mating partners. The molecular basis of these responses was further explored and four odor receptors were identified that mediate responses to fly odors. A model is proposed in which olfactory information flows through these receptors according to a simple logic. Although the full repertoire of pheromones and receptors has yet to be characterized, it is possible that the model may be richly elaborated without undergoing an alteration in its fundamental logic (van der Goes van Naters, 2007).

Transcriptional regulation of odorant receptors; Mechanisms of odor receptor gene choice in Drosophila

A remarkable problem in neurobiology is how olfactory receptor neurons (ORNs) select, from among a large odor receptor repertoire, which receptors to express. Computational algorithms and mutational analysis were used to define positive and negative regulatory elements that are required for selection of odor receptor (Or) genes in the proper olfactory organ of Drosophila, and an element was identified that is essential for selection in one ORN class. Two odor receptors are coexpressed by virtue of the alternative splicing of a single gene, and dicistronic mRNAs were identified that each encode two receptors. Systematic analysis reveals no evidence for negative feedback regulation, but provides evidence that the choices made by neighboring ORNs of a sensillum are coordinated via the asymmetric segregation of regulatory factors from a common progenitor. Receptor gene choice in Drosophila also depends on a combinatorial code of transcription factors to generate the receptor-to-neuron map (Ray, 2007).

Sequences that dictate expression in a specific ORN lie close to an Or gene: In order to identify sequences that dictate the expression of Or genes in particular ORNs, the minimal promoter regions that are sufficient to drive faithful expression of an Or gene were defined. This analysis with the Or85e gene. It had been found that the 3.1 kb genomic sequence upstream of the Or85e translational start site was capable of driving faithful expression of the yeast GAL4 gene in pb2A ORNs, as indicated by the expression of UAS-lacZ and UAS-GFP reporter genes that are activated by GAL4 (Ray, 2007).

A series of deletions of this 3.1 kb region were generated, each deletion progressively removing an additional ~500 bp from the 5' end of the region. Each truncated Or85e-GAL4 construct was then used to drive expression of UAS-lacZ and UAS-GFP reporters, and at least five independent lines were examined for each deletion construct. A construct containing only 450 bp upstream of the Or85e translational start site was found, and all larger constructs in the series, gave expression patterns similar to that of the initial 3.1 kb construct. However, a construct containing only 350 bp of upstream DNA showed no expression. To determine whether the cells labeled by the Or85e-GAL4 construct containing 450 bp of upstream DNA are in fact of the pb2A class, a double-labeling experiment was carried out. It was found that all GFP⁺ cells were also labeled with an in situ hybridization probe for Or85e RNA; moreover, nearly all cells labeled with the Or85e RNA probe were also GFP⁺. The simplest interpretation of these results is that 450 bp of DNA upstream of the Or85e translational start site contain the information that dictates specific expression in the pb2A neurons (Ray, 2007).

It was surprising that the information dictating expression in a single ORN class was so economically packed. To determine whether the organization of the Or85e gene is representative of other Or genes, another Or gene, Or46a, which is expressed in the ORN that neighbors pb2A, pb2B, were analyzed. Consistent with the results found for Or85e, it was found that 400 bp upstream of the Or46a translational start site is sufficient to confer specific expression in the pb2B class of ORN (Ray, 2007).

To determine the distance between the transcriptional start site and the translational start site, rapid amplification of 5' cDNA ends (5' RACE) from maxillary palp RNA was carried out for both Or85e and Or46a, as well as for two other Or genes, Or59c and Or71a. In all cases, the predicted transcriptional start sites, as determined by the longest RACE products, lie within 50 bp of the predicted translational start sites (Ray, 2007).

To investigate the mechanism by which the selection of an Or gene in an olfactory organ is restricted to an organ-specific subset of Or genes, it was asked whether there are regulatory elements that are shared among maxillary palp Or genes but not antennal Or genes. Such elements might dictate the organ-specific expression of the maxillary palp genes, perhaps by binding maxillary palp-specific transcription factors. Focused was initially placed on the 500 bp upstream of the translational start sites, as regions of this size were sufficient to confer faithful GAL4 expression patterns in the cases analyzed. As a first means of searching for sequence elements shared among maxillary palp Or genes sequence alignments were generated, but this analysis revealed remarkably little conservation among the 500 bp regions upstream of the seven maxillary palp Or genes, much less than has been observed upstream of mammalian OR genes. Therefore, some powerful computational algorithms were used to identify sequence motifs shared among maxillary palp Or genes. Algorithms were used that detect both of the major types of short DNA elements to which transcription factors bind: unipartite, or oligonucleotide, motifs and bipartite motifs. Specifically, OLIGO-ANALYSIS, which searches for oligonucleotide motifs of 6-8 nucleotides shared by coregulated genes, and DYAD-ANALYSIS, which searches for shared bipartite sequence motifs consisting of either two trimers or two tetramers, separated by 1-20 nucleotides, was used. These algorithms were used to seek motifs that are overrepresented upstream of maxillary palp Or genes, as compared to upstream of all annotated Drosophila genes, but that are not overrepresented upstream of antennal Or genes (Ray, 2007).

A bipartite motif, termed Dyad-1, was identified that consists of two trimers, CTA and TAA, separated by nine nucleotides, that is, CTA(N)₉TAA. This motif was of special interest in that all seven maxillary palp Or genes contain at least one iteration of this sequence in the 500 bp region upstream of the predicted translational start site, and most of these genes contain more than one. The frequency of occurrence upstream of maxillary palp Or genes exceeds that found upstream of the ensemble of Drosophila genes by a factor of 7.2. By contrast, its frequency upstream of 32 Or genes shown to be expressed in the antenna exceeds that of the ensemble of Drosophila genes by a factor of only 1.2 (Ray, 2007).

A heptamer sequence, CTTATAA, which was termed Oligo-1, was identifed. This motif attracted attention, in part because it contains a 6 bp palindromic core sequence, which is characteristic of many transcription factor binding sites. The frequency of occurrence of Oligo-1 in the 500 bp upstream region of maxillary palp Or genes exceeds that of the ensemble of Drosophila genes by a factor of 10.0. By contrast, upstream of the antennal Or genes, it is overrepresented by a factor of only 1.3. The degree of overrepresentation was calculated using a different approach: the iteration frequency in the 500 bp upstream region of each maxillary palp Or gene was compared to that in the entirety of Drosophila noncoding genomic DNA. It was found that by this measure, Dyad-1 is overrepresented by a factor of 13.7 upstream of the maxillary palp genes, and Oligo-1 is overrepresented by a factor of 10.0. By contrast, these elements are overrepresented by factors of only 2.0 and 1.0, respectively, upstream of antennal Or genes (Ray, 2007).

Thus, these two sequence elements are present at unexpectedly high frequencies upstream of maxillary palp Or genes, but not antennal Or genes, suggesting the possibility that these sequences play a role in the process by which the choice of Or genes by ORNs is restricted to an organ-specific subset (Ray, 2007).

The function of Dyad-1 elements was tested in the context of three Or genes using two approaches. First, a series of deletions in the Or71a locus was generated by imprecise excision of a P element located 1.0 kb upstream of the Or71a translational start site. Chromosomes retaining 170 bp or more of the most proximal upstream DNA sequences continued to express Or71a, as determined by in situ hybridization with an Or71a probe. Chromosomes that contain 121 bp or less of this sequence (Δ3, Δ4, and Δ5) did not express Or71a. There are two Dyad-1 elements upstream of Or71a; the chromosomes that retained one of these elements therefore retained Or71a expression, but those that retained no Dyad-1 elements lost expression (Ray, 2007).

Second, the Dyad-1 elements located upstream of Or46a were mutated. Specifically, a 1.9 kb region of DNA upstream of Or46a has previously been shown to drive expression of GAL4 in pb2B ORNs of the maxillary palp. Within this 1.9 kb region are two Dyad-1 elements, located 5 bp apart. A construct that carries alterations in the sequences of both elements and the spacing between them was tested, and it was found that this mutated construct no longer drives expression in the maxillary palp (Ray, 2007).

Third, the function of the Dyad-1 elements at Or85e was tested using both ablation and deletion approaches. The 0.45 kb Or85e-GAL4 construct described above was used and base pairs of each of its two Dyad-1 elements were mutated without altering their relative positions. This ablation abolished expression in the maxillary palp. Truncated constructs that do not contain Dyad-1 elements did not drive expression. The shortest of these constructs contains 90 bp of sequence, which is predicted to contain the basal promoter of the Or85e gene (see Promoter prediction program for the neural network promoter prediction algorithm). The two Dyad-1 sequences were added to the 90 bp construct; the addition of these Dyad-1 elements did not restore expression in the maxillary palp. It was demonstated (see below) that the 90 bp sequence drives expression when different elements are added to it (Ray, 2007).

The simplest interpretation of these results is that Dyad-1 is a positive regulatory element that is necessary but not sufficient for expression in the maxillary palp (Ray, 2007).

To investigate the function of the Oligo-1 element, it was mutated in the context of the Or85e promoter. The mutation was found to cause misexpression in the antenna. A marked decrease was observed in labeling of maxillary palp ORNs (Ray, 2007).

These results were confirmed and extended in an analysis of the Or71a gene. There is no Oligo-1 element in the 500 bp upstream of the Or71a gene, but there are two clustered Oligo-1 elements downstream of the gene, located 426 bp and 458 bp downstream from the translational stop site. When the 2.3 kb of upstream region was used alone to drive GAL4 expression, misexpression was found in the antenna. Reduced labeling was also observed in the maxillary palp: only 48% of cells labeled with an Or71a in situ hybridization probe were also GFP⁺ and the intensity of the GFP⁺ labeling appeared weak in a limited experiment (n = 9 maxillary palps). When both the 2.3 kb of upstream DNA and a 1.4 kb region of downstream DNA that contains both Oligo-1 elements were used, the antennal misexpression was not observed. Moreover, expression in the maxillary palp was increased: nearly 100% of cells labeled with an Or71a in situ hybridization probe were also GFP⁺ (n = 9 maxillary palps), and the intensity of the GFP⁺ labeling appeared strong. To determine whether the repression of antennal labeling and the enhancement of maxillary palp labeling were in fact due to the presence of the Oligo-1 elements in the downstream sequences, a third construct was generated, similar to the second but in which both Oligo-1 sequences were mutated. This construct again produced misexpression in the antenna, and reduced expression in the maxillary palp: again only 48% of cells labeled with an Or71a in situ hybridization probe were also GFP⁺. Moreover, expression was highly variable, and the intensity of the GFP⁺ labeling appeared weak (n = 9 maxillary palps) (Ray, 2007).

The simplest interpretation of these results is that the Oligo-1 elements repress expression of maxillary palp Or genes in the antenna and enhance their expression in the maxillary palp. Moreover, Oligo-1 elements appear capable of acting either upstream or downstream of an Or gene (Ray, 2007).

The Dyad-1 and Oligo-1 elements were identified in a search for sequences that act in the process of receptor gene choice by dictating in which organ an Or gene is expressed. Beyond organ-specific regulation, however, Or genes require an additional level of control to generate the receptor-to-neuron map: they must contain information dictating their precise expression in a single ORN class (Ray, 2007).

In an effort to identify a neuron-specific regulatory element, advantage was taken of the discovery that one maxillary palp ORN, pb2A, coexpresses two unlinked Or genes, Or85e and Or33c. It was reasoned that because both Or genes are expressed in the same neuron, they are likely to share regulatory elements that dictate expression in this neuron. Accordingly, the 500 bp upstream regions of both genes were examined for sequence elements of at least 6 bp in length that are shared by these two genes, but not by other maxillary palp Or genes (Ray, 2007).

Two elements were identified that meet these criteria: a 12 bp element, TTTATTTGCATA, which was designated the pb2A-1 element, and an 8 bp element, AGTTTTTA, which was designated pb2A-2. pb2A-1 is located at −320 bp relative to the translational start site of Or85e and at −206 bp relative to Or33c; pb2A-2 is located at −102 bp relative to Or85e and −274 bp relative to Or33c. It was noted that although these elements were identified by examining the 0.5 kb upstream regions, their specificity extends farther: they are not found in the 1 kb region upstream of any other maxillary palp Or gene, nor in the 0.5 kb downstream region of any maxillary palp Or gene (Ray, 2007).

To test the function of these elements, they were mutated in the context of the 450 bp Or85e promoter. No effect of mutating pb2A-1 was found: the mutant construct produced a pattern of GFP expression that appeared identical to that of the wild-type control construct. Moreover, it was confirmed that these GFP⁺ cells are in fact pb2A cells by showing that they hybridize to an Or85e probe in a double-label experiment. Mutation of pb2A-2, however, abolished expression in pb2A cells, indicating that the pb2A-2 element is necessary for expression in pb2A. It was also found that when seven copies of pb2A-2 and two copies of Dyad-1 were fused to the 90 bp basal promoter of Or85e, expression was observed in the maxillary palp. As a further test, seven copies of pb2A-2 and two copies of Dyad-1 were inserted upstream of a second minimal promoter; in this case, the pb2A-2 elements were 45 bp upstream of a TATA box. Again, expression was found in the maxillary palp. Although expression was too weak to allow double-label in situ hybridization, in each case at least some of the cells could be identified as neurons by the presence of dendrites, and their patterns of expression and distribution on the maxillary palp are consistent with those of pb2A. These results indicate that not only is pb2A-2 necessary for Or85e expression but that artificial promoters containing pb2A-2 sequences and Dyad-1 sequences can drive expression in maxillary palp ORNs. It was noted that two Dyad-1 sequences alone did not drive expression of Or85e, as indicated above (Ray, 2007).

Two functional odor receptors from one alternatively spliced Or gene: One mechanism by which an ORN may select two Or genes thus appears to depend on the location of common ORN-specific elements, such as pb2A-2, upstream of two different Or genes. Another instance was found of receptor coexpression that occurs through a different mechanism. This coexpression was revealed by a detailed analysis of the Or46a locus, which has been shown to be expressed in pb2B cells (Ray, 2007).

The Or46a locus has been proposed to contain two coding regions, Or46aA and Or46aB, separated by less than 100 bp and expressed by alternative splicing. It was confirmed that the locus produces two alternatively spliced mRNAs; both mRNAs were identified in multiple independent experiments. These two splice forms were observed at comparable levels in RT-PCR analysis, and were the major products detected; the possibility that minor species of functional significance may also be produced cannot be excluded. A detailed analysis of the two major mRNAs revealed surprising structures. The shorter mRNA is spliced from an internal position within exon 3 to exon 5, bypassing exon 4, and contains the Or46aA coding region. The longer mRNA lacks the splice between exons 3 and 5 and is a dicistronic message, containing two coding regions. The first of these two coding regions, which was termed Or46a1, is identical to that of Or46aA except at the 3' end, which encodes C-terminal residues that lie immediately beyond the seventh predicted transmembrane domain. Or46a1 encodes 18 terminal amino acids that show no identity to the 17 terminal amino acids of Or46aA. The second of the two coding regions, Or46aB, shares only 36% amino acid identity with Or46aA and 31% identity with Or46a1 (Ray, 2007).

This unexpected splicing pattern predicts that multiple receptors from this locus may be coexpressed in the same cell. A double-label in situ hybridization experiment was carried out using probes for Or46aA, consisting of sequences from within exon 1, and Or46aB, consisting of sequences within exon 4. It was found that all cells labeled by Or46aA sequences were also labeled by Or46aB sequences; thus all cells that express the short transcript also express the long transcript. Likewise, all cells labeled with Or46aB were also labeled by Or46aA, but this latter result is less informative, on account of the expected crosshybridization between Or46aA and Or46a1. Thus, from this experiment alone, it cannot be determined whether there are any cells that express the long transcript but not the short transcript. In any case, these experiments reveal that pb2B cells express sequences corresponding to more than one Or open reading frame (Ray, 2007).

In order to determine whether more than one of the distinct ORFs in fact encodes a functional odor receptor, an in vivo expression system, the 'empty neuron' system, was used. Using this method, it was found that Or46aA and Or46aB both encode functional odor receptors that respond to phenols, while Or46a1 did not impart responses to any odor (Ray, 2007).

The Or46a locus thus encodes three predicted odor receptors. Their expression appears limited to a single class of neuron, pb2B. To investigate the significance of this one receptor gene-multiple receptor organization, it was asked whether it is conserved in evolution. The genomes of five additional Drosophila species were examined: D. simulans, D. yakuba, D. erecta, D. pseudoobscura, and D. grimshawi. It was found that orthologs of the ORFs encoding Or46aA, Or46a1, and Or46aB are present in all species and thus have been maintained for tens of millions of years. Moreover, the organization of the locus is well conserved; for example, the distance between the stop codon of Or46a1 and the start codon of Or46aB is in all cases between 69 bp and 164 bp. Double-label in situ hybridization with D. pseudoobscura sequences representing the orthologs of Or46aA and Or46aB showed coexpression in cells of the D. pseudoobscura maxillary palp (Ray, 2007).

In the mosquito Anopheles gambiae, the genes most closely related to Or46aA and Or46aB are AgOr34 and AgOr37, whose predicted products both show 22% amino acid identity to Or46aB and 19% and 20% identity to Or46aA, respectively. Interestingly, these two mosquito genes are also tightly linked, but in inverted fashion, with only 1.5 kb of intervening noncoding DNA, suggesting the possibility that they may share common regulatory sequences and hence be coexpressed (Ray, 2007).

It is noted, finally, that in order to express a functional Or46aB protein from a dicistronic RNA, an internal ribosome entry site (IRES) is required. Although there is precedent for functional IRES sequences in Drosophila, they have not been well defined. IRES sequences are poorly conserved across phylogeny, but dicistronic RNAs identified from polio virus and the human ornithine decarboxylase gene share a common UUUC sequence approximately 26-34 bp upstream of the distal AUG. The presence was noted of a UUUC sequence 38 bp upstream of the AUG of Or46aB, a sequence that is conserved at the same position in D. yakuba and D. ananassae (Ray, 2007).

Mechanisms of Or gene coexpression in the antenna: Evidence has been presented for two distinct mechanisms by which a single maxillary palp ORN can express two odor receptors, one mechanism that depends on the localization of a common cis-regulatory element upstream of two unlinked Or genes, and one that depends on alternative splicing. To determine whether these mechanisms are singularities, unique to the maxillary palp, the analysis was expanded to include the entire olfactory system. All pairs of Or coding regions were examined that are separated by <1 kb of intervening DNA: (Or22a, Or22b), (Or33a, Or33b), (Or33b, Or33c), (Or42a, Or42b), (Or59b, Or59c), (Or65b, Or65c), (Or69aA, Or69aB), and (Or85b, Or85c). Also examined were (Or10a, Gr10a), which are also tightly linked (Ray, 2007).

It was surprising to find a dicistronic message that encodes both Or10a and Gr10a, in both of two strains analyzed, Oregon-R and w¹¹¹⁸. Or10a is an odor receptor for methyl salicylate, while Gr10a is a member of the gustatory receptor gene family. The dicistronic mRNA was identified in each of six independent experiments, each using a different combination of primer pairs. It was noted that there are two UUUC motifs upstream of the Gr10a translational start codon, at positions −32 and −52, suggesting the possibility that one may act as an IRES. These results provide a molecular mechanism to explain the coexpression of these two genes in ab1D ORNs (Ray, 2007).

It was found that although Or22a and Or22b are each encoded by independent transcripts, two transcripts were identified that are likely to have the same 5' ends, based on the positions of the longest cDNAs isolated. These results suggest that Or22a and Or22b can be expressed from the same promoter, which could in principle explain the coexpression of the two genes in ab3A ORNs. In the longer transcript, encoding Or22b, the second intron is not removed, creating a frameshift mutation and a nonsense codon in Or22a; thus a functional Or22a receptor would be encoded only by the shorter transcript. The presence is noted of a UUUC at position −37 upstream of the Or22b translational start codon, suggesting a means by which Or22b could be translated from a long mRNA that also includes an Or22a translational start site. At the same time, there is a TATA box 70 bp upstream of the Or22b translational start codon, and a 1.3 kb region upstream of Or22b was found to drive faithful reporter gene expression, suggesting the existence of a third, Or22b-specific transcript. In the Canton-S strain, Or22a has been shown to be a functional odor receptor, whereas Or22b is nonfunctional. However, in an Oregon-R strain, both genes encode functional odor receptors (Ray, 2007).

Or69a also produces two distinct mRNAs, which contain identical, or nearly identical, 5'ends. The shorter mRNA encodes Or69aA; the longer transcript encodes Or69aB. The mRNA encoding Or69aB contains many of the codons of Or69aA but the Or69aA ORF is terminated by a stop codon following the first splicing event. These results provide a molecular explanation for the coexpression of Or69aA and Or69aB in ORNs of ab9 sensilla; however, a 0.9 kb region upstream of the Or69aB ATG was found to drive reporter gene expression in ab9 sensilla, suggesting the possibility of an additional Or69aB-specific transcript (Ray, 2007).

In addition to these three cases of antennal coexpression, recent mapping studies have identified 6 additional antennal ORN classes that coexpress Or genes among the 36 antennal ORN classes to which Or genes were mapped. Four of these coexpressed Or gene pairs are unlinked: (Or33b, Or85a); (Or33a, Or56a); (Or33b, Or47a); and (Or49a, Or85f), raising the possibility that they might, like (Or33c, Or85e), contain a common regulatory element. Using the same bioinformatics approach used to identify pb2A-1 and pb2A-2, common motifs were identified for each pair. These elements were neuron specific in that they were not found in the 500 bp upstream of any other Or gene in the genome. Shared motifs were likewise identified for (Or22a, Or22b) and (Or69aA, Or69aB), suggesting an additional mechanism by which the upstream and downstream ORFs could be coexpressed. A common element was found for a pair of Or genes that are coexpressed in a larval ORN (Or94a, Or94b). As a control, the same bioinformatics approach was carried on two genes that are not coexpressed, Or85f and Or56a, and no elements were found that met these criteria, that is, no common elements were found unique to these genes. Or19a and Or19b are coexpressed but appear to have duplicated recently and there is extensive identity in their upstream regions (Ray, 2007).

Odor receptor expression is permissive: lack of negative feedback regulation in Drosophila: In mammals, analysis of nonfunctional receptor genes has led to the proposal that the expression of one odor receptor inhibits the expression of others in the same ORN by negative feedback regulation. To investigate directly whether expression of an odor receptor inhibits the expression of any others in Drosophila, each of three receptors was ectopically expressed in the maxillary palp, and the effects on expression of others were tested. Expression of Or85e, which in wild-type is expressed only in pb2A, was driven in all or almost all ORNs of the maxillary palp using the C155-GAL4 driver, which initiates expression before the onset of normal Or expression. Then the expression of other maxillary palp Or genes was tested by in situ hybridization and electrophysiology (Ray, 2007).

It was confirmed by in situ hybridization that Or85e was in fact expressed in most if not all ORNs of the maxillary palp in C155-GAL4; UAS-Or85e flies. Then expression of the Or genes that are normally expressed in each of the other five maxillary palp ORN classes was examined. It was found that each tested gene was expressed in what appeared to be a normal pattern, in the presence of ectopic Or85e expression. Thus, expression of Or85e does not inhibit transcription of other Or genes (Ray, 2007).

It was then asked whether the other Or genes were functionally expressed, by carrying out electrophysiological recordings from C155-GAL4; UAS-Or85e flies, using a diagnostic odor panel that distinguishes between Or85e-expressing ORNs and all other classes of maxillary palp ORNs. Or85e responds strongly to fenchone; the pb1A neuron, by contrast, responds strongly to 2-heptanone, on account of Or42a expression, but not fenchone. When Or85e is overexpressed, neurons were detected that respond strongly to both fenchone and 2-heptanone. The simplest interpretation of these results is that misexpression of Or85e in pb1A cells does not inhibit functional expression of Or42a. Similar physiological evidence was found to indicate that expression of Or85e does not inhibit the functional expression of Or71a, Or46a, Or59c, and Or85d. In a more limited experiment, C155-GAL4 was used to drive the early expression of two other receptors, Or42a and Or10a (an antennal gene). By performing a similar electrophysiological analysis, it was found that in each ORN class in which a novel odor response was conferred by misexpression of Or42a or Or10a, the response conferred by the endogenously expressed Or gene was still present. These results, taken together, indicate that expression of one Or gene does not repress that of others by feedback regulation (Ray, 2007).

Coordination of receptor choice between two neurons of a sensillum: Each sensillum of the maxillary palp contains two ORNs, combined according to a strict pairing rule. For example, each pb1 sensillum contains a pb1A neuron that expresses Or42a, paired with a pb1B neuron that expresses Or71a; for the present analysis, this cellular expression pattern was designated as the (Or42a⁺; Or71a⁺) configuration. Such stereotyped pairing of ORNs has been documented in diverse insects but is not observed in mammals. It raises the problem of how the choice of a receptor in one ORN is coordinated with that in a neighboring ORN. In principle, the choice made by one ORN could induce a specific choice in the neighboring ORN; alternatively, the choices of two neighboring maxillary palp ORNs could be made simultaneously and be coordinated by virtue of the asymmetric segregation of regulatory proteins from a common progenitor cell (Ray, 2007).

In the development of antennal sensilla there is evidence that a single progenitor, or founder cell, recruits three secondary progenitor cells of which one, PIIc, divides to give rise to two neurons. Mastermind (Mam), a nuclear protein in the Notch pathway, is essential for asymmetric cell division of embryonic neuroblasts in the developing Drosophila nervous system: both loss and gain of mam function result in altered identities of the daughter cells. The expression of Or genes was systematically investigated by paired ORNs in maxillary palps that misexpress mastermind (mam) (Ray, 2007).

First UAS-mam was expressed in the developing olfactory organs using the eyeless-GAL4 driver. Then receptor gene expression was examined by double-label in situ hybridization. It was found that the coordination of receptor gene expression between neighboring ORNs was abnormal in all three types of sensilla, pb1, pb2, and pb3. A substantial fraction of sensilla expressing Or42a showed expression of this gene in both neurons: these sensilla contained one ORN that expressed Or42a alone and another that expressed both Or42a and Or71a, designated the (Or42a⁺; Or42a⁺Or71a⁺) configuration. Likewise, sensilla were found in (Or85e⁺; Or85e⁺Or46a⁺) and (Or59c⁺; Or59c⁺Or85d⁺) configurations; in wild-type, these genes are expressed only in (Or85e⁺; Or46a⁺) and (Or59c⁺; Or85d⁺) configurations. No other abnormal configurations were identified: those ORNs that misexpressed an Or gene always misexpressed the Or gene of the neighboring ORN (Ray, 2007).

This abnormal partitioning of receptor expression was confirmed by an independent method, electrophysiological recordings. Specifically, in the wild-type pb2 sensillum, the A cell produces large spike amplitudes and strong responses to fenchone, whereas the B cell produces small spike amplitudes and strong responses to 4-methyl phenol. In eyeless-GAL4; UAS-mam, sensilla were identified containing one neuron with a large spike amplitude and a strong fenchone response, paired with a neuron that produces a small spike amplitude and strong responses to both fenchone and 4-methyl phenol, as expected of (Or85e⁺; Or85e⁺Or46a⁺) sensilla. It was also confirmed the abnormal partitioning of receptor expression in another genotype, by driving a truncated dominant-negative form of mam, UAS-mamH, with the pan-neuronal driver elav-GAL4. Double-label in situ hybridization was carried out with Or85e and Or46a probes and identified (Or85e⁺; Or85e⁺Or46a⁺) sensilla (Ray, 2007).

These results are consistent with a model in which the A and B neurons of a maxillary palp sensillum are siblings that derive from a common progenitor by a Mam-dependent asymmetric cell division. Perturbation of Mam function might lead to the abnormal segregation of a regulatory protein, which in turn leads to misexpression of a receptor (Ray, 2007).

The observation that misexpression in an ORN was restricted to the Or gene of its neighbor, and not to any other Or genes, is consistent with a model in which the progenitor has undergone a restriction that limits its daughter ORNs to the expression of the Or genes of one sensillum type. It is noted that the data are also consistent with a model in which the progenitor, and both daughter ORNs, contain a positive regulatory factor that binds to a site shared by, and specific to, the Or genes expressed in a particular sensillum type. Computational analysis has in fact revealed such elements -- for example, Or42a and Or71a both contain an upstream AAATCAATTA element that is not found adjacent to other Or genes of the maxillary palp or antenna; however, genetic analysis of this element has not revealed a functional requirement for it in receptor gene expression, and no support was found for the existence of a sensillum-specific determinant of receptor gene expression (Ray, 2007).

Thus, these data, taken together, are consistent with a model in which the coordination of receptor gene expression in a sensillum is achieved through the Mam-dependent segregation of regulatory factors. The results underline the importance of identifying regulatory proteins whose proper distribution in the ORNs of the maxillary palp is essential to the proper distribution of receptor gene expression (Ray, 2007).

Different Or genes depend on different combinations of the transcription factors Lz and Acj6: In a complementary analysis of the mechanisms of receptor gene choice, transcription factors were investigated whose expression had been reported in at least one olfactory organ and whose mutations had been shown to cause olfactory defects. One such protein, the Runx domain-containing transcription factor Lozenge, was found had predicted binding sites (RACCRCA, R = purine) adjacent to four maxillary palp Or genes. Specifically, it was found that two maxillary palp Or genes, Or59c and Or85d, had two Lz binding sites, and two genes, Or71a and Or85e, had one Lz binding site, within 1 kb upstream or downstream of the coding region. Lz is required for the specification of cell fate in the eye and for normal numbers of olfactory sensilla in the antenna. In the maxillary palp the numbers of sensilla are normal, but electropalpogram recordings showed large reductions in odor responses (Ray, 2007).

To investigate the possibility that Lz is required for normal receptor gene expression, it was first asked whether it is expressed in ORNs of the maxillary palp. Lz is coexpressed with Elav, indicating that it is expressed in the nuclei of all maxillary palp ORNs. Then the expression of six maxillary palp Or genes was examined, one from each ORN class, in lz³, a strong hypomorphic mutant. The four genes that are flanked by predicted Lz binding sites all showed reduced levels of expression; the two genes that contain two Lz binding sites, Or59c and Or85d, showed particularly severe reductions (of 47% and 87%, respectively) in the number of labeled cells. The mildest reduction, 18%, was observed for Or85e; consistent with this result, a 14% reduction was observed when DNA including the predicted Lz binding site was removed from an Or85e-GAL4 driver (the construct containing 3 kb of upstream DNA labeled 13.4 ± 0.4 cells, whereas the construct containing 0.45 kb labeled 11.5 ± 0.3 cells; n = 12). The two genes that did not contain Lz binding sites did not show a reduction in labeling in lz³. These results demonstrate that lz is required for the expression of a subset of Or genes in the maxillary palp (Ray, 2007).

Next a weaker, temperature-sensitive allele, lz^ts1, was used to investigate the possibility that levels of Or gene expression are susceptible to modulation during the adult stage. It was found that Or85d is expressed in 18% fewer cells (p < 0.05) when lz^ts1 flies are raised at the restrictive temperature (29°) than when raised at the permissive temperature (18°). When flies were raised at the restrictive temperature and then shifted to the permissive temperature for 24 hr, 1 week after eclosion, the number of Or85d-expressing cells showed an increase of 19%, to a level indistinguishable from that of flies that had been cultured continuously at the permissive temperature. These results confirm the finding of a functional role for lz in Or expression, provide direct evidence that levels of Or expression can be altered after eclosion, and invite investigation of epigenetic modulation of odor receptor expression in Drosophila (Ray, 2007).

Only one other transcription factor, the POU domain protein Acj6, has previously been demonstrated to be required for odor receptor expression in Drosophila. Specifically, expression of Or33c, Or42a, Or46a, Or59c, and Or85e was severely reduced by the null allele acj6⁶, whereas expression of Or71a and Or85d was unaffected. It has been shown in this study that expression of Or59c, Or71a, Or85e, and Or85d was reduced by lz³, but expression of Or42a and Or46a was not. Thus, the maxillary palp Or genes can be divided into three classes based on their sensitivity to these mutations: those sensitive to both acj6⁶ and lz³ (Or59c and Or85e), to acj6⁶ alone (Or42a and Or46a), or to lz³ alone (Or71a and Or85d). These results support a model in which Or gene expression depends not only on a combinatorial code of regulatory elements but also on a combinatorial code of transcription factors (Ray, 2007).

In summary, in mammals, it is thought that transcriptional regulatory mechanisms direct expression of OR genes in specific zones of the olfactory epithelium, but that within a zone, OR gene choice is based on a stochastic selection mechanism. A third mechanism, negative feedback, could then operate to limit the number of OR genes expressed in individual neurons (Ray, 2007).

In Drosophila, the process of receptor gene choice achieves a conceptually simple end: it produces a highly stereotyped receptor-to-neuron map. However, the large number of receptors and neurons presents a regulatory problem of great complexity. To achieve such a precise and highly ordered organization, Drosophila has evolved a sophisticated suite of regulatory mechanisms. This study has documented organ-specific and neuron-specific levels of transcriptional control, including both positive and negative mechanisms. A posttranscriptional mechanism, alternative splicing, was identified and the system has even evolved a relatively rare innovation, dicistronic mRNAs (Ray, 2007).

The worm Caenorhabditis elegans has a much larger repertoire of odor receptor genes than Drosophila, but the number of ORNs to which it allocates them is very limited. Thus the number of receptor genes per neuron is increased, but the complexity of the regulatory problem is decreased. In vertebrates, however, the repertoire is very large and the number of receptor genes expressed per neuron is very low. Perhaps as the receptor gene repertoire expanded in vertebrate evolution, the complexity of the regulatory problem eventually exceeded the ability of the system to execute a deterministic plan with sufficient fidelity, and deterministic mechanisms were replaced by a stochastic mechanism and a negative feedback mechanism. In any case, the ultimate result of receptor gene choice in Drosophila is the same as in vertebrates: a spectacular diversity of ORNs that underlie the detection and discrimination of odors (Ray, 2007).

A regulatory code for neuron-specific odor receptor expression

Olfactory receptor neurons (ORNs) must select (from a large repertoire) which odor receptors to express. In Drosophila, most ORNs express one of 60 Or genes, and most Or genes are expressed in a single ORN class in a process that produces a stereotyped receptor-to-neuron map. The construction of this map poses a problem of receptor gene regulation that is remarkable in its dimension and about which little is known. By using a phylogenetic approach and the genome sequences of 12 Drosophila species, regulatory elements were systematically identfied that are evolutionarily conserved and specific for individual Or genes of the maxillary palp. Genetic analysis of these elements supports a model in which each receptor gene contains a zip code, consisting of elements that act positively to promote expression in a subset of ORN classes, and elements that restrict expression to a single ORN class. A transcription factor, Scalloped, was identifed that mediates repression. Some elements are used in other chemosensory organs, and some are conserved upstream of axon-guidance genes. Surprisingly, the odor response spectra and organization of maxillary palp ORNs have been extremely well-conserved for tens of millions of years, even though the amino acid sequences of the receptors are not highly conserved. These results, taken together, define the logic by which individual ORNs in the maxillary palp select which odor receptors to express (Ray, 2008).

The spatial organization was examined of ORN classes in the maxillary palp. First, an anti-Elav antibody was used to illustrate the distribution of the entire population of ORN nuclei of the maxillary palp. Second, a multiple-label experiment was carried out to differentially mark ORNs of the three types of sensilla: ORNs of the pb1A class were labeled in green, pb2B in yellow, and pb3A in red. The three classes of ORNs show extensive spatial overlap. These results are consistent with the intermingling of sensillum types that are observed when recordings are taken from sensillar shafts. The spatial overlap of ORN nuclei indicates that the identity of an ORN and, by extension, its choice of a receptor gene, are not dictated solely by its spatial position in a field (Ray, 2008).

The upstream regions of the two Or genes coexpressed in pb2A have been compared to identify regulatory sequences shared by these two genes, but not by any other maxillary palp Or gene. To identify upstream regulatory elements for the other five maxillary palp Or genes, a different strategy was used based on phylogenetic analysis (Ray, 2008).

D. melanogaster and D. pseudoobscura diverged tens of millions of years ago and contain orthologous receptor genes. The upstream regions of orthologous Or genes were examined for conserved elements shared by the members of each orthologous pair, but not by any of the other maxillary palp Or genes. Accordingly, all conserved upstream sequences were identified greater than 6 base pairs (bp) in length for each pair of orthologs using DOT-PLOT analysis, and from these conserved elements, those were selected that were specific to each gene. The analysis was focused on the 500 bp that are upstream of the translational start site, because in a previous study, this extent of DNA was sufficient to confer faithful expression to a GAL4 reporter gene in the case of each of two maxillary palp Or genes analyzed in detail. One pair of orthologs, Or85d and its D. pseudoobscura counterpart, was exceptionally well-conserved in the 500-bp upstream region, showing 80% identity. To identify discrete conserved elements within the region upstream of Or85d, the analysis was expanded to include a more divergent species, D. virilis (Ray, 2008).

Conserved, gene-specific elements were identified for each of the five Or genes analyzed. The number of such elements varies: Or59c contains one, whereas Or42a contains six. In the special case of Or85d, two elements are shared by D. virilis and D. melanogaster upstream of Or85d, but are not found upstream of any other maxillary palp Or gene (Ray, 2008).

To identify the best candidate for a regulatory element for each of these receptor genes, a powerful bioinformatic approach was used that takes advantage of the recent sequencing of the genomes of ten other Drosophila species: D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. persimilis, D. willistoni, D. virilis, D. mojavensis, and D. grimshawi. The upstream regulatory regions of the orthologous receptor genes from all 12 species were aligned using the genome browser at the University of California Santa Cruz, and each of the elements was mapped onto the alignment. Using this approach, it was possible to identify the gene-specific element with the highest sequence conservation for each of the receptor genes; in the case of Or42a, two elements were nearly identical in their extent of conservation, and both were analyzed (Ray, 2008).

To determine whether the evolutionarily conserved, gene-specific elements have a regulatory function, they were tested in vivo using two complementary approaches, one based on a loss of function and one on a gain of function. For each gene, the element was analyzed with the highest sequence conservation. Or85d elements were not analyzed because no faithful Or85d-GAL4 driver was available (Ray, 2008).

Or46a is expressed in the pb2B neuron, and its upstream region contains two conserved, gene-specific elements. One of these elements, 46a1, is more highly conserved. It is 10 bp long, its sequence shows 93% identity across the 12 species, and its position is conserved. A 1.9-kb region of DNA upstream of Or46a drives faithful expression of a GAL4 reporter in pb2B. However, when the 46a1 element is mutated, the 1.9-kb region no longer drives expression. In most cases, no cells are labeled; in rare cases, a single ORN is labeled. The simplest interpretation of these results is that the 46a1 element is necessary for Or46a expression in pb2B (Ray, 2008).

It was then asked whether the 46a1 element can drive expression in the context of a minimal promoter. Four copies of 46a1 upstream were placed of a TATA box, and it was found that this small construct can in fact drive expression in maxillary palp cells. Many, if not all, of the cells could be identified as ORNs, because they contain dendrites and axons; their identity is considered further below. Expression from this artificial promoter could also be detected in a small subset of neurons in the main gustatory organ, the labellum (Ray, 2008).

Or71a is expressed in pb1B. Its upstream region contains multiple gene-specific elements, of which the longest and best conserved is 71a3, consisting of 16 bp and showing 97% sequence identity. This element was tested in the context of the Or71a 5' + 3' construct, which contains sequences both upstream and downstream of Or71a. This construct drives faithful expression of GAL4 when the 71a3 element is intact, but not when it is mutated. When multiple copies of 71a3 were placed upstream of a TATA box, the construct drove GAL4 expression in maxillary palp cells that can be identified as ORNs by virtue of their dendrites and axons. Low levels of expression could also be detected in a small subset of cells in the labellum (Ray, 2008).

Or59c is expressed in pb3A, and its upstream region contains a single gene-specific conserved element, 59c1, which is 11 bp long and shows 97% sequence identity across nine species; the region containing the 59c1 sequences could not be identified in three of the most distantly related species, D. virilis, D. mojavensis and D. grimshawi. Its function was tested by placing multiple copies upstream of a TATA box and it was found that this minimal promoter drives robust expression of GAL4 in the maxillary palp. Expression was not detected in the labellum (Ray, 2008).

Earlier studies have shown that the expression of a subset of the maxillary palp Or genes requires the POU domain transcription factor Acj6, which is expressed in all ORNs of the maxillary palp. Acj6 also controls axon targeting specificity of a subset of maxillary palp ORNs . The 46a1, 71a3, and 59c1 elements do not contain predicted Acj6 binding sites, and the transcription factors that act on these sequences are unknown. To test whether the factors that act on these neuron-specific elements are dependent on acj6, the expression of the minimal promoter constructs was examined in an acj6⁶ background (Ray, 2008).

In the acj6⁶ mutant, although the expression of the Or46a-GAL4 driver is lost, which is consistent with the loss of Or46a mRNA observed previously, the expression of the 46a1 minimal promoter construct is still strong. These results suggest that the factors that direct expression from the 46a1 motif are independent of acj6 for their expression and function. An alternative possibility is that another transcription factor can compensate for the loss of acj6 (Ray, 2008).

Expression of the Or71a-GAL4 driver can be detected in acj6, and the expression of the 71a3 minimal promoter construct can also be detected. These results suggest that the factors binding to 71a3 do not require acj6 for their expression or function (Ray, 2008).

In the case of Or59c, it was found that acj6 is required both for expression of the gene and for the minimal promoter. These results suggest that acj6 is required directly or indirectly for the expression of the 59c1 binding factor or for its function at the 59c1 site (Ray, 2008).

Or42a is expressed in pb1A, and 4.1 kb of upstream DNA drives faithful expression of GAL4 in maxillary palp ORNs. Two elements are nearly identical in their high conservation: 42a4 (98%) and 42a6 (98%), and the function of both elements was tested in vivo. 42a6 maps only three bp from 42a5. A small deletion was constructed that eliminates both 42a6 and 42a5 elements, and no effect was found on Or42a-GAL4 expression (Ray, 2008).

The longer of the two most highly conserved elements at Or42a, 42a4, contains an inverted repeat: AGTGTAAAAGTTTACACTT. Surprisingly mutation of this element led to a 2-fold increase in the number of labeled maxillary palp cells, from 18.2 ± 1.8 to 33.2 ± 3.7. The simplest interpretation of this result is that 42a4 is a negative regulatory element that represses Or42a in a subset of ORNs. To test this interpretation, a double-label experiment was carried out using probes for the endogenous Or42a mRNA and for the green fluorescent protein (GFP) that is driven by the mutant promoter via GAL4. It was found that all Or42a⁺ cells express GFP, but that GFP is also expressed in an additional subset of cells (Ray, 2008).

To identify the cells that ectopically express GFP, a series of additional double-label experiments was undertaken. It was found that the GFP⁺ cells do not express Or59c mRNA, indicating that they are not pb3A neurons, nor are they paired with cells that express Or59c mRNA, indicating that they are not pb3B neurons. In another experiment, GFP⁺ cells did not label with an Or33c probe, indicating that they are not pb2A neurons; however, GFP⁺ cells were often found paired with Or33c⁺ cells, indicating that many GFP⁺ cells are pb2B neurons. The identity of these GFP⁺ cells as pb2B neurons was confirmed directly in another double-label experiment using a probe for Or46a mRNA (Ray, 2008).

The simplest interpretation of these results is that positive regulatory elements in the Or42a upstream region are capable of driving expression not only in the pb1A neuron but also in the pb2B neuron. The 42a4 element represses expression in pb2B neurons, thereby restricting expression to a single ORN class, pb1A (Ray, 2008).

The ectopic expression of an Or42a promoter in Or46a⁺ neurons suggested a relationship between these two genes. Further evidence for a relationship came from analysis of the minimal promoter containing multiple copies of 46a1. This promoter drove GFP expression in more ORNs than could be accounted for by Or46a⁺ neurons alone. A double-label experiment showed that while most of the GFP⁺ cells are in fact Or46a⁺, some are Or42a⁺ (Ray, 2008).

The reciprocal relationship between Or42a and Or46a misexpression suggests that Or42a may contain an unidentified positive regulatory element, 42ax, that is similar in sequence to 46a1, with both sites able to bind a transcription factor present in both pb1A and pb2B. To test this interpretation, the 500 bp upstream region of Or42a was examined for an element similar, but not identical, to 46a1 (GACATTTTAA). A sequence, TATATTTTAA, was identified identical to 46a1 at the 8 underlined positions, at -455 bp. Moreover, these two sequences share an ATTTTA core, which has been shown to function as a binding site for basic helix-loop-helix transcription factors at other loci. TATATTTTAA is not found upstream of any other maxillary palp Or genes. This 42ax sequence is conserved in sequence (80% identity) and location in seven of the 12 Drosophila species. It will be interesting to identify the transcription factor that binds 46a1 and then test directly its binding to 42ax (Ray, 2008).

When DNA upstream of Or59c was fused to GAL4, expression of the reporter GFP was not faithful; the same result was obtained when upstream regions of varying lengths were used (either 2.1 kb, which extends to the next upstream gene, or 5.2 kb, which includes upstream coding sequences). Double-label experiments using an Or59c probe revealed misexpression in many Or59c^– cells; moreover, many Or59c⁺ cells did not express GFP. Some of the misexpressing cells are the neighboring pb3B neurons, which can be seen to be paired with Or59c⁺ pb3A cells. To identify the other ORNs that ectopically express the Or59c-GAL4 construct, double-label experiments were carried out with other Or genes. Misexpression was also observed in pb1A cells, which express Or42a, but not in the pb1B cells, nor in the pb2A or B cells. In summary, misexpression is specific to pb1A and pb3B (Ray, 2008).

Because neither of the varying lengths of upstream DNA sequences were sufficient to restrict GAL4 expression to the Or59c⁺ cells, 3' sequences were added to the construct. Initially, 500 bp of DNA taken directly from the region immediately downstream from the Or59c stop codon was added downstream of the GAL4 coding region. Between the downstream sequences of Or59c and the GAL4 coding region was the Hsp70 3' untranslated region (UTR), which is present in the GAL4 vector and which is often present in promoter-GAL4 analysis (Ray, 2008).

This Or59c 5' + 3' construct showed much less misexpression in Or59c⁻ cells. The total number of GFP⁺ cells declined from 49.7 ± 1.3 to 27.3 ± 2.1. However, some misexpression remained, and only 62% of the Or59c⁺ neurons were GFP⁺. Then the Hsp70 3' UTR sequences were removed, such that the Or59c downstream sequences were in close proximity to the 3' end of the GAL4 coding region and the Or59c 3' UTR is used. This construct drove faithful expression. Thus, there is a negative regulatory element downstream of Or59c that restricts expression of this gene to pb3A neurons, and either there is a requirement that the native 3' UTR be used, or else there is a regulatory factor that acts on this element in a context-dependent fashion in order to achieve this negative regulation. It is noted with interest that the inclusion of the downstream sequences, without the Hsp70 sequences, also drove expression in Or59c⁺ neurons that had previously failed to express the reporter, suggesting that the downstream sequences are required for positive as well as negative regulation of Or59c (Ray, 2008).

Inspection of the sequences downstream of Or59c that repressed misexpression revealed a binding site for the transcription factor Scalloped (Sd), AAATATTT. This site is well-conserved among a number of other species. Sd has been shown to be expressed in olfactory organs. To confirm and extend the description of sd expression an enhancer trap line, sd^ETX4 was used, and it was confirmed that sd is expressed in a subset of cells in the maxillary palp (Ray, 2008).

To test whether sd represses Or59c, in situ hybridizations were carried to the maxillary palp of a hypomorphic sd mutant, sd¹. A 40% increase was found in the number of Or59c⁺ neurons. By contrast, there was no increase in the number of Or42a⁺ neurons. There was, however, an increase in the number of Or85d⁺ cells, and it is noted with interest that there is another type of Sd binding site, TAAAATTA, 737 bp downstream from the stop codon of Or85d (Ray, 2008).

The Or59c-GAL4 construct that contains only upstream sequences, Or59c 5', misexpresses in two ORN classes, the neighboring pb3B cell (Or85d⁺) and pb1A (Or42a⁺). It was asked whether sd is expressed in these two ORN classes. Using an Or59c probe, which labels the pb3A cell, it was found that sd is in fact expressed in neighboring cells, but not in pb1A cells, which express Or42a. These results suggest that Sd may repress the Or59c gene in pb3B. If so, it would be expected that in an sd mutant, cells would be observed that coexpress Or59c and Or85d. This possibility was tested by carrying out double-label in situ hybridizations in two different hypomorphic alleles of sd, sd¹, and sd^SG29.1. In both alleles, Or59c⁺ Or85d⁺ cells were found, but not Or59c⁺ Or42a⁺. Thus repression of Or59c in the neighboring pb3B cell requires both a Sd binding site and Sd (Ray, 2008).

Since Sd represses Or59c in pb3B, why doesn't Sd also repress Or85d in pb3B, given that both Or genes have Sd binding sites? The simplest explanation is that the two Sd binding sites are distinct. There are several potential interacting partners with which Sd may interact to form a functional transcription factor, and the pb3B cell may contain a partner necessary for repression at the Or59c binding site but not a partner necessary for repression at the Or85d binding site. If a faithful Or85d-GAL4 construct becomes available, it will be interesting to replace the Or85d-type Sd binding site with the Or59c-type Sd binding site, to determine whether the Or59c-type site confers repression in the pb3B cell (Ray, 2008).

It is noted that Or85d-GAL4 constructs containing only the 5' regions of Or85d, which lack the Sd binding site, drive misexpression in a number of non-neuronal cells of the maxillary palp. Most of the labeled cells lack dendrites and axons, and when labeled with a membrane-bound GFP, as opposed to with RNA probes that label the cell bodies, these cells appear larger than ORNs. These results suggest that Sd may interact with a binding partner in non-neuronal cells to repress Or85d expression in these cells (Ray, 2008).

Or42a is expressed in the larval olfactory system as well as in the maxillary palp. The Or42a-GAL4 construct shows expression in one ORN in each of the bilaterally symmetric larval olfactory organs, the dorsal organs. Expression was also observed in two neurons of the labellum, the taste organ on the adult head. To determine whether the conserved elements identified in analysis of maxillary palp receptor choice can act in these other chemosensory organs, Or42a-GAL4 constructs were examined in which these elements were mutated. A mutation that affects both 42a6 and 42a5, which did not affect expression in the maxillary palp, had no effect on expression in these other organs. However, mutation of 42a4, which relieved repression of Or42a in other maxillary palp ORNs, also relieved repression of Or42a-GAL4 in the larval olfactory organs and the labellum: in both cases supernumerary neurons were labeled. In the labellum, ~8-10 pairs of neurons were labeled. These results suggest that the molecular mechanisms underlying receptor gene choice in the maxillary palp overlap with those specifying receptor expression in other chemosensory organs (Ray, 2008).

This study has identified and functionally characterized a number of regulatory elements that operate in directing the formation of the receptor-to-neuron map of D. melanogaster. Because the newly defined elements analyzed in this study are conserved in sequence and position among Drosophila species, it is predicted that the programmed regulation leading to the formation of receptor-to-neuron maps would be conserved as well. To test this prediction, a physiological analysis of the D. pseudoobscura maxillary palp was carrie out . Although each of the seven Or genes expressed in the maxillary palp has an ortholog expressed in the D. pseudoobscura maxillary palp, it is expected that their odor response profiles would have diverged a great deal over the course of tens of millions of years. It was not known a priori whether it would be possible to correlate D. pseudoobscura ORNs with D. melanogaster counterparts (Ray, 2008).

It was surprising to find that the profiles of the maxillary palp ORNs are remarkably well conserved between these two species. Despite the tens of millions of years of separation, each ORN class in D. melanogaster has a counterpart in D. pseudoobscura, and their responses to a panel of ten diverse odorants are strikingly similar. Not only are the magnitudes of the responses well conserved, but the modes of the responses, i.e., excitation versus inhibition, are conserved. For example, both the pb2B ORN of D. melanogaster and its D. pseudoobscura counterpart are excited by 4-methyl phenol and inhibited by 3-octanol. The orthologous receptors show amino acid identity as low as 59% in the case of Or71a, and in no case exceeded 84%, the identity determined for Or42a. Thus pb1B in D. melanogaster, which expresses Or71a, shows the same specificity for 4-methyl phenol and 4-propyl phenol as the corresponding ORN in D. pseudoobscura, although Or71a is only 59% identical between the two species (Ray, 2008).

The conservation of odor response spectra allows determination of whether the stereotyped pairing of ORNs is also conserved in the two species. The results suggest that not only are the response spectra of the odor receptors conserved with respect to a diverse panel of odorants, but that the program of receptor gene expression is also conserved between these distantly related species (Ray, 2008).

Given the success in identifying gene-specific elements required for the expression of individual Or genes in individual classes of ORNs, it was asked whether the same approach could be used to identify sensillum-specific elements required uniquely by the Or genes that are expressed in the neighboring ORNs of a common sensillum. Sensillum-specific elements were sought conserved in the upstream regions of D. melanogaster and D. pseudoobscura Or genes. Only one element, AAATCAATTA, was found upstream of all orthologs expressed in a particular sensillum type. Mutational analysis of this element in the Or42a promoter did not, however, appear to affect expression. Furthermore, expression was not affected by mutation of the more proximal of the two copies of this element in the Or71a upstream region. These results suggest that this element is not required for expression in the pb1 sensillum (Ray, 2008).

This study has concentrated on receptor gene choice in the maxillary palp, on account of its numerical simplicity. Does a system of molecular zip codes also underlie the process of receptor gene choice across the entire odor receptor repertoire? In addition to the seven maxillary palp receptors, the Or gene family contains 53 other members expressed in the antenna or the larval olfactory system. Using a comparative bioinformatic approach, a large-scale analysis was performed of sequence conservation in the 500 bp upstream of each of 42 Or genes across all 12 Drosophila species. Great diversity was found in the number, lengths, and distribution of highly conserved upstream regions. Within the most highly conserved of these regions a variety of elements were identified that are shared among subsets of Or genes. This analysis, then, reveals a combinatorial structure to the organization of shared elements upstream of these receptor genes. This pattern supports a model in which a combinatorial code of positive and negative regulatory elements dictates the proper expression of each Or gene (Ray, 2008).

What kind of proteins accomplish this regulation? In C. elegans, several kinds of transcription factors have been elegantly shown to play roles in specifying ORN identity and receptor expression. In the mouse, a LIM-homeodomain protein, Lhx2, is required for normal ORN differentiation and expression of OR genes. In Drosophila the POU domain protein Acj6 is required for the expression of a subset of Or genes. This study has shown that Sd, a TEA domain-containing transcription factor, is critical in restricting the expression of some Or genes to their proper ORNs. Sd has been shown to act as a repressor in other systems and in fact is required for normal taste behavior in both larvae and adults. Another aspect of receptor gene choice depends on proteins of the Notch pathway: receptor choice in neighboring ORNs of a sensillum appears to be coordinated via asymmetric segregation of regulatory factors from a common progenitor (Ray, 2008).

Some elements that are essential to odor receptor gene choice are also located upstream of genes required for axon guidance and sorting. The presence and positions of these elements have been conserved for tens of millions of years of evolution. The presence of Or regulatory elements upstream of ORN axon-guidance genes could reflect a relationship between receptor gene choice and axon targeting. In addition to selecting particular Or genes for expression, ORNs send axons to particular glomeruli in the antennal lobe of the brain. ORNs that express the same Or gene send axons to the same glomerulus. Thus the olfactory system contains both a stereotyped receptor-to-neuron map and a stereotyped connectivity map in the antennal lobes. The tight coordination between receptor gene choice and axonal projection could in principle arise in part from overlap in the mechanisms underlying these processes. In mammals, odor receptors play a role in ORN targeting. In Drosophila, ORN targeting does not require the receptors, but could require the regulatory apparatus used to express the receptors. Acj6 provides an example of a link between the two processes: it acts both in receptor expression and ORN axon targeting. Moreover, it was found that Acj6 is required for the activity of one of the regulatory elements identified in this study (Ray, 2008).

This study found a remarkable similarity of function between the maxillary palp ORNs of two species that diverged more than tens of millions of years ago. It had been expected that over this time interval, the odor specificities of the ORNs would have diverged markedly to serve differing needs of the two evolving species. Instead, every ORN class showed strikingly similar responses, with few exceptions. The results show that two odor receptors can differ a great deal in amino acid sequence and still exhibit a very similar odor specificity (Ray, 2008).

The organization of the organ in the two species is also identical, in that corresponding ORNs are combined according to the same pairing rules. This high degree of conservation suggests a critical role for the maxillary palp in odor coding and in the generation of olfactory-driven behavior. The conservation of regulatory elements and organization also suggests that the two species use common mechanisms to specify the receptor-to-neuron map (Ray, 2008).

The regulatory challenge confronted by the Drosophila olfactory system represents an extreme among problems of gene regulation. It requires the storage and deployment of a great deal of information. These data support a model in which Or gene expression is controlled by a system of molecular zip codes. Each Or gene contains elements that dictate expression in the proper olfactory organ, positive regulatory elements that specify expression in a subset of ORN classes, and negative regulatory elements that restrict expression to a single ORN class. This logic and the components that execute it have solved such a challenging problem with such efficiency that they have apparently been well conserved for tens of millions of years (Ray, 2008).

Related sites:

• Abnormal chemosensory jump 6, a regulator of odorant receptor expression
• Lush, an odorant binding protein
• no receptor potential A codes for an enzyme involved in odor signal transduction in maxillary palps
• Odor coding in the Drosophila maxillary palp
• Genes expressed in the antennal portion of the eye-antennal disc
• Retrieve publications on olfactory receptors or on olfaction from PubMed that have been published within the last 90 days.

REFERENCES

Ache, B. W. and Young, J. M. (2005). Olfaction: diverse species, conserved principles. Neuron 48: 417-430. PubMed Citation: 16269360

Banerjee, S., et al. (2006). Compensation of inositol 1,4,5-trisphosphate receptor function by altering sarco-endoplasmic reticulum calcium ATPase activity in the Drosophila flight circuit. J. Neurosci. 26: 8278-8288. PubMed Citation: 16899722

Barth, A. L., Justice, N. J., and Ngai, J. (1996). Asynchronous onset of odorant receptor expression in the developing zebrafish olfactory system. Neuron 16(1): 23-34. Medline abstract: 8562087

Benton, R., Vannice, K. S. and Vosshall, L. B. (2007). An essential role for a CD36-related receptor in pheromone detection in Drosophila. Nature 450: 289-293. PubMed Citation: 17943085

Benton, R., Vannice, K. S., Gomez-Diaz, C. and Vosshall, L. B. (2009). Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell 136(1): 149-62. PubMed Citation: 19135896

Buck, L. and Axel, R. (1991). A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65(1): 175-87. Medline abstract: 1840504

Buck, L. B. (1996). Information coding in the vertebrate olfactory system. Annu. Rev. Neurosci. 19: 517-44. Medline abstract: 8833453

Chiu, J., et al. (1999). DeSalle R, Lam H, Meisel L, Coruzzi G. Molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals diverged. Mol. Biol. Evol. 16: 826-838. PubMed Citation: 10368960

Clyne, P. J., et al. (1999a). A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22(2): 327-38. Medline abstract: 10069338

Clyne, P. J., et al. (1999b). The odor specificities of a subset of olfactory receptor neurons are governed by Acj6, a POU-domain transcription factor. Neuron 22(2): 339-47. Medline abstract: 10069339

Couto, A., Alenius, M. and Dickson, B. J. (2005). Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr. Biol. 15: 1535-1547. PubMed Citation: 16139208

Dubos, C., et al. (2003). Huggins D, Grant GH, Knight MR, Campbell MM. A role for glycine in the gating of plant NMDA-like receptors. Plant J. 35: 800-810. PubMed Citation: 12969432

Hallem, E. A., Ho, M. G. and Carlson, J. R. (2004). The molecular basis of odor coding in the Drosophila antenna. Cell 117(7): 965-79. 15210116

Kain, P., Chakraborty, T. S., Sundaram, S., Siddiqi, O., Rodrigues, V. and Hasan, G. (2008). Reduced odor responses from antennal neurons of G_qalpha, phospholipase Cβ, and rdgA mutants in Drosophila support a role for a phospholipid intermediate in insect olfactory transduction. J. Neurosci. 28(18): 4745-55. PubMed Citation: 18448651

Kalidas, S. and Smith, D. P. (2002). Novel genomic cDNA hybrids produce effective RNA interference in adult Drosophila. Neuron 33: 177-184. PubMed Citation: 11804566

Lam, H. M., et al. (1998). Glutamate-receptor genes in plants. Nature 396: 125-126. PubMed Citation: 9823891

Lee, Y. J., Dobbs, M. B., Verardi, M. L. and Hyde, D. R. (1990). dgq: a Drosophila gene encoding a visual system-specific G alpha molecule. Neuron 5: 889-898. PubMed Citation: 2125225

Littleton, J. T. and Ganetzky, B. (2000). Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron 26: 35-43. PubMed Citation: 10798390

Malnic, B., et al. (1999). Combinatorial receptor codes for odors. Cell 96(5): 713-23. Medline abstract: 10089886

Matsunami, H. and Buck, L. B. (1997). A multigene family encoding a diverse array of putative pheromone receptors in mammals. Cell 90(4): 775-84. Medline abstract: 9288756

Mayer, M. L., et al. (2006). Crystal structures of the kainate receptor GluR5 ligand binding core dimer with novel GluR5-selective antagonists. J. Neurosci. 26: 2852-2861 . PubMed Citation: 16540562

Naur, P., et al. (2007). Ionotropic glutamate-like receptor delta2 binds D-serine and glycine. Proc. Natl. Acad. Sci. 104: 14116-14121. PubMed Citation: 17715062

Qi, Z., Stephens, N. R. and Spalding, E. P. (2006). Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile. Plant Physiol. 142: 963-971. PubMed Citation: 17012403

Ratnaparkhi, A., Banerjee, S. and Hasan, G. (2002). Altered levels of Gq activity modulate axonal pathfinding in Drosophila. J. Neurosci. 22: 4499-4508. PubMed Citation: 12040057

Ray, A., van Naters, W. G., Shiraiwa, T. and Carlson, J. R. (2007). Mechanisms of odor receptor gene choice in Drosophila. Neuron 53(3): 353-69. Medline abstract: 17270733

Ray, A., van der Goes van Naters, W. and Carlson, J. R. (2008). A regulatory code for neuron-specific odor receptor expression. PLoS Biol 6: 1069-1083. PubMed Citation: 18846726

Robertson, H. M. (1998). Two large families of chemoreceptor genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae reveal extensive gene duplication, diversification, movement, and intron loss. Genome Res. 8(5): 449-63. Medline abstract: 9582190

Sato, K., Pellegrino, M., Nakagawa, T., Nakagawa, T., Vosshall, L. B. and Touhara, K. (2008). Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452(7190): 1002-6. PubMed citation: 18408712

Scott, K., Becker, A., Sun, Y., Hardy, R. and Zuker, C. (1995). Gq alpha protein function in vivo: genetic dissection of its role in photoreceptor cell physiology. Neuron 15: 919-927. PubMed Citation: 7576640

Sullivan, S. L. et al. (1996). The chromosomal distribution of mouse odorant receptor genes. Proc. Natl. Acad. Sci. 93(2): 884-8. Medline abstract: 8570653

Talluri, S., Bhatt, A. and Smith, D. P. (1995). Identification of a Drosophila G protein alpha subunit (dGq alpha-3) expressed in chemosensory cells and central neurons. Proc. Natl. Acad. Sci. 92: 11475-11479. PubMed Citation: 8524786

Troemel, E. R., et al. (1995). Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans. Cell 83(2): 207-18. Medline abstract: 7585938

van der Goes van Naters, W. and Carlson, J. R. (2007). Receptors and neurons for fly odors in Drosophila. Curr. Biol. 17(7): 606-12. Medline abstract: 17363256

Vosshall, L. B., et al. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96: 725-736. Medline abstract: 10089887

Wicher, D. et al. (2008). Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452(7190): 1007-11. PubMed citation: 18408711

Yao, C. A., Ignell, R. and Carlson, J. R. (2005). Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna. J. Neurosci. 25: 8359-8367. PubMed Citation: 16162917

date revised: 30 November 2009

Zygotically transcribed genes

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