The Interactive Fly
Zygotically transcribed genes
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).
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).
(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.
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).
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, lzts1, 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 lzts1 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).
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date revised: 30 May 2008
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