Odorant receptor 22a and Odorant receptor 22b: Biological Overview | Developmental Biology | Effects of Mutation | References
Gene name - Odorant receptor 22a and Odorant receptor 22b
Cytological map positions - 22A2--3 and 22A2
Function - Odorant receptor
Symbol - Or22a and Or22b
Genetic map position -
Classification - G-protein coupled receptor
Cellular location - surface
Animals are able to sense and discriminate among a remarkable number of odors. Olfactory information is received and encoded by olfactory receptor neurons (ORNs). These neurons encode the quality and intensity of odors, as well as aspects of their spatiotemporal distribution. The code is in the form of action potentials and is based on the differential responses of ORNs to different olfactory stimuli. The signals generated by ORNs are transmitted to the brain, where processing takes place (Dobritsa, 2003).
ORNs vary in their odor specificity, sensitivity, and response dynamics. The cellular basis of the odor code has been explored in detail in Drosophila, whose relatively simple olfactory system allows precise physiological measurements of individual ORNs in vivo. Flies contain two olfactory organs, the antenna and the maxillary palp, which contain ~1200 and ~120 ORNs, respectively (Stocker, 1994; Shanbhag, 1999, 2000). These ORNs are compartmentalized in olfactory sensilla, which divide into morphologically distinct classes, including large basiconic sensilla, small basiconic sensilla, trichoid sensilla, and coeloconic sensilla. Each sensillum contains up to four neurons, whose activities can be defined by extracellular electrophysiological recordings (Dobritsa, 2003 and references therein).
In Drosophila, extensive recordings have revealed that ORNs fall into distinct functional classes based on their odor response spectra. Sixteen functional classes of ORNs, each with a unique response spectrum to a panel of 47 odors, were identified from recordings of antennal basiconic sensilla (De Bruyne, 2001). These ORNs exhibit diverse response dynamics, including excitatory and inhibitory responses, and various modes of termination kinetics. The 16 ORN classes are found in stereotyped combinations in seven functional types of basiconic sensilla, each mapping to a defined subregion of the antennal surface (Dobritsa, 2003).
Functional differences among ORN classes are believed to arise from the expression of different odor receptors. A family of at least 60 seven-transmembrane-domain receptor genes, the Or genes, was discovered in Drosophila and proposed to encode odor receptors (Clyne, 1999a; Gao, 1999; Vosshall, 1999, 2000). Individual Or genes are expressed in different subsets of ORNs. A mutation that alters the expression of a subset of Or genes alters the odor specificity of a subset of ORNs (Clyne, 1999b), and direct evidence (Störtkuhl and Kettler, 2001; Wetzel, was recently found for the involvement of one Or gene in olfactory signaling (Dobritsa, 2003).
The isolation of Or genes and the functional identification of discrete ORN classes by physiological analysis now allows a critical problem to be explored in Drosophila: the integration of the molecular and cellular maps of the olfactory system. Three means of mapping the receptor repertoire to the neuronal repertoire are demonstrated in this study. Moreover, since the odor specificities of the neurons are defined, the results by extension map receptor space to odor space. This approach allows an integrated molecular and cellular definition of the basis of odor coding (Dobritsa, 2003).
Individual receptors are demonstrated to map to individual neuronal classes through a genetic and molecular analysis of two Or genes, Or22a and Or22b. There are three functional types of large basiconic sensilla, ab1, ab2, and ab3, defined on the basis of electrophysiological recordings from the ORNs they contain. The sensilla expressing Or22a and Or22b contain an A neuron whose strongest responses are to ethyl butyrate, pentyl acetate, and ethyl acetate and a B neuron whose strongest responses are to heptanone, hexanol, and octenol. The Or22a receptor is shown to map to the ab3A neuron, by using the Or22a promoter and the GAL4-UAS system to drive expression of GFP or the cell death gene reaper, followed by physiological recordings from individual sensilla. The Or22a receptor is thereby linked to the odor ethyl butyrate, to which ab3A is highly sensitive (Dobritsa, 2003).
Analysis of a mutant lacking Or22a, together with rescue experiments using an Or22a transgene, confirm the mapping of Or22a to the ab3A neuron. This genetic analysis provides direct evidence that an Or gene is required in vivo for normal odor detection (Dobritsa, 2003).
Evidence is provided that Or22b is coexpressed with Or22a in the same cell but that Or22b is neither necessary nor sufficient for ab3A function; rather, the broad response spectrum of the ab3A neuron is accounted for by a single receptor. Ectopic expression of another receptor, Or47a, in the mutant ab3A neuron is used to identify the ORN from which Or47a derives and to determine its odor specificity. These results show that the odor response spectrum of an ORN in Drosophila depends on the Or gene that it expresses. Expression of Or47a in a wild-type ORN shows that two receptors are able to function in a single cell (Dobritsa, 2003).
Finally, the possibility of a developmental role for Or genes is addressed, a possibility that has not been systematically investigated by expression analysis and that can be rigorously determined only by functional analysis. The ab3A ORN is able to navigate toward its target in the CNS if Or22a and Or22b are deleted or substituted by other receptor genes (Dobritsa, 2003).
The demonstration that deletion mutants lacking Or22a and Or22b are defective in odor response and that the response is restored upon introduction of an Or22a transgene provide direct evidence that Drosophila Or genes are in fact critical components of olfactory signal transduction. The effect of the deletion mutation is specific: the mutation has a profound effect on the ab3A neuron but no other ORN among the large basiconic sensilla. The response of the ab3A cell is eliminated for all odors tested. Most ab3A neurons in mutant flies exhibited spontaneous electrical activity, albeit at a low rate, and thus the absence of Or22a/b does not lead to the immediate death of these neurons. The loss of Or22a/b does not appear to lead to the de novo expression of another functional receptor, suggesting that the process of receptor gene choice does not include a receptor-mediated negative feedback mechanism (Dobritsa, 2003).
A central problem in odor coding concerns the distribution of odor receptors among ORNs. A priori, there are several ways of distributing n receptor types among m functional classes of ORNs. Each receptor type could be expressed in a single ORN class, or, by contrast, in multiple, distinct ORN classes. If expressed in a single ORN class, then a receptor, Ri, could in principle be the only receptor expressed in its class, or it could be one of multiple receptors, e.g., (Ri, Rj), that are invariably coexpressed in that class. If a receptor is expressed in multiple, distinct ORN classes, then the ORN classes may be functionally distinct either because they express different combinations of receptors, e.g., (Ri, Rj) versus (Ri, Rk), or, conceivably, because each expresses the same receptor in different molecular contexts, (Ri; X) versus (Ri; Y), containing ORN class-specific differences in local OBPs, RAMPs, or heterodimerization partners, for example (Dobritsa, 2003).
The expression of the Or22a receptor was found to be limited to a single morphological subtype of olfactory sensillum (LB-I), a single functional type of sensillum (ab3), and a single class of ORN (ab3A). The mapping of Or22a to a single functional type of neuron argues against a model in which different neuronal classes acquire their diverse identities through the combinatorial expression of different receptors, or through the expression of a single receptor in different molecular contexts; according to these models, an individual receptor would be expressed in multiple, distinct neuronal classes (Dobritsa, 2003).
ORNs vary in the breadth of their odor response spectrum. Physiological recordings from individual ORNs have shown that some are narrowly tuned, whereas others are broadly tuned with respect to a panel of test odors (De Bruyne, 1999, 2001). For example, the ab5A cell responds to only one of 47 odors tested at relatively high doses, whereas the ab3A neuron responds to a variety of esters, alcohols, ketones, and other odors of varying chain lengths. The broad tuning specificity of ORNs such as ab3A could in principle be due either to the expression of multiple receptors or to the expression of a single receptor that is broadly tuned. The finding that deletion of Or22a and Or22b eliminates response to all tested odors, and that the full response spectrum can be rescued by Or22a alone, suggests that the broad response spectrum documented for ab3A can be attributed to one receptor, Or22a (Dobritsa, 2003).
The results with Or47a are also consistent with a model in which a single receptor accounts for the odor response profile of a particular ORN class. When expression of a single Or gene, Or47a, is driven in an ab3A cell lacking expression of Or22a and Or22b, Or47a confers an odor response profile like that of ab5B. These results support an interpretation in which the odor response profile of ab5B derives from the expression of a single Or gene, Or47a. Functional analysis of other Or genes will be required to determine the generality of these results, but they are consistent with observations made with mammalian ORNs, which are able to respond to diverse odors, apparently by virtue of expression of a single odor receptor in many (Malnic, 1999; Araneda, 2000; Bozza, 2002), if not all (Rawson, 2000; Spehr, 2002), cases (Dobritsa, 2003).
While these results are consistent with a model in which Or22a is the only receptor that functions in the ab3A neuron, the data force consideration of the possibility that Or22a is not the only receptor that is expressed in the ab3A neuron. Both Or22a-GAL4 and Or22b-GAL4 drive expression of GFP in ab3 sensilla and of rpr in ab3A neurons. The driver constructs were designed to mimic the expression of the endogenous Or22a or Or22b genes, respectively. In each construct, GAL4 coding sequences replace the coding sequences of the respective Or genes, and they include a substantial amount of DNA upstream of either Or22a or Or22b (8.2 and 10.3 kb, respectively). While it is formally possible that the expression of Or22b-GAL4 does not mimic the expression of Or22b in vivo, there is no evidence for expression of Or22b in ORNs of the fly other than ab3A: (1) in situ labeling with Or22b probes revealed no labeling outside the region of the antenna that contains ab3 sensilla; (2) immunolabeling showed no staining of sensilla other than LB-I in the wild-type antenna, nor the maxillary palp nor the larval olfactory organ. At the same time, it is clear that the antibody can recognize an Or22b product, since immunolabeling is observed in antennae lacking Or22a but expressing Or22b. Moreover, Or22b transcripts have been amplified from the antenna by RT-PCR or found in antennal/maxillary palp cDNA libraries in multiple laboratories (Clyne, 1999a; Vosshall, 1999). The simplest interpretation of all these data, taken together, is that Or22b is coexpressed with Or22a in ab3A neurons, but that Or22b is neither necessary nor sufficient for response to the odors used in this study (Dobritsa, 2003).
A functional role for Or22b is nonetheless suggested by the observation that an Or22b ortholog is present in D. simulans, which diverged from D. melanogaster ~2.5 million years ago. Most important, in neither species has the gene accumulated stop codons, frameshift mutations, or deletions. One possibility is that Or22b recognizes odors not tested in this study, such as pheromones. Another possibility is that it functions only under a specific set of epigenetic, e.g., environmental, conditions (Dobritsa, 2003).
Does the odor response spectrum of a cell depend exclusively on its receptor expression or on a more complex molecular context? In the case of Or47a, the substitution of Or47a for Or22a and Or22b engenders a transformation of the response spectrum from that of ab3A to that of ab5B. These results are consistent with evidence from other organisms that the odor response spectrum of an ORN depends on the odor receptor gene that it expresses (Dobritsa, 2003).
ORNs in insects are intimately associated with each other in sensillar compartments. In this study the activity of an ORN was measured following genetic manipulation of its neighbor. When the function of ab3A was severely compromised, either by mutation of Or22a and Or22b or by expression of the cell death gene rpr, the neighboring ab3B cell showed strong responses to odors. Thus, the ability of ab3B to respond to odors does not depend absolutely on the presence of a functional neighboring cell (Dobritsa, 2003).
At the same time, however, in both cases a large increase was noted in the response of ab3B to pentyl acetate. It is formally possible that this effect may arise to some extent from difficulties in counting the small ab3B spikes during intense activity of the ab3A cell. However, another interpretation is that in wild-type the activity of ab3A inhibits the response of ab3B; when this inhibition is relieved, ab3B exhibits an increased response. In some insects, neighboring ORNs in certain sensilla have been shown to respond to odors whose behavioral significance is related (Wojtasek, 1998; Grant, 1998). It seems plausible that information transmission between adjacent ORNs represents an early step in the processing of information carried by neighboring ORNs (Dobritsa, 2003 and references therein).
An emerging body of evidence in mammals indicates that odor receptor expression is essential to normal axonal pathfinding in the vertebrate olfactory system. The results of this study indicate, however, that the ab3A neuron finds its normal glomerular target in a mutant that lacks Or22a and Or22b expression, demonstrating that these receptors are not required for targeting. Moreover, ectopic expression of either of two other receptors did not cause alterations in the targeting. The possibility that Or22a and Or22b play a subtle role cannot be excluded, or that ab3A expresses an additional Or gene that plays a role in pathfinding, such as Or83b, which is expressed widely among ORNs (Vosshall, 1999, 2000) and whose function is unknown. However, the simplest interpretation of these results is that ab3A finds its glomerular targets through mechanisms independent of Or expression (Dobritsa, 2003).
One striking difference between the insect and mammalian olfactory systems is that in mammals, but not insects, ORNs are regenerated throughout adult life. Thus, pathfinding of mammalian ORNs toward their target glomeruli occurs during adult life, whereas in insects axonal pathfinding occurs only during development. Insect ORN pathfinding likely depends on a system of navigational cues that are expressed in an orchestrated temporal and spatial program. Perhaps the mammalian dependence on Ors, which are expressed both during development and during adult life, reflects the evolution of a mechanism designed to operate independently of signals that occur only transiently in development (Dobritsa, 2003).
A second difference between insect and mammalian olfactory systems is the greater numerical complexity of mammals. The number of ORNs and glomeruli in mammals exceeds that of Drosophila by more than an order of hmagnitude, and it is likely that the mammalian olfactory system accordingly requires more information to specify the larger number of connections. Perhaps the use of extant Ors to provide developmental cues may have been the most economical means of expanding the informational content of the navigational system during evolution (Dobritsa, 2003).
Many of the Drosophila OR 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, 1999a).
Demonstrating the expression of a single receptor gene in individual sensory neurons within the antenna is difficult since this olfactory organ expresses 32 members of the DOR gene family in ~1000 sensory cells. The lateral distal domain of the antenna contains about 300 neurons, 50 of which express Or47b. This same domain expresses 15 other receptors, including Or47a, Or23a, Or67a, Or43a, Or88a, Or49b, Or98a, and Or83c. In pairwise experiments, antennal sections were annealed with an Or47b antisense RNA probe and either individual or mixed probes for these eight additional receptors. RNA probes were labeled with either fluorescein or digoxigenin and visualized with antibodies that distinguish the two types of probes. Or47b is expressed in a subpopulation distinct from that expressing any of these eight receptors. A similar experiment was performed to examine DOR gene expression in the medial proximal region of the antenna. Or22a probes were labeled with fluorescein and a mix of probes complementary to five additional genes expressed in this domain (Or7a, Or56a, Or85b, Or42b, and Or59b) was labeled with digoxigenin. Despite the interspersion of Or22a cells with cells expressing other DOR genes in the domain, there is no overlap in the expression of Or22a with any of these five DOR genes. Taken together, these data provide strong support for a model reminiscent of the mammalian olfactory system, in which individual sensory neurons express only a single receptor. This conclusion must be tempered by the previous description of an odorant receptor gene, Or83b, that is expressed in all olfactory sensory neurons in both the antenna and maxillary palp. If Or83b does indeed recognize odorous ligands, then this data would indicate that all sensory neurons express two receptors rather than one: Or83b and one additional gene from the family of DOR genes (Vosshall, 2000).
These experiments suggest that there are 39 distinct neuronal cell types within the Drosophila olfactory organs. In the mammalian olfactory system, neurons expressing the same receptor project their axons to two spatially invariant glomeruli. It was therefore determined whether cells expressing a given receptor in Drosophila converge upon spatially defined loci in the antennal lobe, the first relay station for olfactory information in the fly brain. Axons from the antenna and maxillary palp have been found to synapse with the dendrites of projection neurons in the antennal lobe. There are 43 morphologically distinct synaptic structures or glomeruli in the antennal lobe that are invariant in position and size in individual flies. The number of antennal lobe glomeruli dedicated to olfactory input (41) approximates the number of different sensory neurons identified in the two olfactory organs (39), suggesting that olfactory neurons expressing a given receptor may converge on a single glomerulus (Vosshall, 2000).
Genetic experiments were performed that permit the visualization of the axonal projections from neurons expressing a given receptor. Transgenic flies were generated in which DOR gene promoters direct the expression of the yeast transcriptional activator, Gal4. These flies were then crossed with stocks bearing a transgene in which the Gal4-responsive promoter, UAS, drives the expression of either beta-galactosidase (LacZ) or a C-terminal fusion of green fluorescent protein (GFP) to neuronal synaptobrevin (nsyb-GFP). The expression of LacZ in specific subpopulations of sensory neurons allows the visualization of cell bodies, whereas the expression of nsyb-GFP, which selectively labels synaptic vesicles in nerve terminals, allows the visualization of terminal axonal projections (Vosshall, 2000).
Two to eight kilobases of DNA immediately upstream of the putative translation start from five DOR genes were fused to the coding sequence of Gal4. To demonstrate that these transgenes recapitulate the expression of the endogenous gene, the DOR-Gal4 strains were crossed with UAS-lacZ responders. The progeny of these crosses express LacZ in spatially defined subsets of cells in the antenna or maxillary palp that mirror the pattern of expression of the endogenous receptor. To confirm that these cells are sensory neurons, it was demonstrated that cells expressing LacZ are also labeled with an antibody that recognizes the neuron-specific RNA binding protein Elav. As a further test for the fidelity of expression of the promoter fusions, it was demonstrated that all cells expressing LacZ from the transgenes also express the corresponding endogenous odorant receptor RNA. In these experiments, RNA in situ hybridization was used to detect endogenous receptor RNA; immunofluorescence localized the expression of LacZ protein. Each of five DOR promoter fusions recapitulates the pattern of expression of the endogenous receptor gene (Vosshall, 2000).
The DOR-Gal4 transgenes were used to drive the expression of UAS-nsyb-GFP to visualize the projections of different populations of sensory neurons. Flies carrying five different DOR-Gal4 transgenes were crossed with animals bearing a UAS-nsyb-GFP transgene, and the brains of the adult progeny were then examined for localization of GFP. Immunofluorescence was performed on whole-mount brain preparations with antibody directed against GFP and with the monoclonal antibody, nc82, which labels neuropil in the fly brain and identifies the individual glomeruli in the antennal lobe. Four DOR-Gal4 promoter fusions (Or47a, Or47b, Or22a, and Or23a) are expressed in subpopulations of antennal neurons. When these flies are crossed with flies carrying the UAS-nsyb-GFP transgene, GFP labeling is seen in spatially invariant subsets of glomeruli within the antennal lobe. Or47a, for example, is expressed in 20 lateral distal neurons and their axonal projections converge on a single bilaterally symmetric glomerulus located at the dorsal medial limit of the antennal lobe. Or47b is expressed in 50 lateral distal neurons and these cells project axons that converge on a single large glomerulus that lies at the ventral lateral edge of the antennal lobe. Neurons expressing Or22a converge upon one dorsal glomerulus. The projections of one of the seven different subpopulations of sensory neurons from the maxillary palp have also been visualized. Sensory neurons expressing the palp receptor, Or46a, project to a single glomerulus that resides ventrally in the antennal lobe (Vosshall, 2000).
These four subpopulations of olfactory sensory neurons therefore project axons to single spatially invariant, bilaterally symmetric glomerulus within the antennal lobe. Cells expressing Or23a, however, send processes to two glomeruli. Or23a fibers enter the brain and initially converge upon a small dorsal glomerulus. Axons are seen extending more ventrally into the anterior of the antennal lobe where they converge upon a larger glomerulus. These two Or23a glomeruli are bilaterally symmetric and positionally invariant in multiple independent transgenic lines. It is not possible at present to determine whether individual fibers branch to form synapses within the two glomeruli or if Or23a-expressing neurons sort such that individual subpopulations project to either one of the two glomeruli (Vosshall, 2000).
The topographic map of all five populations of olfactory sensory projections in the antennal lobe is unrelated to spatial domains of receptor expression in the antenna. For instance, while the 20 neurons that express Or22a in a proximal medial domain of the antenna converge upon a dorsal medial glomerulus, the Or47a glomerulus is situated directly adjacent to the Or22a glomerulus but receives input from cells in the lateral distal domain of the antenna. These precise patterns of glomerular convergence were observed in at least 20 individuals derived from multiple independent transgenic lines obtained for each of the four constructs. At a low frequency (1/100 flies), Or47a-expressing olfactory neurons project to two bilaterally symmetric medial glomeruli in addition to the OR47a glomerulus. The basis for this variation in axon targeting is unknown (Vosshall, 2000).
In order to test the hypothesis that Or22a and Or22b encode odor receptors, and to examine the relationship between Or expression and sensillum type, the cellular and subcellular distribution of the Or22a and Or22b proteins were examined. For this purpose a polyclonal antibody was raised against a 16 amino acid sequence common to the N terminus of both proteins; this region was selected because it was predicted by bioinformatic analysis to generate effective antibodies. If Or22a and Or22b are in fact odor receptors, they would be expected to be localized to the dendritic membranes of ORNs. It was of interest to determine whether the labeled sensilla all belonged to the same morphological type of olfactory sensilla. In situ hybridization with Or22a and Or22b probes show labeling of cells in the dorso-medial region of the antenna but is of insufficient resolution to determine with precision the morphological types of sensilla associated with the labeled cells (Dobritsa, 2003).
The antibody to Or22a and Or22b common sequence stains a subset of the large basiconic sensilla in the dorso-medial region of the antenna. Labeling is clearly visible in the sensillum shaft, where the dendrites of ORNs are located. No labeling was observed in cell bodies or axons. No labeling was detected in the maxillary palp, the other adult olfactory organ, or in the larval antenno-maxillary complex, which mediates both larval olfaction and taste. Sexual dimorphism was observed: although both males and females show similar spatial patterns of labeling, the number of labeled sensilla in females, 29 ± 2, is greater than that in males, 18 ± 2 (Dobritsa, 2003).
Immunoelectron microscopy confirms that the labeled sensilla are large basiconic sensilla and allows a precise definition of their morphological subtype, LB-I. Sensilla of this subtype are characterized by a relatively thin cuticle, and they contain two ORNs per sensillum. In agreement with these findings, this subtype exhibits sexual dimorphism, with 33 LB-I sensilla present in females and 19 in males (Shanbhag, 1999). Immunoelectron microscopy further reveals that the label is distributed on the surfaces of dendrites, consistent with the expression of membrane receptors (Dobritsa, 2003).
Having determined that Or22a and Or22a localize to a particular morphological type of sensillum, the large basiconic sensilla, attempts were made to localize them to a functional type of sensillum. The seven well-characterized functional types of antennal basiconic sensilla show overlap in their distributions, but each type is restricted to a particular spatial domain on the antennal surface (De Bruyne, 2001). There are three functional types of large basiconic sensilla, ab1, ab2, and ab3, defined on the basis of electrophysiological recordings from the ORNs they contain. ab1 sensilla are easily recognized in such recordings because they contain four ORNs with distinct spike amplitudes or shapes, whereas ab2 and ab3 each house two ORNs (De Bruyne, 2001). All three sensillum types can be distinguished by measuring the response of their neurons to a panel of odors. Throughout this study ORN activity was monitored by recording action potentials, which provide a direct measure of ORN signaling (Dobritsa, 2003).
To determine in which functional type or types of sensilla Or22a/b are expressed, physiological recordings were made from live flies in which the Or22a/b-expressing sensilla were labeled with GFP. In brief, two strains of transgenic flies were generated in which the presumed promoters of Or22a or Or22b drive expression of the yeast transcription factor GAL4, which in turn drives expression of GFP. Recordings were then made from GFP-labeled sensilla, which allows the correlation of a particular receptor gene with a particular sensillum type (Dobritsa, 2003).
Specifically, to drive GAL4 under the control of the Or22a promoter, an 8.2 kb region upstream of the Or22a translational start codon was isolated and fused to the coding sequence of GAL4 to generate a construct referred to as 22a-GAL4. To drive GAL4 under the control of the Or22b promoter, a 10.3 kb region upstream of Or22b was isolated to generate 22b-GAL4. These 10.3 kb include the 8.2 kb upstream of Or22a, the Or22a coding sequence, and the intergenic region between Or22a and Or22b. Flies carrying the 22a-GAL4 or 22b-GAL4 transgenes were crossed to flies carrying UAS-GFP to yield progeny in which GAL4 binds to a UAS and activates transcription of GFP. The particular GFP derivative used was mCD8-GFP, which contains sequences of the mouse lymphocyte surface marker CD8 and which accordingly localizes to membranes. This derivative is hereafter referred to as 'GFP' for simplicity (Dobritsa, 2003).
It was found that 22a-GAL4 drives expression of GFP in a subset of large basiconic sensilla in the dorso-medial region of the antenna, and 22b-GAL4 shows an indistinguishable pattern. To confirm that GFP expression recapitulates the endogenous Or22a/b expression pattern, double-labeling experiments were performed with anti-Or22a/b and antibodies directed against GFP. All sensilla expressing GFP were found also to express Or22a/b in the case both of the 22a-GAL4 driver and the 22b-GAL4 driver (Dobritsa, 2003).
The GFP-labeled sensilla are visible in live animals, thereby allowing the sensilla to be distinguish and allowing them to be electrophysiologically recorded. Recordings from labeled sensilla of 22a-GAL4; UAS-GFP flies revealed that these sensilla house two neurons, as expected of ab2 or ab3, but not ab1. The labeled sensilla were tested with a diagnostic set of 11 odors that distinguish among the different types of large basiconic sensilla. The sensilla labeled with 22a-GAL4 are homogeneous in their response spectrum. They contain an A neuron whose strongest responses are to ethyl butyrate, pentyl acetate, and ethyl acetate (like the A neuron of the ab3 sensillum) and a B neuron whose strongest responses are to heptanone, hexanol, and octenol (like the B neuron of the ab3 sensillum). In the 22b-GAL4; UAS-GFP flies, the labeled sensilla are also homogeneous: they contain two ORNs each, and the ORNs yield a response pattern similar to that of ab3 (Dobritsa, 2003).
These results indicate that both 22a-GAL4 and 22b-GAL4 drive expression in the ab3 sensillum. It is noted further that the total number of ab3 sensilla estimated for the male antenna in the physiological studies of De Bruyne (2001) is 18, a number that agrees well with the number of sensilla labeled in males with the anti-Or22a/b antibody, 18 ± 2, and the number (19) of LB-I sensilla in males as determined in ultrastructural studies (Dobritsa, 2003).
Although all sensilla expressing GFP reacted with the anti-Or22a/b antibody, some anti-Or22a/b-reactive sensilla did not show expression of GFP (24.6% for 22a-GAL4; 9.6% for 22b-GAL4). A simple interpretation of this observation is that these two GAL4 lines are not completely expressive, an interpretation also drawn from experience with several other Or-GAL4 lines. In support of this interpretation, no physiological differences were found between GFP+ and GFP- ab3 sensilla in either 22a-GAL4; UAS-GFP or 22b-GAL4; UAS-GFP lines (Dobritsa, 2003).
To increase the resolution of mapping from sensillum type to neuron type, a modified strategy was adopted. Or promoter-GAL4 constructs and single-unit electrophysiology were used, but rather than using GAL4 to drive GFP, GAL4 was used to drive the cell death gene reaper (rpr). Specifically, it was asked whether Or22a-GAL4; UAS-rpr or Or22b-GAL4; UAS-rpr antennae lack a particular ORN (Dobritsa, 2003).
Recordings from ab3 sensilla of Or22a-GAL4; UAS-rpr flies do not show the large spikes characteristic of the ab3A neuron. By contrast, the small spikes characteristic of ab3B are present. To characterize the effect of rpr expression more fully, both Or22a-GAL4; UAS-rpr and Or22b-GAL4; UAS-rpr flies were tested with the entire panel of odors. In both genotypes, there is no response of the ab3A neuron to any tested odor. The ab3B neuron, however, responds strongly to all of the odors that elicit a response from a control line, Or22a-GAL4; UAS-GFP. The ORNs of the other types of large basiconic sensilla appeared normal in limited testing. These results indicate that both Or22a and Or22b drivers direct expression in the ab3A neuron (Dobritsa, 2003).
It is noted with interest that for some odors, most notably pentyl acetate, mean response of the ab3B neuron in the rpr lines is greater than that in the control. Further investigation will be needed to establish whether this effect reflects an inhibitory role of the ab3A cell on the activity of the neighboring ab3B cell in wild-type sensilla (Dobritsa, 2003).
The Deltahalo mutant, which is deleted for Or22a and Or22b, was used to investigate the relationship between Or expression and ORN response spectrum. One simple model is that the odor response spectra of ORNs are dictated solely by the Or genes they express. Alternatively, the response spectrum might be determined by an ensemble of molecules that includes not only an Or protein but also other proteins that are differentially distributed among the various sensilla and ORNs of the system. Such molecules could include odor binding proteins (OBPs) that might bind and deliver odors to the Or, other GPCRs that might heterodimerize with the Or, or other molecules such as RAMPs (receptor activity-modifying proteins) that might modulate the ligand specificity of the Or. To address this issue, the ab3A neuron of the Deltahalo mutant was used as a recipient for the expression of other Or genes (Dobritsa, 2003).
An antennal gene, Or47a, was expressed under the control of 22a-GAL4 in the Deltahalo mutant so as to drive its expression in ab3A cells. Large basiconic sensilla that contain ORNs with a response spectrum characteristic of the adjacent ab3B cell were then identified. The response of the cell neighboring the ab3B cell was analyzed and it was found that it responded to a subset of 11 odors tested. This neighboring cell responds most strongly to pentyl acetate, followed by 2-heptanone, a pattern similar to that of the ab5B neuron, which has been defined previously (De Bruyne, 2001). To test further the cell's similarity to ab5B, its response was measured to a panel of odors structurally related to pentyl acetate and to 3-methylthio-1-propanol, which elicits a much stronger response from ab5B than from any other characterized ORN on the antenna. The neuron adjacent to ab3B in flies ectopically expressing Or47a was found to exhibit a response spectrum similar to that of ab5B and distinct from that of ab3A. The simplest interpretation of all these results is that Or47a is expressed in ab5B and that it confers a response pattern similar to that of ab5B in the ab3A neuron when substituted for Or22a/b (Dobritsa, 2003).
Having established that Or47a is capable of functioning when driven by 22a-GAL4 in a Deltahalo background, i.e., in a cell that apparently contains no other functional receptors, it was asked whether Or47a can function when driven by 22a-GAL4 in a wild-type background, i.e., in a cell containing at least one other functional receptor, Or22a. Accordingly, a UAS-47a construct was expressed under the control of 22a-GAL4 and then response was tested across a broad concentration range to ethyl butyrate, which elicits a strong response from Or22a but a much weaker response from Or47a, and pentyl acetate, which elicits a strong response from Or47a but a weaker response from Or22a (Dobritsa, 2003).
Recordings from cells designed to express both Or22a and Or47a yielded a dose-response curve for ethyl butyrate that is identical to that of cells expressing Or22a but not Or47a. These results suggest that the expression of Or47a does not interfere with the expression and function of Or22a. Responses to pentyl acetate are greater in the cells expressing Or47a and Or22a than in the cells expressing Or22a alone, indicating that Or47a is able to function in a cell that is also expressing Or22a. The response to pentyl acetate of cells expressing both Or47a and Or22a is lower than that in cells expressing Or47a alone; one interpretation of this finding is that Or22a, which has a moderate response to pentyl acetate, competes with Or47a, which has a strong response to pentyl acetate, and the integrated signal sent by the cell in the form of action potentials is lower than in cells expressing only Or47a. In any case, the differing results observed in the cell when stimulated with ethyl butyrate versus pentyl acetate are informative: in the former case, the expression of Or47a appears to have no effect on Or22a, indicating that the introduction of Or47a does not block expression of Or22a or inhibit the cell nonspecifically, whereas in the latter case it appears to engender a response greater than that of Or22a alone, indicating that Or47a is expressed in a form that is capable of contributing to olfactory signaling, despite the presence of another receptor (Dobritsa, 2003).
To investigate the function of the Or22a and Or22b genes directly, a synthetic deletion that removes both genes was used, a deletion referred to hereafter as Deltahalo. Immunostaining of the mutant antenna with the anti-Or22a/b antibody reveals no labeling (Dobritsa, 2003).
Electrophysiological recordings from the large basiconic sensilla in mutant flies revealed that the ab3A neurons are unresponsive to all odors of the test panel. By contrast, the ab3B neurons in the mutant sensilla show a response spectrum similar to that of wild-type ab3B neurons, with the exception of an unexpectedly large response of the ab3B cell to pentyl acetate in the mutant, as observed in the rpr ablation experiments. All other neuronal classes in large basiconic sensilla appear normal as well, as judged by testing the four neuronal classes of ab1 and the two neuronal classes of ab2 with the odors to which they respond most strongly (ethyl acetate, 2,3-dibutanone, CO2, methyl salicylate, ethyl acetate, 1-hexanol, and ethyl butyrate). Thus, deletion of Or22a and Or22b has a profound effect on the ab3A cell, consistent with the mapping of Or22a and Or22b to ab3A in the rpr-ablation experiments (Dobritsa, 2003).
To test the possibility that ab3A expresses an additional odor receptor that functions independently of Or22a or Or22b, ab3 sensilla were challenged in the deletion mutant and in wild-type with odors of complex natural food sources: banana, orange, and apple. These odor mixtures elicited strong activity in the wild-type from both ab3A and ab3B neurons, but in the mutant they elicit a response only from the ab3B neuron. Thus, in the absence of Or22a and Or22b, the ab3A cell does not respond to any of a wide variety of tested odors (Dobritsa, 2003).
In addition to the severe loss of odor response in ab3A cells lacking Or22a and Or22b, a second physiological phenotype was observed: an abnormality in the temporal pattern of those spikes that are observed at low frequency in mutant ab3A cells. Although two of ten ab3A neurons examined in Deltahalo were entirely silent, the others showed a low level of activity that consisted largely of bursts of action potentials. Bursts typically contained three or four action potentials, with an interspike interval of 14 ± 0.8 ms. These bursts occurred at ~10 s intervals in the absence of odor stimulation. The frequency of bursts increased during responses of the neighboring ab3B neuron, but the overall frequency of firing was still very low: for example, when ab3B was stimulated with 2-heptanone, 18 ± 3.9 impulses s-1 were recorded from ab3A (Dobritsa, 2003).
A mutant deleted for Or22a and Or22b suffers a loss of odor response and abnormal firing of the ab3A neuron. The deletion, Deltahalo, is a synthetic deficiency that combines Df(2L)dp79b and Dp(2;2)dppd21, thereby removing a fragment of ~100 kb in cytogenetic region 22A. Do the physiological phenotypes documented arise from loss of Or22a, Or22b, both, or neither? This question is of special interest in light of the broad response spectrum of the ab3A neuron and the relatively high degree of sequence identity between Or22a and Or22b. It was of special interest when testing the hypotheses that (1) Or22a mediates response to a subset of the odors to which ab3A responds, while Or22b mediates response to a different subset; (2) Or22a and Or22b form an obligate heterodimer; (3) Or22a and Or22b, which are closely related, are functionally redundant; (4) one receptor mediates all the odor responses of ab3A (Dobritsa, 2003).
To distinguish among these hypotheses, transformation rescue experiments were carried out with transgenic constructs carrying Or22a, Or22b, both, or neither. It was reasoned that if each transgene rescued a portion of the response, then hypothesis 1 was likely correct; if rescue required cotransformation with both receptors, then hypothesis 2 was likely correct; if response was rescued by either receptor, then hypothesis 3 was likely correct; and if the response was rescued by one receptor, but not the other, then hypothesis 4 was likely correct (Dobritsa, 2003).
Accordingly, four transgenic constructs were generated, referred to as 22a+22b+, 22a+22b-, 22a-22b+, and 22a-22b-. The 22a+22b+ construct contains 12 kb of genomic DNA carrying wild-type copies of both Or22a and Or22b as well as all the upstream sequences contained in the 22a-GAL4 and 22b-GAL4 constructs. The 22a+22b- construct is identical, except that stop codons were inserted into the predicted transmembrane domain 1 (TM1) of Or22b. In the 22a-22b+ construct, a frameshift mutation was introduced to create a stop codon in TM1 of Or22a. 22a-22b- contains both the Or22a and Or22b mutations. The four constructs were then introduced separately into Deltahalo mutants, and transgenic lines were tested for electrophysiological responses to the diagnostic set of odors (Dobritsa, 2003).
The 22a+22b+ transgene rescues the activity of the ab3A neuron, indicating that the mutant phenotype is in fact caused by the absence of one or both Or genes. As expected, the 22a-22b- construct did not rescue the phenotype. The 22a+22b- construct rescued the response of ab3A, to the same extent as 22a+b+, as if 22a+ alone is sufficient to rescue. By contrast, the 22a-b+ construct provided no appreciable rescue of the odor response in either males or females. The bursting phenotype also was not rescued. Thus, rescue is provided only by those constructs containing an intact Or22a gene, suggesting that Or22a is necessary for rescue, with no rescue provided by the addition of Or22b. A caveat in this analysis is that little immunoreactivity was observed in the line carrying the 22a-22b+ rescue construct, and therefore attempts were made to test the function of Or22b by other means (Dobritsa, 2003).
To test further the roles of Or22a and Or22b in ab3A response, Or22a and Or22b were expressed in the Deltahalo background using the GAL4-UAS system. Specifically, either Or22a or Or22b was placed under the control of a UAS and expressed using the 22a-GAL4 driver. It was found that the expression of Or22a by this method rescued the response of ab3A, while expression of Or22b did not, in either of two independent insertion lines, either in males or in females. Expression of Or22b in this neuron conferred no appreciable response to any of 87 tested odors, including a wide variety of alcohols, aldehydes, acetate esters, organic acids, ketones, and terpenes. Nor was the bursting phenotype affected by the introduction of UAS-Or22b. Expression of Or22b in neurons in the expected region of the antenna was observed in this line , in a pattern comparable to that in other transgenic rescue lines The simplest interpretation of all the rescue results, taken together, is that a single Or gene, Or22a, is necessary and sufficient for the odor response of the ab3A neuron (Dobritsa, 2003).
Is Or22b expressed in the wild-type antenna? Or22b probes, like Or22a probes, label the antenna by in situ hybridization, although the possibility that at least some of this labeling is due to crosshybridization to Or22a RNA cannot be excluded. Also, evidence has been found for expression of Or22b in each of three independent preparations of antennal RNA from RT-PCR analysis, using multiple sets of primers, followed by sequence analysis. Finally, cDNA clones corresponding to both Or22a and Or22b have been isolated from an antennal/maxillary palp cDNA library. Thus, Or22b is transcribed in wild-type, and Or22b RNA encodes a stable protein that shows the localization expected of an odor receptor (Dobritsa, 2003).
Since no function was identified for Or22b, the possibility was considered that it has lost function over evolutionary time. In this case, the absence of premature stop codons or other mutations in Or22b suggests that such a loss of function would have occurred recently, perhaps following a recent duplication event. To investigate this possibility, it was asked whether another Drosophila species, D. simulans, contains orthologs of Or22a and Or22b (Dobritsa, 2003).
Orthologs were identified for both Or22a and Or22b in D. simulans. These genes, which are tentatively called DsOr22a and DsOr22b, are 94% and 86% identical in amino acid sequence to their D. melanogaster counterparts. Neither of the D. simulans genes contains premature stop codons or other mutations. The evolutionary conservation of these genes, and of Or22b in particular, argues in favor of a functional role for Or22b (Dobritsa, 2003).
It is noted further that the two D. simulans genes are tightly clustered, like their D. melanogaster counterparts: they are located within 778 bp of each other. Moreover, there is extensive sequence identity between the intergenic regions of the two species, consistent with the possibility that the two Or genes arose from a duplication that occurred before the two species diverged. The anti-Or22a/b antibody shows staining in the dorso-medial region of the D. simulans antenna, in a pattern similar to that seen in D. melanogaster. Physiological recordings from large basiconic sensilla in this region of the D. simulans antenna have revealed the presence of a sensillum whose ORNs have response spectra similar to those of ab3, consistent with a conservation of function between orthologous receptors (not shown) (Dobritsa, 2003).
Studies of olfactory receptors in mammals have provided a growing body of evidence that they participate in axon guidance of ORNs toward their glomerular targets in the olfactory bulb. In Drosophila, the organization of ORN projections is similar to mammals, in that ORNs expressing the same odor receptor project to the same, topographically invariant glomeruli in the antennal lobe, the structural and functional equivalent of the vertebrate olfactory bulb (Dobritsa, 2003 and references therein).
Little is known about the developmental expression of Or genes. Few if any Or genes have been systematically examined for expression throughout olfactory system development to determine whether they might play a role in ORN axon guidance or synapse formation. Moreover, expression studies are difficult to interpret because of the limited sensitivity of in situ hybridization; for example, only a small fraction of the Gr family of 7 transmembrane domain chemosensory receptors are detectable by in situ hybridization. In any case, the possibility of a developmental role for Or genes can be rigorously addressed only by functional testing (Dobritsa, 2003).
To determine whether Or22a and Or22b are required for axonal pathfinding of the ab3A cells, their projection patterns were compared in wild-type and in Deltahalo mutants. Axons of the ab3A neurons were labeled using 22a-GAL4 and UAS-GFP. In wild-type, the ab3A neurons project to a single dorso-medial glomerulus; this glomerulus has been identified as DM2. In the mutant, the neurons project to the same glomerulus, and no gross abnormalities are observed in the projections. Moreover, when Or47a or another odor receptor, Or33c, were substituted for Or22a/b in ab3A neurons, the axons were again observed to project to DM2. In wild-type animals, ORNs expressing Or47a have been shown to target a distinct glomerulus. It is concluded that the targeting of the ab3A neuron to the DM2 glomerulus does not depend on normal Or gene expression (Dobritsa, 2003).
Search PubMed for articles about Drosophila Odorant receptor 22a or Drosophila Odorant receptor 22b
Araneda, R., Kini, A. and Firestein, S. (2000). The molecular receptive range of an odorant receptor. Nat. Neurosci. 3: 1248-1255. 11100145
Bozza, T., Feinstein, P., Zheng, C. and Mombaerts, P. (2002). Odorant receptor expression defines functional units in the mouse olfactory system. J. Neurosci. 22: 3033-3043. 11943806
Clyne, P. J., et al. (1999a). A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22(2): 327-38.
Clyne, P. J., et al. (1999b). The odor specificities of a subset of olfactory receptor neurons are governed by Acj6, a POU-domain transcription factor. Neuron 22(2): 339-47.
De Bruyne, M., Clyne, P. J. and Carlson J. R. (1999). Odor coding in a model olfactory organ: the Drosophila maxillary palp. J. Neurosci. 19: 4520-4532. 10341252
De Bruyne. M., Foster. K. and Carlson. J. (2001). Odor coding in the Drosophila antenna. Neuron 30: 537-552. 11395013
Dobritsa, A. A., et al. (2003). Integrating the molecular and cellular basis of odor coding in the Drosophila antenna. Neuron 37: 827-841. 12628173
Gao, Q. and Chess, A. (1999). Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. Genomics 60: 31-39. 10458908
Gao, Q., Yuan, B. and Chess, A. (2000). Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe. Nat. Neurosci. 3: 780-785. 10903570
Grant, A., Riendeau, C. and O'Connell, R. (1998). Spatial organization of olfactory receptor neurons on the antenna of the cabbage looper moth. J. Comp. Physiol. [A] 183: 433-442
Malnic, B., et al. (1999). Combinatorial receptor codes for odors. Cell 96(5): 713-23.
Rawson, N., et al. (2000). Expression of mRNAs encoding for two different olfactory receptors in a subset of olfactory receptor neurons. J. Neurochem. 75: 185-195. 10854261
Shanbhag, S., Muller, B. and Steinbrecht, A. (1999). Atlas of olfactory organs of Drosophila melanogaster. 1. Types, external organization, innervation and distribution of olfactory sensilla. Int. J. Insect Morphol. Embryol. 28: 377-397
Shanbhag. S., Muller. B. and Steinbrecht. A. (2000). Atlas of olfactory organs of Drosophila melanogaster. 2. Internal organization and cellular architecture of olfactory sensilla. Int. J. Insect Morphol. Embryol. 29: 211-229
Spehr, M., Wetzel, C. H., Hatt, H. and Ache, B. W. (2002). 3-phosphoinositides modulate cyclic nucleotide signaling in olfactory receptor neurons. Neuron 33: 731-739. 11879650
Stocker R. (1994). The organization of the chemosensory system in Drosophila melanogaster: a review. Cell Tissue Res. 275: 3-26. 8118845
Störtkuhl, K. and Kettler, R. (2001). Functional analysis of an olfactory receptor in Drosophila melanogaster Proc. Natl. Acad. Sci. 98: 9381-9385. 11481495
Vosshall, L. B., et al. (1999). A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96: 725-736.
Vosshall, L. B., Wong, A. M. and Axel, R. (2000). An olfactory sensory map in the fly brain. Cell 102: 147-159.
Wetzel, C., Behrendt, H., Gisselmann, G., Störtkuhl K., Hovemann, B. and Hatt, H. (2001). Functional expression and characterization of a Drosophila odorant receptor in a heterologous cell system. Proc. Natl. Acad. Sci. 98: 9377-9380. 11481494
Wicher, D. et al. (2008). Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452(7190): 1007-11. PubMed citation: 18408711
Wojtasek, H., Hansson, B. and Leal, W. (1998). Attracted or repelled? A matter of two neurons, one pheromone binding protein, and a chiral center. Biochem. Biophys. Res. Comm. 250: 217-222. 9753610
date revised: 30 May 2008
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