Olfactory receptor gene expression

seminars in

CELL & DEVELOPMENTAL BIOLOGY, Vol 8, 1997: pp 189–195

Olfactory receptor gene expression Heinz Breer and J¨org Strotmann

compounds depends on the specificity with which odorants interact with appropriate olfactory neurons. Originally, the ill-defined broad response spectra of olfactory neurons appeared to be in favor of rather nonspecific mechanisms, such as lipid modulation. However, the elucidation of G protein-coupled second-messenger transduction cascades in olfactory signalling strongly supported the concept of specific molecular reception via stereospecific protein receptors. These receptors were presumed to be members of the seven-transmembrane-domain receptor class, known to be universally coupled to G proteins. Based on this assumption, an intensive search led to the discovery of a novel family of genes which encode G protein-linked receptors and are expressed in the olfactory epithelium.1 Meanwhile, putative odorant receptors have been cloned from various vertebrate species ranging from fish to man.2-7

Recognition and discrimination of odorous molecules are determined by heptahelical G-protein-coupled receptor proteins localized primarily in the ciliary membrane of olfactory sensory neurons. The discovery of a large multigene family encoding odorant receptors allows us to approach various facets concerning the molecular basis of olfactory chemospecificity, ranging from chromosomal localization and control of expression of olfactory receptor genes to temporal and spatial expression patterns of various receptor types in the nasal neuroepithelium. The target-independent onset of receptor expression and its topographical organization suggest a precommited functional identity of olfactory neurons. Key words: chemospecificity/odorant receptor/olfaction/ olfactory neuron / olfactory receptor ©1997 Academic Press Ltd

THE CAPABILITY of mammals to recognize and discriminate thousands of odors with high sensitivity and specificity is mediated by the olfactory neurons residing in the specialized neuroepithelium that lines the convoluted turbinates in the posterior cavity of the nose. It is the task of these chemosensory cells, which carry cilia that protrude into the mucus covering the nasal epithelium, to recognize certain volatile compounds and convert these chemical stimuli into electrical signals that are transmitted via the olfactory bulb to higher brain regions. Chemo-electrical signal transduction, the primary process in olfaction, is mediated by second messenger cascades, that amplify the signal and lead to changes in membrane conductance resulting in excitation or inhibition of the sensory neurons.

Receptor family Genomic analysis revealed a surprisingly large number of genes encoding odorant receptors. The predicted size of this receptor family has been proposed as perhaps 500–1000 members in the rat,1 whereas in fish there may be only about 100.2 Thus, the olfactory receptor multigene family comprises more members than all the other identified G protein-coupled receptor types and is one of the largest gene families known to date. Olfactory receptor genes are located at multiple loci in the genome; in the mouse more than 10 gene clusters located on seven different chromosomes have been identified.8 A cluster of about 20 receptor genes within a contiguous stretch of 400 kb of DNA at the telomeric end of human chromosome 17 has been studied in detail.7 The identified genes display a relatively high degree of intra-cluster diversity; it has therefore been suggested that the gene cluster did not arise from repeated duplication and modifications of a single primordial gene, but rather

Reception of odorous compounds The accuracy of discrimination between odorous From the University Stuttgart-Hohenheim, Institute of Zoophysiology, Stuttgart, 70593, Germany ©1997 Academic Press Ltd 1084-9521/97/020189 + 07 $25.00/0/sr960135

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H. Breer and J. Strotmann segregated in defined regions of the olfactory epithelium. The availability of molecular probes for distinct receptor types has allowed us to explore whether sensory neurons expressing distinct receptors are indeed spatially segregated within the olfactory epithelium employing in-situ hybridization approaches15-18 It was found that a given olfactory receptor gene is expressed only in a small fraction (0.1–1%) of the sensory neurons, supporting the simplifying assumption that each neuron expresses only one or very few receptor types out of a repertoire of several hundred. Such a ‘clonal exclusion’ was originally proposed for the olfactory system by analogy with the immune system.19 Cells expressing receptors are located in the middle layer of the pseudostratified nasal neuroepithelium; they are considered as mature neurons. Although cells expressing a distinct receptor type can be found throughout all layers of the olfactory epithelium, a quantitative analysis revealed that the majority of this cell population is positioned in a particular laminar zone and small subsets are arranged at regular horizontal distances.20 Interestingly, olfactory neurons that are retrogradely labelled upon injection of horseradish peroxidase into distinct glomeruli of the bulb are also found in distinct levels of the epithelium arranged with regular spacing.21 At a higher level of organization, it was found that in mammals, there is a broad organization of odorant receptor gene expression into spatial zones. Detailed analyses have demonstrated that the expression of each receptor subtype is restricted to well circumscribed but rather broad zones within the olfactory epithelium, each zone being composed of separate bands extending all along the anterior–posterior axis, parallel to the turbinates. The various bands display bilateral symmetry in the two nasal cavities and are virtually identical in different individuals and even in different rodent species.22 These observations seem to indicate that the olfactory epithelium is subdivided into several chemotopic zones. Within a given zone, neurons expressing the same receptor gene seem to be randomly distributed; thus, a topographical unit is not composed of patches of sensory neurons endowed with distinct receptor types, but rather represents a mosaic of sensory neurons with different receptors and thus likely responding to different odorants. The dispersed distribution of receptor-expressing neurons in large longitudinal zones is difficult to reconcile with the local ‘hot spots’ of physiological responsiveness.14,23,24 It has therefore been hypothe-

have been generated by the duplication of an ancestral gene cluster, which existed early in evolution.9

Response profile of receptors and sensory cells The enormous diversity of olfactory receptors has probably evolved to facilitate the discrimination of extraneous molecules, which supposedly relies on the differential binding specificities of individual receptor types. In organisms with large olfactory receptor repertoires, an odorant may be recognized by numerous receptor types with a smooth gradation of affinities or thresholds.10 In view of the vast variety of odorous molecules it seems a formidable task to determine the exact ligand specificity of individual receptors. However, heterologous expression studies revealed that, following transfection with cDNA encoding a distinct receptor type, surrogate cells gained responsiveness to certain odorants. Graded responses to only a subset of odorous compounds out of a large collection indicate that individual odorant receptors may display a selective but rather broad ligand specificity (ref 6 and Meinken, unpublished results). Studies examining the reaction spectrum of individual olfactory neurons revealed that a given cell is responding to a broad set of odorants but the response profile is dependent on odorant concentration.11,12 The basis for the apparent broad reaction spectrum of individual sensory neurons may be based on multiple receptor types or due to a single broadly tuned receptor type interacting with specific molecular features of odorous compounds, such as shape, charge distribution, hydrophobicity or functional groups. Although in-situ studies suggest that individual olfactory neurons may express only one, or at most a few, receptor types, this issue is still unclear.

Spatial pattern of receptor expression Recordings from the surface of the rat olfactory epithelium have indicated that specific odors elicit defined spatial patterns of activity.13,14 The typical response pattern was described as a localized region of peak sensitivity (‘hot spot’) surrounded by less sensitive regions that show diminishing responsiveness with distance from the peak. This topographical pattern of responsiveness suggested that sensory neurons having a similar response spectrum, i.e. cells endowed with the same receptors types, may be 190

Olfactory receptors tissue- and cell-specific genes determining the specification of sensory neurons defined by their receptor types, transduction machinery, cell surface markers and synaptic targeting. It has been proposed that olfactory neurons may first establish synaptic contacts in the bulb and are subsequently instructed by signals from the synaptic partner to make a particular receptor choice; such retrograde influences have previously been implicated in the control of neuronal phenotype in other systems.29 Alternatively, receptor choice may precede synaptic targeting; i.e. functional identity of the neuron is established early in development and precise connectivity is accomplished by pathfinding and recognition molecules together with activity-dependent mechanisms.30 It is also possible that patterns of connectivity may evolve from a temporal coordination of development between different bulb regions and population of sensory neurons, i.e. neurons expressing different receptor types would appear at different times during ontogeny. Recent studies exploring the onset and pattern of olfactory receptor gene expression in the developing olfactory epithelium have provided some preliminary insights. In-situ hybridization experiments indicate that in the developing rat or mouse embryo, the onset of receptor expression occurs early in development, between E12 and E14 in the rat and E11.5 in mice.31,32 These observations seem to indicate that receptor expression occurs prior to synaptogenesis. The discrepancy with a previous study, which based on PCR analyses suggested that receptor expression occurs later in development and may coincide with synaptogenesis,33 is probably due to the higher resolution and sensitivity of in-situ hybridization approaches. Expression of receptor genes in olfactory neurons before the establishment of functional connections in the bulb was recently also observed in birds.4,5 In addition, elements of the transduction cascade appear to be also expressed rather early in development,32,34 which may render the olfactory system functional. This is in contrast to the visual cascade which develops only postnatally35 but consistent with the notion of in-utero olfactory learning in rats.36 The observation that between E16 and E18/19 reactive sensory cells of the rat olfactory epithelium indiscriminately respond to many odorants whereas later cells become selectively responsive to a more restricted set of odorants37 may be indicative for changes in receptor expression. Since receptors are expressed much earlier, the non-selective responsiveness of E16 neurons is probably not due to non-receptor mechanisms, like changes in

sized that in addition to the widely dispersed type of distribution, an additional group of receptors may be expressed in neurons segregated in highly restricted areas.14 During the course of the in-situ hybridization studies, one receptor type was found which exhibits a unique clustered type of expression pattern. Neurons expressing the receptor type OR37 occupy only a small area on the tip of endoturbinate II and ectoturbinate 3.17,25 The proportion of OR37-expressing cells is very high in the center of this region and decays towards the periphery. This concentric distribution pattern for neurons endowed with a distinct receptor type is consistent with the observations in multi-site electrophysiological recordings showing that the ‘hot spots’ for particular odorants display a central region of peak responsiveness surrounded by regions with decaying sensitivity.14 It is unclear whether the topographical distribution of receptor types has any functional implications for encoding the odor information; it is difficult to imagine how expression of an olfactory receptor type in one zone rather than in another may influence the odor image conveyed to the brain. In fact, it is still elusive why a strictly regulated spatial topography exists in the olfactory system which does not physically map the external world into the brain, like other sensory systems, but rather deals with signals which have no inherent spatial order. Studies examining the extent of spatial topography in the axonal projection patterns onto second-order neurons in the olfactory bulb indicate that it lacks point-to-point topography as seen in other sensory systems: however, different zones project to different domains in the olfactory bulb.26 Thus, the initial organization into broad zonal sets seems to be maintained in the projections from the olfactory epithelium to the bulb.15,16,18 Moreover, recent studies indicate that neurons expressing the same receptor type converge their axons onto the same glomerulus,27,28,28a suggesting that sensory information which is broadly organized in the nose is refined in the olfactory bulb, processing the information from sensory neurons with the same receptor type in a distinct glomerulus.

Onset and temporal pattern of receptor expression The complex functional organization of the mammalian olfactory system requires a variety of subtle mechanisms for coordinated expression of many 191

H. Breer and J. Strotmann Table 1. Total numbers of neurones expressing distinct odorant receptor subtypes in the developing rat olfactory epithelium

been made to define common DNA sequences among the control regions for the olfactory genes and to identify proteins which bind to these putative cisacting regulatory sites.38,39 Based on characteristic sites originally identified in the OMP gene40 it was found that olfactory-specific genes appear to contain at least one homologous site which binds a protein factor (Olf-1).39 Recent cloning approaches revealed that Olf-1 belongs to a distinct family of transcription factors,41 that is supposed to control the expression of distinct genes encoding cell-specific proteins including the highly specialized elements of the transduction cascade.42 Expression of olfactory receptors requires an additional dimension of regulation. How do individual olfactory sensory neurons choose to express a distinct receptor subtype from a family of about 1000 genes? One level of control emerged from the spatial pattern of receptor expression in distinct zones.15-18 Restricted expression of odorant receptors to only one topological zone is likely to result from positional information within the olfactory epithelium. Within a given zone, the distribution of neurons expressing a given receptor is random. This suggests that the choice among permitted receptors within a given zone is stochastic and not governed by positional information, i.e. when an olfactory neuron chooses which odorant receptor gene(s) to express, it is restricted to a single zonal gene set, but may choose a gene out of the permitted set via stochastic mechanisms.15 Recent studies have demonstrated that only one allelic array of receptor encoding genes is active in an individual neuron.43 The stochastic expression of a single receptor gene from a linked array could result from cis-regulatory elements that can activate only one gene from the array; this type of regulation may be due to single locus control elements. Alternatively, it has been proposed that there may be a single expression site; into this site one of the ‘silent’ genes may be introduced by a gene conversion event. If this DNA rearrangement occurs in a stochastic fashion, cells expressing a distinct receptor would be randomly distributed. If under the control of a spatial factor only a subset of the receptor genes is accessible for transposing into the expression locus, a defined zonal expression would be achieved. Thus, a hierarchy of controls, including positional information, allelic inactivation and cis-regulation may assure that individual olfactory neurons express only one or a small set of distinct receptor types.43

Development stage Receptor subtype OR5 OR14 OR124 OR37

E14

E16

E18

531±72 512±48 417±38 121±41

1384±215 1612±217 1105±230 1354±127

3787±332 3599±396 2977±237 3100±311

membrane fluidity as previously suggested, but rather may reflect changes in receptor expression during maturation. Also, it is unlikely that the non-selective response is due to multiple receptor types in immature cells since the percentage of cells expressing a distinct receptor type is quite constant during development and in adult life.18 However, it may be possible that during the early phase of development cells expressing broadly tuned receptors predominate, whereas cells differentiating during later stages express narrowly tuned receptors, which may outnumber the broadly tuned cells during the course of development. In rodents all receptor types analysed appeared concomitantly at the same stage of development, suggesting a general onset of receptor expression; this would imply that pattern formation is probably not due to temporal asynchrony in the expression for different receptor genes. In fact, it was found that from the earliest time receptor expression can be assessed, receptor genes are expressed in typical zonal pattern;31,32 i.e. the typical patterning of the olfactory epithelium is evident prior to the connectivity with the bulb. Therefore, spatial segregation of olfactory receptor expression is apparently not imposed by retrograde signals from the brain.

Control of olfactory receptor gene expression The functional properties of mature olfactory neurons rely on the coordinated expression of specific genes during differentiation, especially the distinct receptor subtype(s) and olfactory-specific elements of the sensory transduction cascade. Regulation of gene expression that leads to the final phenotype of mammalian cells is often mediated by tissue-specific transcription factors controlling target genes through interactions with specific DNA sequences in the promotor regions. Tremendous effort has recently 192

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H. Breer and J. Strotmann 9. Lancet D, Ben-Arie N (1993) Olfactory receptors. Curr Biol 3:668-674 10. Lancet D, Sadovsky E, Seidemann E (1993) Probability model for molecular recognition in biological receptor repertoires: significance to the olfactory system. Proc Natl Acad Sci USA 90:3715-3719 11. Sicard G, Holley A (1984) Receptor cell responses to odorants: similarities and differences among odorants. Brain Res 292:283-296 12. Firestein S, Picco C, Menini A (1993) The relation between stimulus and response in olfactory receptor cells of the tiger salamander. J Physiol (Lond.) 468:1-10 13. Kent PF, Mozell MM (1992) The recording of odorantinduced mucosal activity patterns with a voltage-sensitive dye. J Neurophysiol 68:1804-1819 14. Mackay-Sim A, Kesteven S (1994) Topographic patterns of responsiveness to odorants in the rat olfactory epithelium. J Neurophysiol 71:150-160 15. Ressler KJ, Sullivan SL, Buck LB (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73:597-609 16. Vassar R, Ngai J, Axel R (1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 74:309-318 17. Strotmann J, Wanner I, Helfrich T, Beck A, Meinken C, Kubick S, Breer H (1994a) Olfactory neurones expressing distinct odorant receptor subtypes are spatially segregated in the nasal neuroepithelium. Cell Tiss Res 276:429-438 18. Strotmann J, Wanner I, Helfrich T, Breer H (1994b) Rostrocaudal patterning of receptor-expressing olfactory neurones in the rat nasal cavity. Cell Tiss Res 278:11-20 19. Lancet D (1986) Vertebrate olfactory reception. Annu Rev Neurosci 9:329-355 20. Strotmann J, Konzelmann S, Breer H (1996) Laminar segregation of odorant receptor expression in the olfactory epithelium. Cell Tiss Res 282:347-354 21. Pedersen PE, Jastreboff PJ, Stewart WB, Shepherd GM (1986) Mapping of an olfactory receptor population that projects to a specific region in the rat olfactory bulb. J Comp Neurol 250:93-108 22. Strotmann J, Beck A, Kubick S, Breer H (1995a) Topographic patterns of odorant receptor expression in mammals: a comparative study. J Comp Physiol A 177:659-666 23. Thommesen G, Doving KB (1977) Spatial distribution of the EOG in the rat; a variation with odour quality. Acta Physiol Scand 99:270-280 24. Edwards DA, Mather RA, Dodd GH (1988) Spatial variation in response to odorants on the rat olfactory epithelium. Experientia 44:208-211 25. Strotmann J, Wanner I, Krieger J. Raming K, Breer H (1992) Expression of odorant receptors in spatially restricted subsets of chemosensory neurons. NeuroReport 3:1053-1056 26. Schoenfeld TA, Clancy AN, Forbes WB, Macrides F (1994) The spatial organization of the peripheral olfactory system of the hamster. 1. Receptor neuron projections to the main olfactory bulb. Brain Res Bull 34:183-210 27. Ressler KJ, Sullivan SL, Buck LB (1994) Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79:1245-1255 28. Vassar R, Chao SK, Sitcheran R, Nunez JM, Vosshall LB, Axel R (1994) Topographic organization of sensory projections to the olfactory bulb. Cell 79:981-991 28a. Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Mendelsohn M, Edmondson J, Axel R (1996) Visualizing an olfactory sensory map. Cell 87:675-686 29. Patterson PH, Nawa H (1993) Neuronal differentiation factors/cytokines and synaptic plasticity. Cell 72:123-137

Conclusion The identification of genes encoding receptors for odorous compounds has opened new avenues to explore some of the mysteries underlying our sense of smell, ranging from chemospecificity of individual olfactory neurons to the organizational strategies underlying olfactory information processing. Initial studies unraveled a surprising degree of chemotopical complexity, such as zonal patterning and precise axonal projection. Generating the olfactory neuronal network undoubtedly requires a well coordinated execution of a complex developmental program. The study of olfactory neurogenesis is still in its infancy, but the complexity of this process becomes already apparent. Progress in this area will not only be of great interest for better understanding the neural basis of olfactory perception but in addition, there is great expectation for the applicability of olfactory neurogenic mechanisms to other systems.

Acknowledgements The work from this laboratory was supported by the Deutsche Forschungsgemeinschaft and the Human Frontier Science Program.

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