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1.  The Olfactory Transcriptomes of Mice 
PLoS Genetics  2014;10(9):e1004593.
The olfactory (OR) and vomeronasal receptor (VR) repertoires are collectively encoded by 1700 genes and pseudogenes in the mouse genome. Most OR and VR genes were identified by comparative genomic techniques and therefore, in many of those cases, only their protein coding sequences are defined. Some also lack experimental support, due in part to the similarity between them and their monogenic, cell-specific expression in olfactory tissues. Here we use deep RNA sequencing, expression microarray and quantitative RT-PCR in both the vomeronasal organ and whole olfactory mucosa to quantify their full transcriptomes in multiple male and female mice. We find evidence of expression for all VR, and almost all OR genes that are annotated as functional in the reference genome, and use the data to generate over 1100 new, multi-exonic, significantly extended receptor gene annotations. We find that OR and VR genes are neither equally nor randomly expressed, but have reproducible distributions of abundance in both tissues. The olfactory transcriptomes are only minimally different between males and females, suggesting altered gene expression at the periphery is unlikely to underpin the striking sexual dimorphism in olfactory-mediated behavior. Finally, we present evidence that hundreds of novel, putatively protein-coding genes are expressed in these highly specialized olfactory tissues, and carry out a proof-of-principle validation. Taken together, these data provide a comprehensive, quantitative catalog of the genes that mediate olfactory perception and pheromone-evoked behavior at the periphery.
Author Summary
The sense of smell in mice involves the detection of odors and pheromones by many hundreds of olfactory and vomeronasal receptors. The genes that encode these receptors account for around 5% of the whole gene catalog, but they are poorly understood because they are very similar to each other, and are thought to be turned on randomly in only a small number of cells. Here we use multiple gene expression technologies to curate and measure the activity of all the genes involved in the detection of odors and find evidence of many new ones. We show that most genes encoding olfactory and vomeronasal receptors have complex, multi-exonic structures that generate different isoforms. We find that some receptors are consistently more abundant in the nose than others, which suggests they are not turned on randomly. This may explain why mice are particularly sensitive to some odors, but less attuned to others. We find that overall males and females differ very little in gene expression, despite having altered behavioral responses to the same odors. Thus diversity in receptor expression can explain differences in odor sensitivity, but does not appear to dictate whether sex pheromones are differentially detected by males or females.
doi:10.1371/journal.pgen.1004593
PMCID: PMC4154679  PMID: 25187969
2.  Conserved repertoire of orthologous vomeronasal type 1 receptor genes in ruminant species 
Background
In mammals, pheromones play an important role in social and innate reproductive behavior within species. In rodents, vomeronasal receptor type 1 (V1R), which is specifically expressed in the vomeronasal organ, is thought to detect pheromones. The V1R gene repertoire differs dramatically between mammalian species, and the presence of species-specific V1R subfamilies in mouse and rat suggests that V1R plays a profound role in species-specific recognition of pheromones. In ruminants, however, the molecular mechanism(s) for pheromone perception is not well understood. Interestingly, goat male pheromone, which can induce out-of-season ovulation in anestrous females, causes the same pheromone response in sheep, and vice versa, suggesting that there may be mechanisms for detecting "inter-species" pheromones among ruminant species.
Results
We isolated 23 goat and 21 sheep intact V1R genes based on sequence similarity with 32 cow V1R genes in the cow genome database. We found that all of the goat and sheep V1R genes have orthologs in their cross-species counterparts among these three ruminant species and that the sequence identity of V1R orthologous pairs among these ruminants is much higher than that of mouse-rat V1R orthologous pairs. Furthermore, all goat V1Rs examined thus far are expressed not only in the vomeronasal organ but also in the main olfactory epithelium.
Conclusion
Our results suggest that, compared with rodents, the repertoire of orthologous V1R genes is remarkably conserved among the ruminants cow, sheep and goat. We predict that these orthologous V1Rs can detect the same or closely related chemical compound(s) within each orthologous set/pair. Furthermore, all identified goat V1Rs are expressed in the vomeronasal organ and the main olfactory epithelium, suggesting that V1R-mediated ligand information can be detected and processed by both the main and accessory olfactory systems. The fact that ruminant and rodent V1Rs have distinct features suggests that ruminant and rodent V1Rs have evolved distinct functions.
doi:10.1186/1471-2148-9-233
PMCID: PMC2758851  PMID: 19751533
3.  The genomic basis of vomeronasal-mediated behaviour 
Mammalian Genome  2013;25(1-2):75-86.
The vomeronasal organ (VNO) is a chemosensory subsystem found in the nose of most mammals. It is principally tasked with detecting pheromones and other chemical signals that initiate innate behavioural responses. The VNO expresses subfamilies of vomeronasal receptors (VRs) in a cell-specific manner: each sensory neuron expresses just one or two receptors and silences all the other receptor genes. VR genes vary greatly in number within mammalian genomes, from no functional genes in some primates to many hundreds in rodents. They bind semiochemicals, some of which are also encoded in gene families that are coexpanded in species with correspondingly large VR repertoires. Protein and peptide cues that activate the VNO tend to be expressed in exocrine tissues in sexually dimorphic, and sometimes individually variable, patterns. Few chemical ligand–VR–behaviour relationships have been fully elucidated to date, largely due to technical difficulties in working with large, homologous gene families with high sequence identity. However, analysis of mouse lines with mutations in genes involved in ligand–VR signal transduction has revealed that the VNO mediates a range of social behaviours, including male–male and maternal aggression, sexual attraction, lordosis, and selective pregnancy termination, as well as interspecific responses such as avoidance and defensive behaviours. The unusual logic of VR expression now offers an opportunity to map the specific neural circuits that drive these behaviours.
doi:10.1007/s00335-013-9463-1
PMCID: PMC3916702  PMID: 23884334
4.  Dynamic evolution of V1R putative pheromone receptors between Mus musculus and Mus spretus 
BMC Genomics  2009;10:74.
Background
The mammalian vomeronasal organ (VNO) expresses two G-protein coupled receptor gene families that mediate pheromone responses, the V1R and V2R receptor genes. In rodents, there are ~150 V1R genes comprising 12 subfamilies organized in gene clusters at multiple chromosomal locations. Previously, we showed that several of these subfamilies had been extensively modulated by gene duplications, deletions, and gene conversions around the time of the evolutionary split of the mouse and rat lineages, consistent with the hypothesis that V1R repertoires might be involved in reinforcing speciation events. Here, we generated genome sequence for one large cluster containing two V1R subfamilies in Mus spretus, a closely related and sympatric species to Mus musculus, and investigated evolutionary change in these repertoires along the two mouse lineages.
Results
We describe a comparison of spretus and musculus with respect to genome organization and synteny, as well as V1R gene content and phylogeny, with reference to previous observations made between mouse and rat. Unlike the mouse-rat comparisons, synteny seems to be largely conserved between the two mouse species. Disruption of local synteny is generally associated with differences in repeat content, although these differences appear to arise more from deletion than new integrations. Even though unambiguous V1R orthology is evident, we observe dynamic modulation of the functional repertoires, with two of seven V1Rb and one of eleven V1Ra genes lost in spretus, two V1Ra genes becoming pseudogenes in musculus, two additional orthologous pairs apparently subject to strong adaptive selection, and another divergent orthologous pair that apparently was subjected to gene conversion.
Conclusion
Therefore, eight of the 18 (~44%) presumptive V1Ra/V1Rb genes in the musculus-spretus ancestor appear to have undergone functional modulation since these two species diverged. As compared to the rat-mouse split, where modulation is evident by independent expansions of these two V1R subfamilies, divergence between musculus and spretus has arisen more by mutations within coding sequences. These results support the hypothesis that adaptive changes in functional V1R repertoires contribute to the delineation of very closely related species.
doi:10.1186/1471-2164-10-74
PMCID: PMC2644715  PMID: 19203383
5.  Molecular Organization of Vomeronasal Chemoreception 
Nature  2011;478(7368):241-245.
The vomeronasal organ (VNO) plays a key role in mediating the social and defensive responses of many terrestrial vertebrates to species- and sex-specific chemosignals1. Over 250 putative pheromone receptors have been identified in the mouse VNO2,3, but the nature of the signals detected by individual VNO receptors has not yet been elucidated. In order to gain insight into the molecular logic of VNO detection leading to mating, aggression, or defensive responses, we sought to uncover the response profiles of individual vomeronasal receptors to a wide range of animal cues. We describe here the repertoire of ethological and physiological stimuli detected by a large number of individual vomeronasal receptors, and define a global map of vomeronasal signal detection. We demonstrate that the two classes of vomeronasal receptors V1Rs and V2Rs use fundamentally different strategies to encode chemosensory information, and that distinct receptor subfamilies have evolved towards the specific recognition of certain animal groups or chemical structures. The association of large subsets of vomeronasal receptors with cognate, ethologically and physiologically relevant stimuli establishes the molecular foundation of vomeronasal information coding, and opens new avenues for further investigating the neural mechanisms underlying behavior specificity.
doi:10.1038/nature10437
PMCID: PMC3192931  PMID: 21937988
6.  Distinct Evolutionary Patterns between Chemoreceptors of 2 Vertebrate Olfactory Systems and the Differential Tuning Hypothesis 
Molecular Biology and Evolution  2008;25(8):1593-1601.
Most tetrapod vertebrates have 2 olfactory systems, the main olfactory system (MOS) and the vomeronasal system (VNS). According to the dual olfactory hypothesis, the MOS detects environmental odorants, whereas the VNS recognizes intraspecific pheromonal cues. However, this strict functional distinction has been blurred by recent reports that both systems can perceive both types of signals. Studies of a limited number of receptors suggest that MOS receptors are broadly tuned generalists, whereas VNS receptors are narrowly tuned specialists. However, whether this distinction applies to all MOS and VNS receptors remains unknown. The differential tuning hypothesis predicts that generalist MOS receptors detect an overlapping set of ligands and thus are more likely to be conserved over evolutionary time than specialist VNS receptors, which would evolve in a more lineage-specific manner. Here we test this prediction for all olfactory chemoreceptors by examining the evolutionary patterns of MOS-expressed odorant receptors (ORs) and trace amine–associated receptors (TAARs) and VNS-expressed vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs) in 7 tetrapods (mouse, rat, dog, opossum, platypus, chicken, and frog). The phylogenies of V1Rs and V2Rs show abundant lineage-specific gene gains/losses and virtually no one-to-one orthologs between species. Opposite patterns are found for ORs and TAARs. Analysis of functional data and ligand-binding sites of ORs confirms that paralogous chemoreceptors are more likely than orthologs to have different ligands and that functional divergence between paralogous chemoreceptors is established relatively quickly following gene duplication. Together, these results strongly suggest that the functional profile of the VNS chemoreceptor repertoire evolves much faster than that of the MOS chemoreceptor repertoire and that the differential tuning hypothesis applies to the majority, if not all, of MOS and VNS receptors.
doi:10.1093/molbev/msn107
PMCID: PMC2727380  PMID: 18460446
V1R; V2R; OR; TAAR; vomeronasal; olfactory
7.  High-Throughput Microarray Detection of Vomeronasal Receptor Gene Expression in Rodents 
We performed comprehensive data mining to explore the vomeronasal receptor (V1R and V2R) repertoires in mouse and rat using the mm5 and rn3 genome, respectively. This bioinformatic analysis was followed by investigation of gene expression using a custom designed high-density oligonucleotide array containing all of these receptors and other selected genes of interest. This array enabled us to detect the specific expression of V1R and V2Rs which were previously identified solely based on computational prediction from gene sequence data, thereby establishing that these genes are indeed part of the vomeronasal system, especially the V2Rs. One hundred sixty-eight V1Rs and 98 V2Rs were detected to be highly enriched in mouse vomeronasal organ (VNO), and 108 V1Rs and 87 V2Rs in rat VNO. We monitored the expression profile of mouse VR genes in other non-VNO tissues with the result that some VR genes were re-designated as VR-like genes based on their non-olfactory expression pattern. Temporal expression profiles for mouse VR genes were characterized and their patterns were classified, revealing the developmental dynamics of these so-called pheromone receptors. We found numerous patterns of temporal expression which indicate possible behavior-related functions. The uneven composition of VR genes in certain patterns suggests a functional differentiation between the two types of VR genes. We found the coherence between VR genes and transcription factors in terms of their temporal expression patterns. In situ hybridization experiments were performed to evaluate the cell number change over time for selected receptor genes.
doi:10.3389/fnins.2010.00164
PMCID: PMC3024560  PMID: 21267422
vomeronasal receptor; V1R; V2R; microarray; gene expression profiling; developmental dynamics; bioinformatics; neuroscience
8.  Integrated action of pheromone signals in promoting courtship behavior in male mice 
eLife  2014;3:e03025.
The mammalian vomeronasal organ encodes pheromone information about gender, reproductive status, genetic background and individual differences. It remains unknown how pheromone information interacts to trigger innate behaviors. In this study, we identify vomeronasal receptors responsible for detecting female pheromones. A sub-group of V1re clade members recognizes gender-identifying cues in female urine. Multiple members of the V1rj clade are cognate receptors for urinary estrus signals, as well as for sulfated estrogen (SE) compounds. In both cases, the same cue activates multiple homologous receptors, suggesting redundancy in encoding female pheromone cues. Neither gender-specific cues nor SEs alone are sufficient to promote courtship behavior in male mice, whereas robust courtship behavior can be induced when the two cues are applied together. Thus, integrated action of different female cues is required in pheromone-triggered mating behavior. These results suggest a gating mechanism in the vomeronasal circuit in promoting specific innate behavior.
DOI: http://dx.doi.org/10.7554/eLife.03025.001
eLife digest
Pheromones are chemicals that are given off by living things and they lead to a range of social responses in others of the same species. These chemical signals, for example, can let an animal know when a suitable mate is near and trigger the release of hormones that encourage the animal to mate.
In mammals, an organ found between the roof of the mouth and the nose detects pheromones. This organ contains more than 300 different receptors for these chemicals. However, only a few of these receptors have been matched with the pheromones that they detect. One example is a chemical released by male mice that interacts with a specific nasal receptor and causes a female mouse to arch her back in a way that signals she is ready to mate.
One reason that more pheromone-receptor pairs are not known is that pheromones are released in very small quantities, which makes them hard to detect. In an effort to identify more pairs, Haga-Yamanaka et al. took tissue slices from the organ that detects pheromones in mice and then looked for cells that responded to the urine of female mice. Two previously unknown pheromone-receptor pairs were found. One helps male mice detect when a female is present, while the other lets him know if she is ready to mate. Together these two chemicals alert a male mouse to a potential mate and cause him to mount her in order to mate. However, neither chemical is able to trigger this male courtship behavior on its own.
The techniques developed by Haga-Yamanaka et al. may, in the future, help identify more pheromone-receptor pairs. The next challenge will be to identify the pathways of nerve cells that integrate the information about pheromones and trigger the courtship behaviors.
DOI: http://dx.doi.org/10.7554/eLife.03025.002
doi:10.7554/eLife.03025
PMCID: PMC4107909  PMID: 25073926
olfactory; vomeronasal; pheromone; innate behavior; GCaMP; imaging; mouse
9.  The G protein-coupled receptor subset of the rat genome 
BMC Genomics  2007;8:338.
Background
The superfamily of G protein-coupled receptors (GPCRs) is one of the largest within most mammals. GPCRs are important targets for pharmaceuticals and the rat is one of the most widely used model organisms in biological research. Accurate comparisons of protein families in rat, mice and human are thus important for interpretation of many physiological and pharmacological studies. However, current automated protein predictions and annotations are limited and error prone.
Results
We searched the rat genome for GPCRs and obtained 1867 full-length genes and 739 pseudogenes. We identified 1277 new full-length rat GPCRs, whereof 1235 belong to the large group of olfactory receptors. Moreover, we updated the datasets of GPCRs from the human and mouse genomes with 1 and 43 new genes, respectively. The total numbers of full-length genes (and pseudogenes) identified were 799 (583) for human and 1783 (702) for mouse. The rat, human and mouse GPCRs were classified into 7 families named the Glutamate, Rhodopsin, Adhesion, Frizzled, Secretin, Taste2 and Vomeronasal1 families. We performed comprehensive phylogenetic analyses of these families and provide detailed information about orthologues and species-specific receptors. We found that 65 human Rhodopsin family GPCRs are orphans and 56 of these have an orthologue in rat.
Conclusion
Interestingly, we found that the proportion of one-to-one GPCR orthologues was only 58% between rats and humans and only 70% between the rat and mouse, which is much lower than stated for the entire set of all genes. This is in mainly related to the sensory GPCRs. The average protein sequence identities of the GPCR orthologue pairs is also lower than for the whole genomes. We found these to be 80% for the rat and human pairs and 90% for the rat and mouse pairs. However, the proportions of orthologous and species-specific genes vary significantly between the different GPCR families. The largest diversification is seen for GPCRs that respond to exogenous stimuli indicating that the variation in their repertoires reflects to a large extent the adaptation of the species to their environment. This report provides the first overall roadmap of the GPCR repertoire in rat and detailed comparisons with the mouse and human repertoires.
doi:10.1186/1471-2164-8-338
PMCID: PMC2117022  PMID: 17892602
10.  Mammalian social odours: attraction and individual recognition 
Mammalian social systems rely on signals passed between individuals conveying information including sex, reproductive status, individual identity, ownership, competitive ability and health status. Many of these signals take the form of complex mixtures of molecules sensed by chemosensory systems and have important influences on a variety of behaviours that are vital for reproductive success, such as parent–offspring attachment, mate choice and territorial marking. This article aims to review the nature of these chemosensory cues and the neural pathways mediating their physiological and behavioural effects. Despite the complexities of mammalian societies, there are instances where single molecules can act as classical pheromones attracting interest and approach behaviour. Chemosignals with relatively high volatility can be used to signal at a distance and are sensed by the main olfactory system. Most mammals also possess a vomeronasal system, which is specialized to detect relatively non-volatile chemosensory cues following direct contact. Single attractant molecules are sensed by highly specific receptors using a labelled line pathway. These act alongside more complex mixtures of signals that are required to signal individual identity. There are multiple sources of such individuality chemosignals, based on the highly polymorphic genes of the major histocompatibility complex (MHC) or lipocalins such as the mouse major urinary proteins. The individual profile of volatile components that make up an individual odour signature can be sensed by the main olfactory system, as the pattern of activity across an array of broadly tuned receptor types. In addition, the vomeronasal system can respond highly selectively to non-volatile peptide ligands associated with the MHC, acting at the V2r class of vomeronasal receptor.
The ability to recognize individuals or their genetic relatedness plays an important role in mammalian social behaviour. Thus robust systems for olfactory learning and recognition of chemosensory individuality have evolved, often associated with major life events, such as mating, parturition or neonatal development. These forms of learning share common features, such as increased noradrenaline evoked by somatosensory stimulation, which results in neural changes at the level of the olfactory bulb. In the main olfactory bulb, these changes are likely to refine the pattern of activity in response to the learned odour, enhancing its discrimination from those of similar odours. In the accessory olfactory bulb, memory formation is hypothesized to involve a selective inhibition, which disrupts the transmission of the learned chemosignal from the mating male. Information from the main olfactory and vomeronasal systems is integrated at the level of the corticomedial amygdala, which forms the most important pathway by which social odours mediate their behavioural and physiological effects. Recent evidence suggests that this region may also play an important role in the learning and recognition of social chemosignals.
doi:10.1098/rstb.2006.1931
PMCID: PMC1764843  PMID: 17118924
amygdala; maternal bonding; olfactory bulb; pregnancy block; social recognition; vomeronasal
11.  Evolution and origin of vomeronasal-type odorant receptor gene repertoire in fishes 
Background
In teleost fishes that lack a vomeronasal organ, both main odorant receptors (ORs) and vomeronasal receptors family 2 (V2Rs) are expressed in the olfactory epithelium, and used for perception of water-soluble chemicals. In zebrafish, it is known that both ORs and V2Rs formed multigene families of about a hundred copies. Whereas the contribution of V2Rs in zebrafish to olfaction has been found to be substantially large, the composition and structure of the V2R gene family in other fishes are poorly known, compared with the OR gene family.
Results
To understand the evolutionary dynamics of V2R genes in fishes, V2R sequences in zebrafish, medaka, fugu, and spotted green pufferfish were identified from their draft genome sequences. There were remarkable differences in the number of intact V2R genes in different species. Most V2R genes in these fishes were tightly clustered in one or two specific chromosomal regions. Phylogenetic analysis revealed that the fish V2R family could be subdivided into 16 subfamilies that had diverged before the separation of the four fishes. Genes in two subfamilies in zebrafish and another subfamily in medaka increased in their number independently, suggesting species-specific evolution in olfaction. Interestingly, the arrangements of V2R genes in the gene clusters were highly conserved among species in the subfamily level. A genomic region of tetrapods corresponding to the region in fishes that contains the V2R cluster was found to have no V2R gene in any species.
Conclusion
Our results have indicated that the evolutionary dynamics of fish V2Rs are characterized by rapid gene turnover and lineage-specific phylogenetic clustering. In addition, the present phylogenetic and comparative genome analyses have shown that the fish V2Rs have expanded after the divergence between teleost and tetrapod lineages. The present identification of the entire V2R repertoire in fishes would provide useful foundation to the future functional and evolutionary studies of fish V2R gene family.
doi:10.1186/1471-2148-6-76
PMCID: PMC1601972  PMID: 17014738
12.  Functional promiscuity in a mammalian chemosensory system: extensive expression of vomeronasal receptors in the main olfactory epithelium of mouse lemurs 
The vomeronasal organ (VNO) is functional in most terrestrial mammals, though progressively reduced in the primate lineage, and is used for intraspecific communication and predator recognition. Vomeronasal receptor (VR) genes comprise two families of chemosensory genes (V1R and V2R) that have been considered to be specific for the VNO. However, recently a large number of VRs were reported to be expressed in the main olfactory epithelium (MOE) of mice, but there is little knowledge of the expression of these genes outside of rodents. To explore the function of VR genes in mammalian evolution, we analyzed and compared the expression of 64 V1R and 2 V2R genes in the VNO and the MOE of the gray mouse lemur (Microcebus murinus), the primate with the largest known VR repertoire. We furthermore compared expression patterns in adults of both sexes and seasons, and in an infant. A large proportion (83–97%) of the VR loci was expressed in the VNO of all individuals. The repertoire in the infant was as rich as in adults, indicating reliance on olfactory communication from early postnatal development onwards. In concordance with mice, we also detected extensive expression of VRs in the MOE, with proportions of expressed loci in individuals ranging from 29 to 45%. TRPC2, which encodes a channel protein crucial for signal transduction via VRs, was co-expressed in the MOE in all individuals indicating likely functionality of expressed VR genes in the MOE. In summary, the large VR repertoire in mouse lemurs seems to be highly functional. Given the differences in the neural pathways of MOE and VNO signals, which project to higher cortical brain centers or the limbic system, respectively, this raises the intriguing possibility that the evolution of MOE-expression of VRs enabled mouse lemurs to adaptively diversify the processing of VR-encoded olfactory information.
doi:10.3389/fnana.2014.00102
PMCID: PMC4173931  PMID: 25309343
MOE; VNO; V1R; V2R; TRPC2; olfaction; primate; Microcebus
13.  Genomic Organization and Evolution of the Vomeronasal Type 2 Receptor-Like (OlfC) Gene Clusters in Atlantic Salmon, Salmo salar 
Molecular Biology and Evolution  2009;26(5):1117-1125.
There are three major multigene superfamilies of olfactory receptors (OR, V1R, and V2R) in mammals. The ORs are expressed in the main olfactory organ, whereas the V1Rs and V2Rs are located in the vomeronasal organ. Fish only possess one olfactory organ in each nasal cavity, the olfactory rosette; therefore, it has been proposed that their V2R-like genes be classified as olfactory C family G protein-coupled receptors (OlfC). There are large variations in the sizes of OR gene repertoires. Previous studies have shown that fish have between 12 and 46 functional V2R-like genes, whereas humans have lost all functional V2Rs, and frog sp. have more than 240. Pseudogenization of V2R genes is a prevalent event across species. In the mouse and frog genomes, there are approximately double the number of pseudogenes compared with functional genes. An oligonucleotide probe was designed from a conserved sequence from four Atlantic salmon OlfC genes and used to screen the Atlantic salmon bacterial artificial chromosome (BAC) library. Hybridization-positive BACs were matched to fingerprint contigs, and representative BACs were shotgun cloned and sequenced. We identified 55 OlfC genes. Twenty-nine of the OlfC genes are classified as putatively functional genes and 26 as pseudogenes. The OlfC genes are found in two genomic clusters on chromosomes 9 and 20. Phylogenetic analysis revealed that the OlfC genes could be divided into 10 subfamilies, with nine of these subfamilies corresponding to subfamilies found in other teleosts and one being salmon specific. There is also a large expansion in the number of OlfC genes in one subfamily in Atlantic salmon. Subfamily gene expansions have been identified in other teleosts, and these differences in gene number reflect species-specific evolutionary requirements for olfaction. Total RNA was isolated from the olfactory epithelium and other tissues from a presmolt to examine the expression of the odorant genes. Several of the putative OlfC genes that we identified are expressed only in the olfactory epithelium, consistent with these genes encoding odorant receptors.
doi:10.1093/molbev/msp027
PMCID: PMC2668830  PMID: 19221009
olfactory receptor; G protein-coupled receptor; salmonids
14.  Genomic drift and copy number variation of chemosensory receptor genes in humans and mice 
Cytogenetic and Genome Research  2009;123(1-4):263-269.
Recent studies about the structural variation of genomic sequences have shown that there is a large amount of copy number variations (CNVs) of genes within species. Analyzing Redon et al.'s (2006) crude data on copy number variable regions (CNVRs), we previously showed that CNVs are particularly high for chemosensory receptor genes in human populations. In this paper, we reanalyzed the CNVs of these genes using more refined data by Perry et al. (2008). The results showed that the extent of CNVs is somewhat lower in this dataset than in the previous one, but that the extent is still substantial for olfactory receptor (OR), vomeronasal receptor (VR), and taste receptor (TR) genes. We also studied the CNVs for chemosensory receptor genes in mice, using CNVR data obtained from inbred strains. It was found that the extent of CNVs is quite substantial but is lower than that for human populations. However, because the mouse data came from inbred strains and might be biased, this conclusion should be regarded as tentative. Despite this reservation, the distribution of gene copy number for the OR gene family was approximately normal in both humans and mice, suggesting that genomic drift caused by random duplication and deletion of genes plays important roles in determining the evolutionary change of chemosensation.
doi:10.1159/000184716
PMCID: PMC2920191  PMID: 19287163
15.  Contrasted Evolution of the Vomeronasal Receptor Repertoires in Mammals and Squamate Reptiles 
Genome Biology and Evolution  2013;5(2):389-401.
The vomeronasal organ (VNO) is an olfactory structure that detects pheromones and environmental cues. It consists of sensory neurons that express evolutionary unrelated groups of transmembrane chemoreceptors. The predominant V1R and V2R receptor repertoires are believed to detect airborne and water-soluble molecules, respectively. It has been suggested that the shift in habitat of early tetrapods from water to land is reflected by an increase in the ratio of V1R/V2R genes. Snakes, which have a very large VNO associated with a sophisticated tongue delivery system, are missing from this analysis. Here, we use RNA-seq and RNA in situ hybridization to study the diversity, evolution, and expression pattern of the corn snake vomeronasal receptor repertoires. Our analyses indicate that snakes and lizards retain an extremely limited number of V1R genes but exhibit a large number of V2R genes, including multiple lineages of reptile-specific and snake-specific expansions. We finally show that the peculiar bigenic pattern of V2R vomeronasal receptor gene transcription observed in mammals is conserved in squamate reptiles, hinting at an important but unknown functional role played by this expression strategy. Our results do not support the hypothesis that the shift to a vomeronasal receptor repertoire dominated by V1Rs in mammals reflects the evolutionary transition of early tetrapods from water to land. This study sheds light on the evolutionary dynamics of the vomeronasal receptor families in vertebrates and reveals how mammals and squamates differentially adapted the same ancestral vomeronasal repertoire to succeed in a terrestrial environment.
doi:10.1093/gbe/evt013
PMCID: PMC3590772  PMID: 23348039
vomeronasal organ (VNO); monogenic expression; evolution of sensorial abilities; squamates; snakes; phylogeny
16.  The Microevolution of V1r Vomeronasal Receptor Genes in Mice 
Vomeronasal sensitivity is important for detecting intraspecific pheromonal cues as well as environmental odorants and is involved in mating, social interaction, and other daily activities of many vertebrates. Two large families of seven-transmembrane G-protein–coupled receptors, V1rs and V2rs, bind to various ligands to initiate vomeronasal signal transduction. Although the macroevolution of V1r and V2r genes has been well characterized throughout vertebrates, especially mammals, little is known about their microevolutionary patterns, which hampers a clear understanding of the evolutionary forces behind the rapid evolutionary turnover of V1r and V2r genes and the great diversity in receptor repertoire across species. Furthermore, the role of divergent vomeronasal perception in enhancing premating isolation and maintaining species identity has not been evaluated. Here we sequenced 44 V1r genes and 25 presumably neutral noncoding regions in 14 wild-caught mice belonging to Mus musculus and M. domesticus, two closely related species with strong yet incomplete reproductive isolation. We found that nucleotide changes in V1rs are generally under weak purifying selection and that only ∼5% of V1rs may have been subject to positive selection that promotes nonsynonymous substitutions. Consistent with the low functional constraints on V1rs, 18 of the 44 V1rs have null alleles segregating in one or both species. Together, our results demonstrate that, despite occasional actions of positive selection, the evolution of V1rs is in a large part shaped by purifying selection and random drift. These findings have broad implications for understanding the driving forces of rapid gene turnovers that are often observed in the evolution of large gene families.
doi:10.1093/gbe/evr039
PMCID: PMC3114644  PMID: 21551350
Mus musculus; Mus domesticus; vomeronasal receptor; pheromone detection; V1r; evolution
17.  Identification of two distinct genes at the vertebrate TRPC2 locus and their characterisation in a marsupial and a monotreme 
BMC Molecular Biology  2011;12:39.
Background
The vomeronasal organ (VNO) detects pheromones via two large families of vomeronasal receptors: vomeronasal receptor 1 (V1R) and vomeronasal receptor 2 (V2R). Both VRs have a common receptor activation cascade involving transient receptor potential channel, subfamily C, member 2 (TRPC2).
Results
We characterised the TRPC2 locus in a marsupial, the tammar wallaby (Macropus eugenii), and identified two independently regulated genes not previously recognised as distinct. 3'-located exons comprise bona fide TRPC2 whilst 5'-located exons, previously identified as part of TRPC2, comprise a distinct gene, which we term XNDR (XRCC1 N-terminal domain-related). The two genes show contrasting expression patterns in the tammar: TRPC2 is specifically expressed in adult and developing VNO, whereas XNDR is widely expressed in many tissues suggesting a non-VNO-specific role. Strong expression of TRPC2 was detected only after about day 30 post-partum, suggesting that the VNO may not be functional during early pouch life of the tammar. Similarly restricted expression of TRPC2 and widespread expression of XNDR was also detected in the platypus. Bioinformatic analysis of the genomes of a wide range of species suggests that the identity of XNDR and TRPC2 as distinct genes is conserved among vertebrates. Finally, we analysed the promoter of mammalian TRPC2 and identified a conserved binding site for NHLH1, a transcription factor previously implicated in VNO receptor neuron development.
Conclusions
Two functionally distinct vertebrate genes-XNDR and TRPC2 - occupy a genomic locus that was previously defined as a single gene in the mouse. The former is widely expressed with a putative role in DNA repair, while the latter shows VNO-specific expression under the probable regulation of NHLH1.
doi:10.1186/1471-2199-12-39
PMCID: PMC3170594  PMID: 21854574
18.  Requirement of calcium-activated chloride channels in the activation of mouse vomeronasal neurons 
Nature Communications  2011;2:365-.
In terrestrial vertebrates, the vomeronasal organ (VNO) detects and transduces pheromone signals. VNO activation is thought to be mediated by the transient receptor potential C2 (TRPC2) channel. The aberrant behavioural phenotypes observed in TRPC2−/− mice are generally attributed to the lost VNO function. Recently, calcium-activated chloride channels have been shown to contribute to VNO activation. Here we show that CACCs can be activated in VNO slice preparations from the TRPC2−/− mice and this activation is blocked by pharmacological agents that inhibit intracellular Ca2+ release. Urine-evoked Cl− current is sufficient to drive spiking changes in VNO neurons from both wild-type (WT) and TRPC2−/− mice. Moreover, blocking Cl− conductance essentially abolishes VNO activation in WT neurons. These results suggest a TRPC2-independent signalling pathway in the VNO and the requirement of calcium-activated chloride channels currents to mediate pheromone activation. Our data further suggest that TRPC2−/− mice retain partial VNO function.
The vomeronasal organ detects pheromones, which are thought to activate TRPC2 channels on the surface of vomeronasal neurons. Using TRPC2 knockout mice, the authors show that urinary pheromones can also activate these neurons via calcium-activated chloride channels, suggesting a TRPC2-independent pathway for sensing pheromones.
doi:10.1038/ncomms1368
PMCID: PMC3156823  PMID: 21694713
19.  Formyl Peptide Receptors from Immune and Vomeronasal System Exhibit Distinct Agonist Properties* 
The Journal of Biological Chemistry  2012;287(40):33644-33655.
Background: The role of formyl peptide receptors (Fprs) in the vomeronasal system remains unclear.
Results: Agonist properties of vomeronasal Fprs differ extensively from those expressed in immune cells.
Conclusion: We observe neofunctionalization of vomeronasal Fprs and functional conservation of immune Fprs.
Significance: These findings provide new insight into the sensory function and evolution of Fprs.
The formyl peptide receptor (Fpr) family is well known for its contribution to immune defense against pathogens in human and rodent leukocytes. Recently, several structurally related members of these receptors were discovered in sensory neurons of the mouse vomeronasal organ (VNO), key detectors of pheromones and related semiochemicals. Although the biological role of vomeronasal Fprs is not yet clear, the known contribution of other Fprs to host immune defense suggested that they could contribute to vomeronasal pathogen sensing. Precise knowledge about the agonist properties of mouse Fprs is required to determine their function. We expressed all seven mouse and three human Fprs using an in vitro system and tested their activation with 32 selected compounds by conducting high throughput calcium measurements. We found an intriguing functional conservation between human and mouse immune Fprs that is most likely a consequence of closely similar biological constraints. By contrast, our data suggest a neofunctionalization of the vomeronasal Fprs. We show that the vomeronasal receptor mFpr-rs1 can be activated robustly by W-peptide and structural derivatives but not by other typical ligands of immune Fprs. mFpr-rs1 exhibits a stereo-selective preference for peptides containing d-amino acids. The same peptide motifs are contained in pathogenic microorganisms. Thus, the ligand profile of mFpr-rs1 is consistent with a role in vomeronasal pathogen sensing.
doi:10.1074/jbc.M112.375774
PMCID: PMC3460462  PMID: 22859307
7-Helix Receptor; Calcium Imaging; Immunology; Molecular Pharmacology; Neuroscience; Olfaction
20.  Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation 
Peter Karlson and Martin Lüscher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.
doi:10.3791/3311
PMCID: PMC3369656  PMID: 22157638
Neuroscience;  Issue 58;  Vomeronasal organ;  VNO;  pheromone;  calcium imaging;  tissue slice preparation;  floating immunohistochemistry;  GFP
21.  Evolutionary Patterns and Selective Pressures of Odorant/Pheromone Receptor Gene Families in Teleost Fishes 
PLoS ONE  2008;3(12):e4083.
Background
Teleost fishes do not have a vomeronasal organ (VNO), and their vomeronasal receptors (V1Rs, V2Rs) are expressed in the main olfactory epithelium (MOE), as are odorant receptors (ORs) and trace amine-associated receptors (TAARs). In this study, to obtain insights into the functional distinction among the four chemosensory receptor families in teleost fishes, their evolutionary patterns were examined in zebrafish, medaka, stickleback, fugu, and spotted green pufferfish.
Methodology/Principal Findings
Phylogenetic analysis revealed that many lineage-specific gene gains and losses occurred in the teleost fish TAARs, whereas only a few gene gains and losses have taken place in the teleost fish vomeronasal receptors. In addition, synonymous and nonsynonymous nucleotide substitution rate ratios (KA/KS) in TAARs tended to be higher than those in ORs and V2Rs.
Conclusions/Significance
Frequent gene gains/losses and high KA/KS in teleost TAARs suggest that receptors in this family are used for detecting some species-specific chemicals such as pheromones. Conversely, conserved repertoires of V1R and V2R families in teleost fishes may imply that receptors in these families perceive common odorants for teleosts, such as amino acids. Teleost ORs showed intermediate evolutionary pattern between TAARs and vomeronasal receptors. Many teleost ORs seem to be used for common odorants, but some ORs may have evolved to recognize lineage-specific odors.
doi:10.1371/journal.pone.0004083
PMCID: PMC2605262  PMID: 19116654
22.  A Recent Class of Chemosensory Neurons Developed in Mouse and Rat 
PLoS ONE  2011;6(9):e24462.
In most animal species, the vomeronasal organ ensures the individual recognition of conspecifics, a prerequisite for a successful reproduction. The vomeronasal organ expresses several receptors for pheromone detection. Mouse vomeronasal type-2 receptors (V2Rs) are restricted to the basal neurons of this organ and organized in four families. Family-A, B and D (family ABD) V2Rs are expressed monogenically (one receptor per neuron) and coexpress with either Vmn2r1 or Vmn2r2, two members of family-C V2Rs. Thus, basal neurons are characterized by specific combinations of two V2Rs. To investigate this issue, we raised antibodies against all family-C V2Rs and analyzed their expression pattern. We found that six out of seven family-C V2Rs (Vmn2r2-7) largely coexpressed and that none of the anti-Vmn2r2-7 antibodies significantly stained Vmn2r1 positive neurons. Thus, basal neurons are divided into two complementary subsets. The first subset (Vmn2r1-positive) preferentially coexpresses a distinct group of family-ABD V2Rs, whereas the second subset (Vmn2r2-7-positive) coexpresses the remaining group of V2Rs. Phylogenetic reconstruction and the analysis of genetic loci in various species reveal that receptors expressed by this second neuronal subset are recent branches of the V2R tree exclusively present in mouse and rat. Conversely, V2Rs expressed in Vmn2r1 positive neurons, are phylogenetically ancient and found in most vertebrates including rodents. Noticeably, the more recent neuronal subset expresses a type of Major Histocompatibility Complex genes only found in murine species. These results indicate that the expansion of the V2R repertoire in a murine ancestor occurred with the establishment of a new population of vomeronasal neurons in which coexists the polygenic expression of a recent group of family-C V2Rs (Vmn2r2-7) and the monogenic expression of a recent group of family-ABD V2Rs. This evolutionary innovation could provide a molecular rationale for the exquisite ability in individual recognition and mate choice of murine species.
doi:10.1371/journal.pone.0024462
PMCID: PMC3170373  PMID: 21931725
23.  A common gene exclusion mechanism used by two chemosensory systems 
Sensory coding strategies within vertebrates involve the expression of a limited number of receptor types per sensory cell. In mice, each vomeronasal sensory neuron transcribes monoallelically a single V1R pheromone receptor gene, chosen from a large V1R repertoire. The nature of the signals leading to this strict receptor expression is unknown, but is apparently based on a negative feedback mechanism initiated by the transcription of the first randomly chosen functional V1R gene. We show, in vivo, that the genetic replacement of the V1rb2 pheromone receptor coding sequence by an unrelated one from the odorant receptor gene M71, maintains gene exclusion. The expression of this exogenous odorant receptor in vomeronasal neurons does not trigger the transcription of odorant receptor-associated signaling molecules. These results strongly suggests that despite the very divergent odorant and vomeronasal receptor localization, function and transduction cascades, a common mechanism is used by these chemoreceptors to regulate their expression.
doi:10.1111/j.1460-9568.2009.06630.x
PMCID: PMC3709462  PMID: 19200072
pheromone receptor; olfaction; gene regulation; mouse; monogenic expression
24.  The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms 
BMC Genomics  2014;15(1):720.
Background
Molecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods.
Results
We generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology.
Conclusions
Recent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-720) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-720
PMCID: PMC4152597  PMID: 25159659
Chemosensory genes; Microcebus murinus; Multigene family; Pacific Biosciences; Pheromone detection; Single molecule real-time sequencing
25.  A Candidate Subspecies Discrimination System Involving a Vomeronasal Receptor Gene with Different Alleles Fixed in M. m. domesticus and M. m. musculus 
PLoS ONE  2010;5(9):e12638.
Assortative mating, a potentially efficient prezygotic reproductive barrier, may prevent loss of genetic potential by avoiding the production of unfit hybrids (i.e., because of hybrid infertility or hybrid breakdown) that occur at regions of secondary contact between incipient species. In the case of the mouse hybrid zone, where two subspecies of Mus musculus (M. m. domesticus and M. m. musculus) meet and exchange genes to a limited extent, assortative mating requires a means of subspecies recognition. We based the work reported here on the hypothesis that, if there is a pheromone sufficiently diverged between M. m. domesticus and M. m. musculus to mediate subspecies recognition, then that process must also require a specific receptor(s), also sufficiently diverged between the subspecies, to receive the signal and elicit an assortative mating response. We studied the mouse V1R genes, which encode a large family of receptors in the vomeronasal organ (VNO), by screening Perlegen SNP data and identified one, Vmn1r67, with 24 fixed SNP differences most of which (15/24) are nonsynonymous nucleotide substitutions between M. m. domesticus and M. m. musculus. We observed substantial linkage disequilibrium (LD) between Vmn1r67 and Abpa27, a mouse salivary androgen-binding protein gene that encodes a proteinaceous pheromone (ABP) capable of mediating assortative mating, perhaps in conjunction with its bound small lipophilic ligand. The LD we observed is likely a case of association rather than residual physical linkage from a very recent selective sweep, because an intervening gene, Vmn1r71, shows significant intra(sub)specific polymorphism but no inter(sub)specific divergence in its nucleotide sequence. We discuss alternative explanations of these observations, for example that Abpa27 and Vmn1r67 are coevolving as signal and receptor to reinforce subspecies hybridization barriers or that the unusually divergent Vmn1r67 allele was not a product of fast positive selection, but was derived from an introgressed allele, possibly from Mus spretus.
doi:10.1371/journal.pone.0012638
PMCID: PMC2936562  PMID: 20844586

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