Evolution of vomeronasal receptor 1 (V1R) genes in the common marmoset (Callithrix jacchus).

Pheromones are crucial for eliciting innate responses and inducing social and sexual behaviors in mammals. The vomeronasal receptor 1 genes, V1Rs, encode members of a pheromone receptor family that are mainly expressed in the vomeronasal organ (VNO). The V1R family shows extraordinary variety in gene number among vertebrates owing to successive gene gains and losses during evolution. Such diversity is believed to reflect a degree of dependence on the VNO. We investigated V1R evolution in primate lineages closely related to humans because these VNOs show a trend toward degeneration. We performed extensive phylogenetic analyses for V1Rs from a broad range of primate species. Although the decline of intact genes was evident in anthropoids (hominoids, Old World monkeys and New World monkeys), we found that a certain number of intact genes persist in New World monkeys. In one New World monkey species, the common marmoset (Callithrix jacchus), we examined seven putatively functional V1Rs using in situ hybridization and reverse transcription-PCR. Based on their mRNA expression patterns in the VNO and other organs, two types of V1Rs emerged: the canonical class with VNO-specific expression, and a second group having more ubiquitous expression in various organs as well as VNO. Furthermore, phylogenetic analysis revealed that the class with the more widespread expression had been retained longer in evolution than the VNO-specific type. We propose that the acquisition of a novel non-VNO-related function(s) may have led to the survival of a small but persistent number of V1Rs in anthropoid primates.

Distinct functions of two olfactory marker protein genes derived from teleost-specific whole genome duplication.

BACKGROUND: Whole genome duplications (WGDs) have been proposed to have made a significant impact on vertebrate evolution. Two rounds of WGD (1R and 2R) occurred in the common ancestor of Gnathostomata and Cyclostomata, followed by the third-round WGD (3R) in a common ancestor of all modern teleosts. The 3R-derived paralogs are good models for understanding the evolution of genes after WGD, which have the potential to facilitate phenotypic diversification. However, the recent studies of 3R-derived paralogs tend to be based on in silico analyses. Here we analyzed the paralogs encoding teleost olfactory marker protein (OMP), which was shown to be specifically expressed in mature olfactory sensory neurons and is expected to be involved in olfactory transduction. RESULTS: Our genome database search identified two OMPs (OMP1 and OMP2) in teleosts, whereas only one was present in other vertebrates. Phylogenetic and synteny analyses suggested that OMP1 and 2 were derived from 3R. Both OMPs showed distinct expression patterns in zebrafish; OMP1 was expressed in the deep layer of the olfactory epithelium (OE), which is consistent with previous studies of mice and zebrafish, whereas OMP2 was sporadically expressed in the superficial layer. Interestingly, OMP2 was expressed in a very restricted region of the retina as well as in the OE. In addition, the analysis of transcriptome data of spotted gar, a non-teleost fish, revealed that single OMP gene was expressed in the eyes. CONCLUSION: We found distinct expression patterns of zebrafish OMP1 and 2 at the tissue and cellular level. These differences in expression patterns may be explained by subfunctionalization as the model of molecular evolution. Namely, single OMP gene was speculated to be originally expressed in the OE and the eyes in the common ancestor of all Osteichthyes (bony fish including tetrapods). Then, two OMP gene paralogs derived from 3R-WGD reduced and specialized the expression patterns. This study provides a good example for analyzing a functional subdivision of the teleost OE and eyes as revealed by 3R-derived paralogs of OMPs.

Multiple episodic evolution events in V1R receptor genes of East-African cichlids.

Fish use olfaction to detect a variety of nonvolatile chemical signals, and thus, this sense is key to survival and communication. However, the contribution of the olfactory sense to social-especially reproductive-interactions in cichlids is still controversial. To obtain insights into this issue, we investigated the genes encoding V1Rs-possible candidates for reproductive pheromone receptors-among East-African cichlids. Interestingly, we found an excess of nonsynonymous over synonymous substitutions in four of six V1R genes in multiple cichlid lineages. First, we found that highly dimorphic V1R2 allele groups were shared among the cichlids inhabiting all East-African Great Lakes emerged through the episodic accumulation of the nonsynonymous substitutions prior to the radiation of the Lake Tanganyika species flock. We further detected such episodic events in V1R1 of the tribe Tropheini, and in V1R3 and V1R6 of the tribe Trematocarini. The excess of nonsynonymous substitutions in these examples were indicated as dN/dS > 1, which were all statistically significant by Fisher's exact test. Furthermore, we speculate that the amino acid changes in these episodic events are likely functional switch because they occurred in the putative ligand-binding pocket. Our finding of the occurrence of multiple episodic events and the unexpected gene diversity in one unique gene family is suggestive of the contribution of the V1R to the species diversification and the social interaction in cichlids.

Expression of G proteins in the olfactory receptor neurons of the newt Cynops pyrrhogaster: their unique projection into the olfactory bulbs.

We analyzed the expression of G protein α subunits and the axonal projection into the brain in the olfactory system of the semiaquatic newt Cynops pyrrhogaster by immunostaining with antibodies against Gαolf and Gαo , by in situ hybridization using probes for Gαolf , Gαo , and Gαi2 , and by neuronal tracing with DiI and DiA. The main olfactory epithelium (OE) consists of two parts, the ventral OE and dorsal OE. In the ventral OE, the Gαolf - and Gαo -expressing neurons are located in the apical and basal zone of the OE, respectively. This zonal expression was similar to that of the OE in the middle cavity of the fully aquatic toad Xenopus laevis. However, the Gαolf - and Gαo -expressing neurons in the newt ventral OE project their axons toward the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), respectively, whereas in Xenopus, the axons of both neurons project solely toward the MOB. In the dorsal OE of the newt, as in the principal cavity of Xenopus, the majority of the neurons express Gαolf and extend their axons into the MOB. In the vomeronasal organ (VNO), the neurons mostly express Gαo . These neurons and quite a few Gαolf -expressing neurons project their axons toward the AOB. This feature is similar to that in the terrestrial toad Bufo japonicus and is different from that in Xenopus, in which VNO neurons express solely Gαo , although their axons invariably project toward the AOB. We discuss the findings in the light of diversification and evolution of the vertebrate olfactory system.

Lineage-specific expansion of vomeronasal type 2 receptor-like (OlfC) genes in cichlids may contribute to diversification of amino acid detection systems.

Fish use olfaction to sense a variety of nonvolatile chemical signals in water. However, the evolutionary importance of olfaction in species-rich cichlids is controversial. Here, we determined an almost complete sequence of the vomeronasal type 2 receptor-like (OlfC: putative amino acids receptor in teleosts) gene cluster using the bacterial artificial chromosome library of the Lake Victoria cichlid, Haplochromis chilotes. In the cluster region, we found 61 intact OlfC genes, which is the largest number of OlfC genes identified among the seven teleost fish investigated to date. Data mining of the Oreochromis niloticus (Nile tilapia) draft genome sequence, and genomic Southern hybridization analysis revealed that the ancestor of all modern cichlids had already developed almost the same OlfC gene repertoire, which was accomplished by lineage-specific gene expansions. Furthermore, comparison of receptor sequences showed that recently duplicated paralogs are more variable than orthologs of different species at particular sites that were predicted to be involved in amino acid selectivity. Thus, the increase of paralogs through gene expansion may lead to functional diversification in detection of amino acids. This study implies that cichlids have developed a potent capacity to detect a variety of amino acids (and their derivatives) through OlfCs, which may have contributed to the extraordinary diversity of their feeding habitats.

Existence of Gαi2-expressing axon terminals in the goat main olfactory bulb.

In rodents, Gα(i2)-expressing sensory neurons (SNs) that co-express vomeronasal receptor type 1 (V1R) are specifically found in the vomeronasal organ (VNO) and project their axons to the accessory olfactory bulb (AOB). In goats, however, Gα(i2)/V1R-expressing SNs exist in both the VNO and the olfactory epithelium. Thus, we examined whether the Gα(i2)-expressing axons functionally project to the main olfactory bulb (MOB). We analyzed the expression of Gα(i2) in the olfactory bulb and found small Gα(i2)-immunoreactive clusters in the MOB. The Gα(i2)-immunoreactive axons in these clusters made synaptic contacts with second-order neurons in the MOB. These results suggest that some Gα(i2)-expressing SNs functionally project their axons to the MOB in goats.

Characterization of V1R receptor (ora) genes in Lake Victoria cichlids.

Although olfaction could play a crucial role in underwater habitats by allowing fish to sense a variety of nonvolatile chemical signals, the importance of olfaction in species-rich cichlids is still controversial. In particular, examining whether cichlids rely on olfaction for reproduction is of primary interest to understand the mechanisms of speciation. In the present study, we explored the V1R (also known as ora) genes, which are believed to encode reproductive pheromone receptors in fish, in the genomes of Lake Victoria cichlids. By screening a bacterial artificial chromosome library, we identified all six intact V1R genes (V1R1 to V1R6) that have been reported in other teleost fish. Furthermore, RT-PCR and in situ hybridization analyses showed that all of the V1R genes were expressed in the olfactory epithelium, indicating that these receptors are functional in cichlids. These observations indicate that cichlids use V1R-mediated olfaction in some ways for their social behaviors.

Accessory olfactory bulb neurons are required for maintenance but not induction of V2R vomeronasal receptor gene expression in vitro.

Many mammals detect pheromones by a sensory organ, the vomeronasal organ (VNO). In a previous study using immunoblot and immunocytochemical analyses, we reported that cocultures of VNOs with accessory olfactory bulb (AOB) neurons resulted in the maturation of vomeronasal sensory neurons (VSNs) and a greater expression of V2R family vomeronasal receptors than cultures with VNO alone. To further characterize the V2R expression, we here investigated the time course of the expression of V2R mRNA in the presence or absence of AOB neurons using RT-PCR analysis. The expression of V2R mRNA was already detectable not only in the VNO cocultured with AOB neurons for 3 days in coculture but also in the VNO cultured alone for the same number of days. However, the expression of V2R mRNA in the VNO cultured alone was remarkably decreased during the additional culture period, although that in the cocultured VNO showed sustained expression. Moreover, the application of 2 µM TTX to the cocultured VNO resulted in a marked decrease in the V2R mRNA expression to a level equal to that in the VNO cultured alone for 14 days in coculture. Our previous working hypothesis was that the expression of V2Rs in VSNs was induced by interacting with AOB neurons. However, the present results suggest that the receptor expression in VSNs is independent of the interaction with AOB neurons in the early developmental stage, but is maintained by the active interaction with AOB neurons.

Involvement of Gα(olf)-expressing neurons in the vomeronasal system of Bufo japonicus.

Most terrestrial vertebrates possess anatomically distinct olfactory organs: the olfactory epithelium (OE) and the vomeronasal organ (VNO). In rodents, olfactory receptors coupled to Gα(olf) are expressed in the OE, whereas vomeronasal receptors type 1 (V1R) and vomeronasal receptors type 2 (V2R), coupled to Gα(i2) and Gα(o) , respectively, are expressed in the VNO. These receptors and G proteins are thought to play important roles in olfactory perception. However, we previously reported that only V2R and Gα(o) expression is detected in the Xenopus laevis VNO. As X. laevis spends its entire life in water, we considered that expression of limited types of chemosensory machinery in the VNO might be due to adaptation of the VNO to aquatic life. Thus, we analyzed the expression of G proteins in the VNO and the accessory olfactory bulb (AOB) of the adult Japanese toad, Bufo japonicus, because this species is well adapted to a terrestrial life. By using immunohistochemical analysis in combination with in situ hybridization and DiI labeling, we found that B. japonicus Gα(olf) and Gα(o) were expressed in the apical and middle-to-basal layer of the vomeronasal neuroepithelium, and that the axons of these Gα(olf) - and Gα(o) -expressing vomeronasal neurons projected to the rostral and caudal accessory olfactory bulb, respectively. These results strongly suggest that both the Gα(olf) - and Gα(o) -mediated signal transduction pathways function in the B. japonicus VNO. The expression of Gα(olf) in the B. japonicus VNO may correlate with the detection of airborne chemical cues and with a terrestrial life.

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.

Xenopus V1R vomeronasal receptor family is expressed in the main olfactory system.

To date, over 100 vomeronasal receptor type 1 (V1R) genes have been identified in rodents. V1R is specifically expressed in the rodent vomeronasal organ (VNO) and is thought to be responsible for pheromone reception. Recently, 21 putatively functional V1R genes were identified in the genome database of the amphibian Xenopus tropicalis. Amphibians are the first vertebrates to possess a VNO. In order to determine at which point during evolution the vertebrate V1R genes began to function in the vomeronasal system, we analyzed the expression of all putatively functional V1R genes in Xenopus olfactory organs. We found that V1R expression was not detected in the VNO but was specifically detected in the main olfactory epithelium (MOE). We also observed that V1R-expressing cells in the MOE coexpressed Gi2, thus suggesting that the V1R-Gi2-mediated signal transduction pathway, which is considered to play an important role in pheromone reception in the rodent VNO, exists in the amphibian MOE. These results suggest that V1R-mediated signal transduction pathway functions in Xenopus main olfactory system.

Diverse systems for pheromone perception: multiple receptor families in two olfactory systems.

Traditionally, the olfactory epithelium is considered to recognize conventional odors, while the vomeronasal organ detects pheromones. However, recent advances suggest that vertebrate pheromones can also be detected by the olfactory epithelium. In the vomeronasal organ and the olfactory epithelium, structurally distinct multiple receptor families are expressed. In rodents, two of these receptor families, V1R and V2R, are expressed specifically in the vomeronasal organ and detect pheromones and pheromone candidates. A newly isolated trace amine-associated receptor detects some of the putative pheromones in the mouse olfactory epithelium. In addition, distinct second-messenger pathways and neural circuits are used for pheromone perception mediated by each receptor family. Furthermore, the function of these receptor families in these olfactory organs appears to differ among various vertebrate species. The systems for pheromone perception in vertebrates are far more complex than previously predicted.

Localization of the candidate genes ELOVL5 and SCD1 for 'male effect' pheromone synthesis in goats (Capra hircus).

The 'male effect' is a well-known phenomenon in female sheep and goats, whereby pheromone-induced activation of reproductive function occurs. In a previous study, we showed that the genes for elongation of long-chain fatty acids family member 5 (ELOVL5) and stearoyl-CoA desaturase 1 (SCD1) increased their expression significantly, concomitant with induction of pheromone synthesis. Therefore, these genes were considered to be prime candidate genes for pheromone synthesis. In the present study, we performed in situ hybridization to investigate where these two genes are expressed in goat skin. Strong positive signals were detected for both genes in the head skin of the male goat, which is the main site of pheromone production, and were mainly in the basal layer of the sebaceous gland cells, with the remaining cells showing negligible signals. None of the cells in the rump skin of the male goat or the head skin of the orchidectomized goat, neither of which produce pheromone, exhibited strong positive signals. The present study demonstrates that expression of these two candidate genes for pheromone synthesis is primarily localized in the sebaceous glands of the pheromone-producing skin region.

Stable knock-down of vomeronasal receptor genes in transgenic Xenopus tadpoles.

Xenopus V2R (xV2R), a family of G-protein-coupled receptors with seven transmembrane domains, is expressed in the Xenopus vomeronasal organ (VNO). There are six subgroups of xV2R, one of which, xV2RE, is predominantly expressed in the VNO. To understand the function of xV2R during VNO development, we developed a new method to achieve stable siRNA-suppression of the V2RE genes by introducing siRNA expression transgenes into the genomes of unfertilized eggs. We found that some of the derived transgenic tadpoles lacked VNOs and that their olfactory epithelium was fused. With the exception of one tadpole, expression of xV2RE was not detected in morphologically abnormal mutant tadpoles, although the olfactory marker protein and the olfactory receptors were expressed. These results suggest that we successfully produced transgenic tadpoles in which xV2RE expression was stably suppressed by siRNA, and that xV2RE plays a role in the morphogenesis of olfactory organs.

Expression of vomeronasal receptor genes in Xenopus laevis.

In the course of evolution, the vomeronasal organ (VNO) first appeared in amphibians. To understand the relationship between the VNO and the vomeronasal receptors, we isolated and analyzed the expression of the vomeronasal receptor genes of Xenopus laevis. We identified genes of the Xenopus V2R receptor family, which are predominantly expressed throughout the sensory epithelium of the VNO. The G-protein Go, which is coexpressed with V2Rs in the rodent VNO, was also extensively expressed throughout the vomeronasal sensory epithelium. These results strongly suggest that the V2Rs and Go are coexpressed in the vomeronasal receptor cells. The predominant expression of the Xenopus V2R families and the coexpression of the V2Rs and Go imply that V2Rs play important roles in the sensory transduction of Xenopus VNO. We found that these receptors were expressed not only in the VNO, but also in the posterolateral epithelial area of the principal cavity (PLPC). Electron microscopic study revealed that the epithelium of the PLPC is more like that of the VNO than that of the principal and the middle cavity. These results suggest that in adult Xenopus the V2Rs analyzed so far are predominantly expressed in the vomeronasal and vomeronasal-like epithelium. The analysis of V2R expression in Xenopus larvae demonstrates that V2Rs are predominantly expressed in the VNO even before metamorphosis.

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats.

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.