Exceptional LINE Density at V1R Loci: The Lyon Repeat Hypothesis Revisited on Autosomes

The mammalian olfactory system utilizes three large receptor families: the olfactory receptors (ORs) of the main nose and the vomeronasal type-1 and type-2 receptor genes (V1Rs and V2Rs) of the vomeronasal organ. We find that these loci are among the most long interspersed nuclear element (LINE)-dense regions of mammalian genomes. We investigate two evolutionary models to account for this cohabitation. First, we investigate an adaptive selection model, in which LINEs have contributed to expansions of mouse V1R repertoires. We find that even evolutionarily stable V1R loci are exceptionally LINE-rich compared to other genome loci, including loci containing other large gene clusters. Also, a more detailed analysis of specific V1R duplications does not reveal LINE patterns predicted by common LINE-mediated duplication mechanisms. Next, we investigate neutral models, in which LINEs were tolerated by, but not advantageous for, surrounding V1R genes. We find that V1R loci are exceptionally LINE-rich compared to other regions of similar AT base composition, and that duplicated V1R gene blocks are generally depleted of LINE elements, suggesting that these loci did not become densely populated with LINEs simply as a consequence of targeted integration or passive multiplication along with the genes. Finally, we show that individual LINE repeats of a given age at V1R, V2R, and OR loci exhibit a significantly longer average length than at other autosomal loci, suggesting a reduced tendency for these LINEs to be disrupted. We speculate that LINEs at V1R, V2R, and OR loci might be selectively retained because they contribute to allelic regulation of these three gene families. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A multireceptor genetic approach uncovers an ordered integration of VNO sensory inputs in the accessory olfactory bulb

Pheromone detection by the vomeronasal organ (VNO) is thought to rely on activation of specific receptors from the V1R and V2R gene families, but the central representation of pheromone receptor activation remains poorly understood. We generated transgenic mouse lines in which projections from multiple populations of VNO neurons, each expressing a distinct V1R, are differentially labeled with fluorescent proteins. This approach revealed that inputs from neurons expressing closely related V1Rs intermingle within shared, spatially conserved domains of the accessory olfactory bulb (AOB). Mitral cell-glomerular connectivity was examined by injecting intracellular dyes into AOB mitral cells and monitoring dendritic contacts with genetically labeled glomeruli. We show that individual mitral cells extend dendrites to glomeruli associated with different, but likely closely related, V1Rs. This organization differs from the labeled line of OR signaling in the main olfactory system and suggests that integration of information may already occur at the level of the AOB.     

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.     

The vomeronasal receptor V2R2 does not require escort molecules for expression in heterologous systems

In rodents, many behavioural responses are triggered by pheromones. These molecules are believed to bind and activate two families of G-protein coupled receptors, namely V1Rs and V2Rs, which are specifically expressed in the chemosensory neurons of the vomeronasal organ. V2Rs are homologous with Group 3 of G-protein-coupled receptors, which includes metabotropic glutamate receptors, calcium-sensing receptors, fish olfactory receptors, and taste receptors for sweet molecules and amino acids. The large extracellular region of these receptors is folded as a dimer and, in this form, binds agonists that in many cases are amino acids. It has recently been reported that V2Rs must be physically associated with specific major histocompatibility complex class Ib molecules (MHC) for their expression in both mouse vomeronasal neurons and heterologous cell lines. Here, we show that in contrast to the other V2Rs, V2R2, an atypical member of this receptor family, can be successfully and abundantly expressed by insect cells without the requirement of escort molecules like MHC. Moreover, the extracellular binding domain of V2R2, secreted as a soluble product, forms dimers via cysteine-mediated sulphur bridges. Overall, the data presented in this paper confirm that V2R2 diverges from the other members of the V2R family and suggest a different role for this receptor in pheromonal communication.     

A divergent pattern of sensory axonal projections is rendered convergent by second-order neurons in the accessory olfactory bulb

The mammalian vomeronasal system is specialized in pheromone detection. The neural circuitry of the accessory olfactory bulb (AOB) provides an anatomical substrate for the coding of pheromone information. Here, we describe the axonal projection pattern of vomeronasal sensory neurons to the AOB and the dendritic connectivity pattern of second-order neurons. Genetically traced sensory neurons expressing a given gene of the V2R class of vomeronasal receptors project their axons to six to ten glomeruli distributed in globally conserved areas of the AOB, a theme similar to V1R-expressing neurons. Surprisingly, second-order neurons tend to project their dendrites to glomeruli innervated by axons of sensory neurons expressing the same V1R or the same V2R gene. Convergence of receptor type information in the olfactory bulb may represent a common design in olfactory systems.     

A recent class of chemosensory neurons developed in mouse and rat

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.     

Major cis-regulatory elements for rice bidirectional promoter activity reside in the 5′-untranslated regions

Bidirectional promoters are defined as those that regulate adjacent genes organized in a divergent fashion (head to head orientation) and separated by < 1 kb. In order to dissect bidirectional promoter activity in a model plant, deletion analysis was performed for seven rice promoters using promoter-reporter gene constructs, which identified three promoters to be bidirectional. Regulatory elements located in or close to the 5′-untranslated regions (UTR) of one of the genes (divergent gene pair) were found to be responsible for their bidirectional activity. DNA footprinting analysis identified unique protein binding sites in these promoters. Deletion/alteration of these motifs resulted in significant loss of expression of the reporter genes on either side of the promoter. Changes in the motifs at both the positions resulted in a remarkable decrease in bidirectional activity of the reporter genes flanking the promoter. Based on our results, we propose a novel mechanism for the bidirectionality of rice bidirectional promoters.     

Largest vertebrate vomeronasal type 1 receptor gene repertoire in the semiaquatic platypus

Vertebrate vomeronasal chemoreception plays important roles in many aspects of an organism's daily life, such as mating, territoriality, and foraging. Vomeronasal type 1 receptors (V1Rs) and vomeronasal type 2 receptors (V2Rs), 2 large families of G protein-coupled receptors, serve as vomeronasal receptors to bind to various pheromones and odorants. Contrary to the previous observations of reduced olfaction in aquatic and semiaquatic mammals, we here report the surprising finding that the platypus, a semiaquatic monotreme, has the largest V1R repertoire and nearly largest combined repertoire of V1Rs and V2Rs of all vertebrates surveyed, with 270 intact genes and 579 pseudogenes in the V1R family and 15 intact genes, 55 potentially intact genes, and 57 pseudogenes in the V2R family. Phylogenetic analysis shows a remarkable expansion of the V1R repertoire and a moderate expansion of the V2R repertoire in platypus since the separation of monotremes from placentals and marsupials. Our results challenge the view that olfaction is unimportant to aquatic mammals and call for further study into the role of vomeronasal reception in platypus physiology and behavior.     

Distinct evolutionary patterns between chemoreceptors of 2 vertebrate olfactory systems and the differential tuning hypothesis

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.     

Comparative genomics of odorant and pheromone receptor genes in rodents

We applied a comprehensive data-mining strategy to examine the repertoires of rat and mouse odorant receptors (ORs) and type 1 pheromone receptors (V1Rs) using the mm5 (mouse) and rn3 (rat) genomes. We identified 1576 rat OR genes, including 292 pseudogenes. The rat V1R repertoire is composed of 115 intact genes and 72 pseudogenes. The mouse OR and V1R databases were updated using the new assembly mm5, from which 1375 mouse ORs and 308 V1Rs were identified, with more than 100 putative pseudogenes from mm2 now identified as intact because of the higher sequence quality. With these new data we have conducted a series of genomic analyses of the OR and V1R genes from mouse and rat. Orthologous OR clusters were identified in mouse and rat and comparison analysis was performed at three incremental levels: families, coding sequences, and motifs. At the family level, we found that V1R genes have more species-specific families than OR genes. About 20% of intact V1R genes have no orthologous counterpart in the same family, whereas less than 1% of intact ORs are similarly isolated. At the coding sequence level, OR genes are more conserved between mouse and rat than V1R genes. OR genes share greater similarity with their orthologous counterparts than with their closest neighbor, whereas V1R genes show the opposite tendency. Motifs were identified to obtain biological insights. Motifs specific for species or families were found in OR and V1R genes, which may result in the differential pheromone-dependent behaviors and perception of odors between mouse and rat.     

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.