Evolution of spatially coexpressed families of type-2 vomeronasal receptors in rodents

Abstract

The vomeronasal organ (VNO) is an olfactory structure for the detection of pheromones. VNO neurons express three groups of unrelated G-protein-coupled receptors. Type-2 vomeronasal receptors (V2Rs) are specifically localized in the basal neurons of the VNO and are believed to sense protein pheromones eliciting specific reproductive behaviors. In murine species, V2Rs are organized into four families. Family-ABD V2Rs are expressed monogenically and coexpress with family-C V2Rs of either subfamily C1 (V2RC1) or subfamily C2 (V2RC2), according to a coordinate temporal diagram. Neurons expressing the phylogenetically ancient V2RC1 coexpress family-BD V2Rs or a specific group of subfamily-A V2Rs (V2RA8-10), whereas a second neuronal subset (V2RC2-positive) coexpresses a recently expanded group of five subfamily-A V2Rs (V2RA1-5) along with vomeronasal-specific Major Histocompatibility Complex molecules (H2-Mv). Through database mining and Sanger sequencing, we have analyzed the onset, diversification, and expansion of the V2R-families throughout the phylogeny of Rodentia. Our results suggest that the separation of V2RC1 and V2RC2 occurred in a Cricetidae ancestor in coincidence with the evolution of the H2-Mv genes; this phylogenetic event did not correspond with the origin of the coexpressing V2RA1-5 genes, which dates back to an ancestral myomorphan lineage. Interestingly, the evolution of receptors within the V2RA1-5 group may be implicated in the origin and diversification of some of the V2R putative cognate ligands, the exocrine secreting peptides. The establishment of V2RC2, which probably reflects the complex expansion and diversification of family-A V2Rs, generated receptors that have probably acquired a more subtle functional specificity.

Keywords: chemosensory; evolution; pheromones; phylogeny; rodents; vomeronasal.

Fig. 1.—

Structure and classification of V2Rs in the mouse. (A) Unrooted NJ phylogenetic tree based on multiple alignment of full-length V2R sequences of the mouse. Family-E V2R corresponds to the former subfamily A10. The combinatorial coexpression between subfamilies C1 and C2 and family-ABDE V2Rs is indicated by colors in the tree. (B) Domain and exon boundaries in the primary structure of V2Rs are indicated referring to the amino acid sequence of Vmn2r1. The red segments represent the regions of exon 3 and exon 5 considered for PCR amplification of family A and family C, respectively. (C) Predicted 3D structure of V2Rs. The structural model of mouse Vmn2r1 was constructed with the Phyre program using the coordinates of the extracellular region of the metabotropic glutamate receptor (PDB: 2E4W) and the transmembrane region of squid rhodopsin (PDB: 2ZIY) as templates. Portions corresponding to the ligand-binding 1 (LB1), ligand-binding 2 (LB2), cysteine-rich (CR), and transmembrane (7tm) domains as shown in (B) are represented in different colors. Arrow points to the amino acid position in the CR domain used to differentiate between subfamily C1 and C2.

Fig. 2.—

Evolutionary history of family-A V2Rs in rodents. (A) The phylogenetic tree of Rodentia highlighting the five main suborders (Myomorpha, Anomaluromorpha, Castorimorpha, Hystricomorpha, and Sciuromorpha), the Muroidea and Dipodidea clades, the Muridae family and representative genera in which this order is divided (Hedges, et al. 2006; Blanga-Kanfi et al. 2009). (B) Radial dendrogram showing the evolutionary origin of the A-subfamily. The NJ phylogenetic tree is based on a multiple alignment of family-A DNA sequences (exon 3) from representative species of Rodentia. The mouse V2RA subfamilies are indicated. The tree is rooted using family E (E) as an outgroup. Branches are stained according to species classification as shown in panel A. Only representative sequences for each species are included in the tree. The complete repertoire of the analyzed sequences with branch lengths, tip labels and bootstrap supports are shown in supplementary fig. S5, Supplementary Material online.

Fig. 3.—

Phylogeny of family-BCDE V2Rs in rodents. (A) Rooted NJ phylogenetic tree based on multiple DNA sequence alignments of full-length family-BCDE V2Rs. DNA sequences are reconstructed from the available genome databases. Family-A branches are collapsed. The tree is rooted with rabbit family-C V2Rs as outgroups. Bootstrap value of the subfamily C1 and C2 separation node is shown in bold. Scale bar: mean number of base substitutions per site. (B) Maximum likelihood phylogenetic tree based on the multiple amino acid alignment of family-C V2Rs reconstructed from the available databases of rodents. For species abbreviations refer to Methods. Bootstrap values are shown. Scale bar: mean number of amino acid substitutions per site.

Fig. 4.—

Multiple alignment of family-C V2Rs in rodents. The amino acid substitution (Q/H/D → K) in exon 5 that differentiates subfamily-C1 from subfamily-C2 V2Rs is encased by a red and blue rectangle, respectively. The position of this residue in the 3D structure of the protein is indicated in figure 1C with an arrow. For species abbreviations refer to Methods.

Fig. 5.—

Phylogenetic reconstruction of H2-Mv molecules in rodent species. NJ phylogenetic tree based on protein alignment of Mhc, obtained from BLAST search with H2-Mv protein sequences against the available rodentia sequence databases (Ensembl and nr databases). H2-Mv branches are represented in blue whereas protein hits obtained from J. jaculus and N. galili databases are shown in red. Accession numbers are given for each sequence except for mouse (ms) and rat (rt). For species abbreviations refer to Methods. Bootstrap values are shown. Scale bar: mean number of amino acid substitutions per site.

Fig. 6.—

Tissue distribution and expression of family-C V2Rs in the mouse. (A) RT-PCR with mouse family-C specific primers in the VNO and MOE and (B) in extra-vomeronasal tissues. MOE, main olfactory epithelium; K, kidney; T, testis; L, lung; Cx, cortex, M, midbrain; Cb, cerebellum; OB, olfactory bulb; C, negative control. Vmn2r1 (R1 in the panel) clusters with subfamily C1 V2Rs whereas Vmn2r2/5 (R2/5), Vmn2r3 (R3), Vmn2r4 (R4) and Vmn2r6/7 (R6/7) with subfamily C2 V2Rs. The coding sequence of β-actin was used as a control of amplification. (C) Double label immunohistochemistry on tissue sections of the olfactory epithelium with antibody against Vmn2r1 (subfamily C1) and Vmn2r2 (subfamily C2). Scale bar, 20 µm. (D) Immunohistochemical localization of Vmn2r1 in the olfactory epithelium. Double label immunohistochemistry was performed with an antibody against Vmn2r1 in combination with antibodies against IP3R3 or ChAT. Scale bar, 20 µm.

Fig. 7.—

Origins of the pheromone-related genetic components in the basal VNO neurons. Divergence times, in MYA, are based on Hedges et al. (2006).