Gene cluster lock after pheromone receptor gene choice

Abstract

In mammals, perception of pheromones is based on the expression in each vomeronasal sensory neuron of a limited set of receptor genes, chosen among a large repertoire. Here, we report an extremely tight control of the monogenic and monoallelic transcription of the V1rb2 receptor gene. Combining genetic and electrophysiological approaches, we show that the transcription of a non-functional V1r allele leads to the coexpression of another, functional V1r gene. The choice of this coexpressed gene surprisingly includes genes located on the cluster homologous to the one from which the mutant allele is transcribed. However, V1r genes located in cis relative to the transcribed mutant allele are excluded from the coexpression choice. Our observations strongly suggest a monogenic regulatory mechanism acting (a) at a general level, via the expression of the V1r receptor itself, and (b) at a more local level, defined by the V1r gene cluster.

Figure 1

Size and expression variability of the V1r families in the VNO. (A) A schematic representation of a hemisection through the nasal cavity of a mouse. The main olfactory epithelium (MOE) and its projections to the olfactory bulb (OB) are shown in dark yellow. The Gruneberg organ and its projections are indicated in green. The VNO and its axonal projections to the accessory olfactory bulb (AOB) are shown in orange. (B) Coronal section through a mouse VNO, corresponding to the plane indicated in panel A. Basal sensory neurons expressing V2r receptors are shown in green, while apical, V1r- and Gαi2-expressing VSNs are in orange. (C) A tree depicting the percentage of V1rs recognized by our set of in situ probes. The area occupied by the gray disk on the lower left corresponds to 1%. (D) A representation of the proportion of VSNs, which express V1rs from a given family.

Figure 2

Monogenic expression of V1r genes. (A–D) Representative two-color in situ hybridizations using a probe specific for V1rb2 (green), and probes specific for a given V1r family (red). (E) Empty bars represent the number of VSNs reacting with the V1rb2-specific probe, and potentially with the V1ra-l probes. Dark bars (invisible since no coexpression was found) indicate the number of these V1rb2-expressing VSNs cotranscribing a member of any of the V1r families. (F, G) Monoallelic expression of V1rb2. (F) An in situ hybridization with allele-specific probes for V1rb2 (green and red represent paternal and maternal alleles, respectively) (G) Graph showing the number of VSNs reacting with allele-specific probes for V1rb2 (empty bars), and those coexpressing both alleles (dark bar). Genotypes are shown, in white, in the upper right corner. Scale bar, 30 μm.

Figure 3

Expression of a deletion V1rb2 allele allows for coexpression of another V1r. (A) Two-photon imaging of a wt glomerulus innervated by V1rb2-expressing axons. (B) Similar imaging of fibers emanating from VSNs expressing the V1rb2^dv allele, which enter the accessory olfactory bulb glomerular layer, and apparently innervate random glomeruli. (C–E) In situ hybridizations of VSNs coexpressing the V1rb2^dv allele (green) and a member of the V1ra family (red). (F) The number of VSNs coexpressing the V1rb2^dv allele together with a member of another receptor family (dark bars). Genotypes are shown, in white, in the upper right corner. Scale bar, 20 μm.

Figure 4

Coexpressed V1rs are functional receptors. (A–C) Representative whole-cell patch-clamp recordings of VSNs expressing the V1rb2^dv allele to 5″ pulses of 2-heptanone, α and β-farnesenes, 2,5-dimethylpyrazine or pentyl-acetate (10⁻⁶ M; holding potential, −60 mV). VSNs did not respond to these agonists (A), or responded to α and β-farnesenes (B), or to pentyl-acetate (C). (D) Only 2-heptanone (10⁻⁸ M) induced an inward current in VSNs expressing V1rb2 (V1rb2^vg line). Cell viability was assessed by KCl (100 mM) pulses before and after the perfusion with agonists.

Figure 5

Cluster lock associated with V1r gene expression. (A) A schematic representation of the V1ra/V1rb alleles used. (B–D) In situ hybridizations of V1rab^+/+, V1rab^Δ/+ and V1rab^Δ/Δ VNO sections using V1rb9-V1rb2 probes. V1rb transcripts were identified in V1rab^+/+ and V1rab^Δ/+ animals, while no expression of V1rb genes was observed in V1rab^Δ/Δ mice. (E) in situ hybridization of V1rab^Δ/Δ VNO sections using the V1ri1-V1ri8 probe set; expression of V1ri genes was observed in V1rab^Δ/Δ mice. (F) V1rab^Δ /V1rb2^dv VNO sections hybridized with V1ri1-V1ri8 probes. A single VSN (yellow) coexpresses the V1rb2^dv allele and a V1ri gene. (G) Graph corresponding to in situ hybridizations, indicating an absence of coexpression between the V1rb2^dv allele and V1ra/V1rb genes surrounding it. Expected values of coexpressing VSNs were based on the observed frequencies, for each family, of coexpressing VSNs in V1rb2^dv/dv mice. Genotypes are shown, in white, in the upper right corner.