A common gene exclusion mechanism used by two chemosensory systems

Abstract

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 signalling molecules. These results strongly suggest that despite the different odorant and vomeronasal receptor expression sites, function and transduction cascades, a common mechanism is used by these chemoreceptors to regulate their transcription.

Figure 1

Sequence homology between M71 and V1R receptors. A sequence logo corresponding to all mouse, rat and dog V1R sequences is aligned with V1RB2 and M71. This type of analysis provides the consensus sequence and the frequency of a given amino acid at a given position. The size of the residues correlates with their conservation in rat, mouse and dog. Green, black, red and blue colors represent, respectively, uncharged polar (except for glycine and cysteine), nonpolar, acidic and basic residues. Black dots represent amino acid identities between M71 and V1RB2. Asterisks indicate residues shared between the conserved amino acids in the V1R sequence logo and M71.

Figure 2

Forced expression of the odorant receptor M71 in V1rb2^mv-expressing vomeronasal neurons. (A) Schematic illustrating the mouse knock-in lines used in this study. (B, C) Coronal section of vomeronasal organs from the V1rb2^vt and V1rb2^mv lines. LacZ expression, which indirectly reflects the expression of V1RB2 or M71, is in red. (D) Coronal section of a vomeronasal organ from the V1rb2^mv line. Exogenous expression of the odorant receptor M71 in vomeronasal neurons is shown in red, and is restricted to the dendritic endings. (E) Whole-mount X-Gal staining of vomeronasal organs showing punctate transcription of the V1rb2^mv and V1rb2^vt alleles in vomeronasal sensory neurons. Note the decreased number of labeled cells in 4 month-old V1rb2^mv/mv mice (middle panel) relative to 3.5 week-old animals (upper panel). (F) Graph showing the decrease of labeled cells observed between 3.5 and 14-20 weeks in V1rb2^mv mice. Each circle corresponds to a vomeronasal organ and horizontal lines indicate means (n=19). VNO, vomeronasal organ, MOE, main olfactory epithelium, OB, olfactory bulb, AOB, accessory olfactory bulb. Scale bar: 30 micrometers.

Figure 3

Allelic exclusion between V1rb2^mv and V1rb2^vg-expressing vomeronasal sensory neurons. (A, B) Coronal sections of V1rb2^mv/vg vomeronasal organs. Expression of the V1rb2^vg and V1rb2^mv alleles corresponds to the green and red colors respectively. (A) Coexpression of the V1rb2^vg and V1rb2^mv alleles evaluated by immunohistochemistry. (B) Coexpression of the V1rb2^vg and V1rb2^mv alleles evaluated by in situ hybridizations. The area of the green pies correspond to the number of analyzed neurons expressing the V1rb2^mv allele. The yellow color represents the percentage of neurons coexpressing the V1rb^vg and V1rb2^mv alleles. (C) Axonal projections of vomeronasal neurons expressing the V1rb2^vg and V1rb2^mv alleles in the accessory olfactory bulb form independent glomeruli. Glomeruli corresponding to the V1rb2^vg and V1rb2^mv alleles are in green and red respectively. Scale bar: 30 micrometers.

Figure 4

V1rb2^mv mediated negative feedback. (A) Pies indicating the percentage of vomeronasal neurons coexpressing a V1rb2^mv, V1rb2^vt orV1rb2^dv allele with a member of the V1Ra-j families (data not shown for V1Rk and V1Rl families). (B-D) In situ hybridizations of vomeronasal coronal sections with probes corresponding to V1Ri genes (red) and V1rb2^mv (green), V1rb2^vt (green) and V1rb2^dv (green), alleles. A sensory neuron coexpressing the V1rb2^dv allele and a member of the V1Ri family is visible in (D) (yellow). Numbers from control V1rb2^vt and V1rb2^dv alleles correspond to an addition of novel and previous data (Roppolo et al., 2007). Scale bar: 20 micrometers.

Figure 5

V1rb2^mv-expressing vomeronasal sensory neurons do not acquire odorant sensory neuron characteristics. (A, B) Gαolf and CNGA2 expression (green) in M71/72-expressing neurons (red) in the main olfactory epithelium. (C) Absence of TRPC2 expression (green) in M71/M72-expressing neurons (red) in the main olfactory system. (D, E) Gαolf and CNGA2 are not expressed (green) in vomeronasal neurons expressing the V1rb2^mv allele (red). (F) TRPC2 is expressed in vomeronasal neurons expressing the V1rb2^mv allele (red). Scale bars: 20 micrometers.