Synchronous evolution of an odor biosynthesis pathway and behavioral response

Abstract

Background: Rodents use olfactory cues for species-specific behaviors. For example, mice emit odors to attract mates of the same species, but not competitors of closely related species. This implies rapid evolution of olfactory signaling, although odors and chemosensory receptors involved are unknown.

Results: Here, we identify a mouse chemosignal, trimethylamine, and its olfactory receptor, trace amine-associated receptor 5 (TAAR5), to be involved in species-specific social communication. Abundant (>1,000-fold increased) and sex-dependent trimethylamine production arose de novo along the Mus lineage after divergence from Mus caroli. The two-step trimethylamine biosynthesis pathway involves synergy between commensal microflora and a sex-dependent liver enzyme, flavin-containing monooxygenase 3 (FMO3), which oxidizes trimethylamine. One key evolutionary alteration in this pathway is the recent acquisition in Mus of male-specific Fmo3 gene repression. Coincident with its evolving biosynthesis, trimethylamine evokes species-specific behaviors, attracting mice, but repelling rats. Attraction to trimethylamine is abolished in TAAR5 knockout mice, and furthermore, attraction to mouse scent is impaired by enzymatic depletion of trimethylamine or TAAR5 knockout.

Conclusions: TAAR5 is an evolutionarily conserved olfactory receptor required for a species-specific behavior. Synchronized changes in odor biosynthesis pathways and odor-evoked behaviors could ensure species-appropriate social interactions.

Figure 1. Species- and sex-dependent production of the TAAR5 activator, trimethylamine

(A) Variations in production of the TAAR5 activator across mammalian species. HEK-293 cells were transfected with plasmids encoding CRE-SEAP and TAAR5 (black bars) or CRE-SEAP alone (white bars), incubated with serial dilutions of urine from six species indicated, and assayed for SEAP activity (triplicates ± s.d.). Rodent donors were males, while human donors were non-identifiable males and females. (B) Assays performed as above, except donors were male and female mice (triplicates ± s.e.). (C) A region (2.8 to 3.6 ppm) of the NMR spectra of male and female mouse urine (C57BL/6), trimethylamine, and trimethylamine oxide. Chemical assignments were based on published values [40] or analysis of spiked specimens. (D) qPCR analysis of Fmo3 gene expression in liver cDNA prepared from mice of sexes and ages indicated (triplicates ± s.e.). Copy numbers were calculated in cDNA derived from 10 ng of liver RNA by comparison with PCR reactions involving titrations of an Fmo3-containing plasmid.

Figure 2. Trimethylamine biosynthesis in mouse involves microbial metabolism and a sex-dependent oxidation reaction

Urine was collected from male animals provided with normal chow, choline/methionine-free diet, or antibiotics for 1 week. Trimethylamine levels in these specimens were determined by the TAAR5-mediated reporter gene assay (A, B, triplicates ± s.d.) and by NMR spectroscopy (C, D, E). These data are consistent with a two-step pathway regulating trimethylamine production in mouse (F).

Figure 3. Evolution of sex-dependent trimethylamine biosynthesis and Fmo3 gene repression along the Mus lineage

(A) The phylogenetic relationship of Mus species, with Rattus norvegicus as an outgroup. The figure is adapted from published data [41]. Mus musculus sub-species are shown in the grey box, and bracketed letters indicate species providing specimens for subsequent panels. (B) Urine from males (grey bars) and females (black bars) of species indicated were collected and analyzed using the TAAR5 reporter gene assay. (C, D) Fmo3 gene expression in liver and kidney of various rodents. Liver and kidney cDNA was prepared from species indicated, as coded by bracketed letters in the phylogenetic tree (A), and analyzed for Fmo3 gene expression by qPCR analysis (triplicates ± s.e.). Fmo3 expression levels in male animals (grey bars) and female animals (black bars) were compared. The Y-axis indicates copy number in cDNA derived from 10 ng RNA, and was calculated by comparison with PCR reactions involving titrations of an Fmo3-containing plasmid.

Figure 4. Biphasic valence of mouse behavioral responses to trimethylamine

(A) A cartoon depiction of the mouse behavioral assay. (B) Side preferences of mice in a two-compartment arena containing test odors and water were measured as differential occupancy. Increases in test odor side occupancy were scored as attraction, while decreases were scored as aversion. (*p < 0.05, **p < 0.01, ± s.e.). All experiments involved equal numbers of males and females, except as noted in Experimental Procedures.

Figure 5. Rats avoid a trimethylamine odor source

(A) Motion tracks (red) of individual male rats behaving in the open field paradigm. White circles indicate the location of odor sources (water, 4.2 M trimethylamine). (B, C) Occupancy of quadrants, defined as 25% of the arena, containing odors indicated (n=9-12, *p < 0.05, **p < 0.01, ± s.e.). (D) Dose-dependent trimethylamine responses of HEK-293 cells transfected with reporter gene alone (white squares), reporter gene and mouse TAAR5 (black squares), or reporter gene and rat TAAR5 (grey squares) were measured using the cellular reporter gene assay (triplicates ± s.e.).

Figure 6. TAAR5 and trimethylamine are required for mouse scent attraction

(A) Expression of Taar5 and lacZ in olfactory sensory neurons of Taar5^−/−, Taar5^+/−, and Taar5^+/+ mice visualized by two-color in situ hybridization. Scale bar = 50 μm. (B) Responses of Taar5^+/+ and Taar5^−/− mice to trimethylamine were measured using the two-choice odor compartment assay (mean ± s.e.). Experiments involved equal numbers of males and females, except as noted in Experimental Procedures. (C) Male urine was depleted of trimethylamine by incubation with FMO3-containing microsomes. Effective depletion was shown by an inability of TMA-depleted male urine to activate TAAR5 in HEK-293 cells at dilutions indicated (triplicates ± s.e.). (D) Behavioral responses of mice to stimuli indicated (40 μl) using the two-choice compartment assay (mean ± s.e.). (E) Behavioral responses of female mice (n=8-10, ± s.e.) of genotypes indicated to mock-depleted male urine (40 μl). (*p < 0.05, **p < 0.01)