Abstract
In mammals, olfactory perception is based on the combinatorial activation of G protein–coupled receptors. Identifying the full repertoire of receptors activated by a given odorant in vivo, a quest that has been hampered for over 20 years by technical difficulties, would represent an important step in deciphering the rules governing chemoperception. We found that odorants induced a fast and reversible concentration-dependent decrease in the transcription of genes corresponding to activated receptors in intact mice. On the basis of this finding, we developed a large-scale transcriptomic approach to uncover receptor-ligand pairs in vivo. We identified the mouse and rat odorant receptor signatures corresponding to specific odorants. Finally, we found that this approach, which can be used for species for which no genomic sequence is available, is also applicable to non-vertebrate species such as Drosophila.
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Acknowledgements
We thank E. Nagoshi (University of Geneva) for providing w1118 Drosophila, C. Margot (Firmenich SA) for the gift of lyral, the bioimaging and genomics platforms of the University of Geneva, and the genomic analysis VitalIT platform in Lausanne. This work was supported by grants 31003A_149753 to I.R., CR33I3_143723 to I.R. and A.C., and 31003A_153410 to A.C. from the Swiss National Science Foundation, and ERC-2009-StG-243344-Neurochems to A.C. from the European Research Council.
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B.v.d.W., M.L., D.R., J.T., A.W., J.D.C. and C.K. carried out the experimental studies. B.v.d.W., M.L., D.R., C.K., J.T., A.C., A.W., J.D.C. and I.R. performed data analyses. B.v.d.W., M.L., D.R., J.T., A.W., J.D.C., J.T., A.C. and I.R. planned the experiments. I.R., A.C. and D.R. conceptualized the approach. I.R. wrote the bulk of the manuscript, and all of the authors discussed the results and commented on the manuscript. All of the authors edited the paper.
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An application for a patent covering the DREAM technique has been filed by D.R., A.C. and I.R.
Integrated supplementary information
Supplementary Figure 1 The concentration of olfactory receptor transcripts is reduced following odorant exposure in vivo.
(a) Mice were exposed to 5% ethyl isobutyrate for 30 minutes, and for 2, 5, 12, 24 and 48 hours. Concentration of Olfr167 mRNA was plotted. Olfactory receptor gene transcript levels were evaluated by qPCR, and the ratio between the values obtained in non-exposed versus exposed mice are shown. Each dot represents a single mouse. Means are shown as blue or red horizontal bars and boxes extend from the 25th to the 75th percentile. U = 5.65, P = 1.7x10−2 for 30 mins, U = 5.65, P = 1.7x10−2 for 2h, U = 12.6, P = 3.8x10−4 for 5h, Mann Whitney U test (b) Transcript reduction is dependent on odorant concentration. Mice were exposed to various concentrations of ethyl isobutyrate for 5 hours and the amount of Olfr167 mRNA was evaluated by qPCR. U = 25, P = 5.7x10−7, Mann Whitney U test. (c) Schematic of the odorant exposure and MOE mRNA harvest time point shown in panel (d). (d) Odorant induced OR mRNA decrease is reversible. Mice were exposed to 5% ethyl isobutyrate for 5 hours the MOE mRNA was harvested at different times after exposure. Concentration of Olfr171 and Olfr167 mRNA was plotted. U = 7.52, P = 6.1x10−3 between 0h and 12h, Olfr171, U = 5.33, P = 2.1x10−2 between 12h and 48h, Olfr171, U = 7.52, P = 6.1x10−3 between 0h and 12h, Olfr167, Mann Whitney U test, (e) Direct contact with the odorant source is not required to induce OR mRNA level decrease. Mice were exposed to a cotton swab impregnated with 5% ethyl isobutyrate that were either placed in the cage directly on the bedding (contact) or inside a perforated petri dish (no contact). U = 5.33, P = 2.1x10−2 between Olfr171 and Olfr983, no contact, U = 5.33, P = 2.1x10−2 between Olfr167 and Olfr983, no contact. P-values: * (P < 0.05), ** (P < 0.01), *** (P < 0.001).
Supplementary Figure 2 Population analysis of the DREAM method.
(a) Frequency distribution of the relative fold change of mRNA concentration between non-exposed and exposed mice, determined by qPCR, for all receptors exposed to molecules known to be non-agonists. The distribution is fitted by a Gaussian curve (dotted line). (b) Cumulative probability distributions of the same fold change for receptors exposed to known agonists (red) and for receptors exposed to known non-agonists (black). The concentration of OR mRNA is significantly reduced in mice exposed to known ligands.
Supplementary Figure 3 Distribution of mouse OR gene mRNA fold changes after acetophenone and ethyl isobutyrate exposure.
Grey bars represent the frequency distribution of the log2 mRNA concentration relative fold changes. The black curve shows the normal distribution that was fitted to the data. The black segment below the X axis indicates the confidence interval which comprises 99% of the normal distribution. The red zone beneath the Gaussian curve represents 0.5% OR genes expressed in the RNAseq libraries. (a) Data for acetophenone-exposed mice (864 OR genes) and (b) ethyl isobutyrate-exposed mice (876 OR genes).
Supplementary Figure 4 MOR23+ neurons express pS6 after lyral exposure.
(a) Transgenic mice coexpressing GFP and MOR23 in olfactory sensory neurons were exposed to lyral. Coronal sections of their olfactory epithelium were immunolabeled with pS6 (red) and GFP antibodies (green, to identify MOR23+ neurons), showing a colocalization of the activity marker with GFP (green). Little or no colocalization was observed in non-exposed or acetophenone-exposed mice. Scalebar is 20μm. (b) Quantification of pS6 coexpression after acetophenone (dark grey) or lyral (light grey) exposure. Similar results were obtained when MOR23-expressing neurons were identified by immunohistochemistry or by in situ hybridization. Immuno control compared to lyral: X2 = 685.41, P < 2.2x10−16, acetophenone compared to lyral: X2 = 566.74, P < 2.2x10−16, in-situ control compared to lyral: X2 = 215.41, P < 2.2x10−16, acetophenone compared to lyral: X2 = 230.44, P < 2.2x10−16
Supplementary Figure 5 Neurons whose OR transcript concentrations are reduced by odorant exposure are activated neurons.
(a-c) Receptor transcript-specific in situ hybridization followed by pS6 immunolabeling. Neurons expressing specific olfactory receptors (green) colocalize with the activity reporter pS6 (red) after acetophenone exposure (Olfr983, (a)), or after ethyl isobutyrate exposure (Olfr171 and Olfr60, (b) and (c)). Scalebar is 20μm.
Supplementary Figure 6 Transcriptome-wide evaluation in rats of OR transcript concentrations after and ethyl isobutyrate exposure.
(a) Distribution of rat OR gene mRNA fold changes after ethyl isobutyrate exposure. Grey bars represent the frequency distribution of the log2 mRNA concentration relative fold changes. The distribution of the data is normal (W = 0.98803, P = 0.0002127, Shapiro-Wilk test). The black curve shows the normal distribution that was fitted to the data. The black segment below the X axis indicates the confidence interval which comprises 99% of the normal distribution. The red zone beneath the Gaussian curve represents 0.5% OR genes expressed in the RNAseq libraries. (b) OR genes whose transcript concentrations are reduced after ethyl isobutyrate exposure in the rat. RNAseq values correspond to the mean of 4 animals, and candidates were confirmed by qPCR. As defined on Fig. S2, the 99% confidence interval is represented on the left by a black line.
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von der Weid, B., Rossier, D., Lindup, M. et al. Large-scale transcriptional profiling of chemosensory neurons identifies receptor-ligand pairs in vivo. Nat Neurosci 18, 1455–1463 (2015). https://doi.org/10.1038/nn.4100
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DOI: https://doi.org/10.1038/nn.4100
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