The mammalian olfactory system uses a large family of odorant receptors (ORs) to detect and discriminate amongst a myriad of volatile odor molecules. Understanding odor coding requires comprehensive mapping between ORs and corresponding odors. We developed a means of high-throughput in vivo identification of OR repertoires responding to odorants using phosphorylated ribosome immunoprecipitation of mRNA from olfactory epithelium of odor-stimulated mice followed by RNA-Seq. This approach screened the endogenously expressed ORs against an odor in one set of experiments using awake and freely behaving mice. In combination with validations in a heterologous system, we identified sets of ORs for two odorants, acetophenone and 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), encompassing 69 OR-odorant pairs. We also identified shared amino acid residues specific to the acetophenone or TMT receptors and developed models to predict receptor activation by acetophenone. Our results provide a method for understanding the combinatorial coding of odors in vivo.
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We thank G. Barnea and R. Axel for antibodies to M72, H. Zhuang for some of the OR constructs, D. Marchuk for sharing of equipment, the Duke IGSP Genome Sequencing and Analysis Core Resource for providing Illumina sequencing service, and S. Mukherjee and J. Mainland for helpful discussions and comments on the manuscript. This work was supported by grants from the US National Institutes of Health (DC012095 and DC014423).
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Integrated supplementary information
Supplementary Figure 1 pS6 induction in the OE following exposure to M72 agonists and control odors.
(b) pS6 immunostaining in the OE following exposure to M72 agonists and control odors. Green: Antibody staining for pS6. Magenta: Antibody staining for a known acetophenone receptor, M72. Blue: Bisbenzimide staining showing the nuclei. Arrowheads indicate colocalization of M72 and pS6 signals.Scale bar, 50 μm.
(a) pS6 induction in the OE for 5 known OR–odorant pairs. Green: Antibody staining for pS6. Magenta: RNA FISH for ORs. Blue: Bisbenzimide staining showing the nuclei. Arrowheads indicate colocalization of OR and pS6 signals when stimulated with the cognate ligands. Scale bar, 25 μm.
(b) Time course of pS6 induction following odor exposure. Green: Antibody staining for pS6. Blue: Bisbenzimide staining showing the nuclei. Scale bar, 50 μm.
(a) Representative figures showing pS6 induction in the OE for newly identified acetophenone ORs and a control OR (Olfr1132) following 1% and 100% acetophenone stimulation for 1 hour. Green: Antibody staining for pS6. Magenta: RNA FISH for ORs. Blue: Bisbenzimide staining showing the nuclei. Scale bar, 25 μm.
(b) Quantification of pS6 staining intensity following 1% and 100% acetophenone stimulation.
Heights of letters indicate residue abundance for a given location in the alignment. The positions that are more conserved with the acetophenone ORs than random are marked with stars. Predicted transmembrane domains are labeled in orange.
Dose response curves of 27 human ORs used for external validation of the model.
(a) Scatter plot comparing immunoprecipitated mRNA counts from stimulated sample (100% TMT) versus unstimulated sample. X–axis: mean read counts of genes in unstimulated IP samples (n=4). Y–axis: mean read counts of genes in TMT stimulated IP samples (n=4). Red dots represent ORs. Gray dots represent non–OR genes. Broken line: unit–slope.
(b) Differential enrichment calling of OR mRNA. 43 ORs are enriched in the 100% TMT stimulated group with p–value smaller than 0.05, after adjusting for multiple comparisons across the detected OR repertoire. Broken line: unit–slope.
(c) Differential enrichment calling of OR mRNA. 4 ORs are enriched in the 1% acetophenone stimulated group with p–value smaller than 0.05, after adjusting for multiple comparisons across the detected OR repertoire. Broken line: unit–slope.
(d) Scatter plot comparing p–values of enrichment in 100% TMT versus 1% TMT stimulated samples. Red dashed line: p=0.001. Blue dashed line: p=0.05. Note the absence of ORs in the bottom right corner.
(e) Localization of ORs that were identified to be activated by TMT via RNA in situ hybridization. 20 μm tissue sections of the OE were marked with digoxigenin (DIG)-labeled RNA probe complementary to specific OR mRNA transcripts. Scale bar = 100 μm and applies to all panels.
ROC curves illustrating performance of classifiers using in vivo enrichment p–values to predict whether the OR is called as acetophenone OR by DREAM. Area Under Curve: 0.860, p=4×10−9, Wilcoxon rank–sum test (one tailed against H0: Classifier performance no better than random).
Supplementary Figures 1–7 (PDF 1513 kb)
Comparison of pS6 signal intensities at different odorant concentrations. One-way ANOVA followed by Dunnett's multiple comparisons test. (XLS 44 kb)
Differential expression analysis results for 100% acetophenone, 1% acetophenone, EC50 values and fold of induction for in vitro luciferase assay, summary of identified acetophenone ORs, differential expression analysis results for 100% TMT, 1% TMT, and list of identified TMT ORs. (XLS 997 kb)
In vivo and in vitro data summary for the 22 ORs for acetophenone identified by the DREAM method. (XLSX 58 kb)
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Jiang, Y., Gong, N., Hu, X. et al. Molecular profiling of activated olfactory neurons identifies odorant receptors for odors in vivo. Nat Neurosci 18, 1446–1454 (2015). https://doi.org/10.1038/nn.4104
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