Article Figures & Data
Figures
- Figure 1.
The gene-targeted S100a5–tauGFP mouse strain allows for activity-dependent fluorescent marking of OSNs. A, The design of the targeted mutation of the mouse S100a5 locus, replacing the coding exons with tauGFP. Boxes, Exons; light blue, coding sequence; dark blue, noncoding sequence; triangles, loxP sites; arrow, translation start site. B, Exposure for 4.5 s detects fluorescent OSNs in a 12 μm coronal section at midseptum, showing the mosaic pattern and variable levels of GFP expression in a male mouse, 25 d postnatal, that is wild type for the Cnga2 locus. C, Exposure for 49 s reveals a near absence of fluorescent OSNs in a Cnga2-deficient male mouse, 25 d postnatal. Scale bars, 100 μm. D, FACS of dissociated cells from olfactory mucosae of a wild-type C57BL/6 mouse is used to set sorting gates (circumscribed with magenta lines) for subsequent capture of GFP+ and GFP− cells from S100a5–tauGFP mice. E, F, FACS capture of GFP+ and GFP− cells from a set of four S100a5–tauGFP mice exposed to vehicle (E) and four mice exposed in parallel to muscone (F). G, In the in vivo assay, OSNs that exhibit GFP fluorescence above background are captured in the GFP+ sample. The distribution of each OSN subtype in GFP+ and GFP− FACS samples from S100a5–tauGFP mice exposed to an odorant ligand differs from that of S100a5–tauGFP mice exposed to vehicle only in those few OSN subtypes that express ORs responsive to the odorant ligand, as exemplified by OR6 in this diagram. Odorant-evoked activity in OR6-expressing OSNs results in an increase of the fraction of these OSNs with GFP fluorescence above background, thus causing a redistribution of these OSNs from the GFP− FACS sample into the GFP+ FACS sample. Differing intensities of GFP fluorescence above background exist among OSNs but are not relevant in this assay, which is based on a binary grouping of fluorescence intensities (GFP− vs GFP+).
- Figure 2.
Eugenol exposure has significant effects on three ORs in S100a5–tauGFP mice. A, The distribution of OR mRNAs between GFP+ and GFP− samples is consistent across experiments. GFP+/GFP− ratios from mice exposed to vehicle (mineral oil) from four replications, distinguished by color (red, blue, green, and black), plotted against the mean GFP+/GFP− ratios of these four vehicle treatments. B, Mean OR mRNA GFP+/GFP− ratio values after exposure to eugenol plotted against the mean ratios after exposure to vehicle (n = 4 groups of mice). Three ORs (Olfr961, Olfr958, and Olfr960) show significant elevation (FDR < 0.10) after eugenol exposure. To illustrate the change, their GFP+/GFP− ratio values in this experiment (green diamonds) are compared with the GFP+/GFP− ratio coordinates in other experiments that did not use eugenol (blue circles). C, Chemical structure of eugenol. D, Mean GFP+/GFP− ratios and corresponding FDR probabilities for the seven ORs with FDR probabilities <0.5.
- Figure 3.
Heterologous expression assay data for ORs responsive to eugenol. A–C, Eugenol concentration–response relationships for Olfr961, Olfr958, and Olfr960, previously identified eugenol-responsive ORs that gave significant differences in the in vivo assay. D, Eugenol concentration–response relationship for Olfr73, the first eugenol-responsive OR to be identified (Kajiya et al., 2001).
- Figure 4.
Muscone exposure has significant effects on five ORs in S100a5–tauGFP mice. A, Chemical structure of muscone. B, Mean OR mRNA GFP+/GFP− ratios after exposure to muscone plotted against the mean ratios after exposure to vehicle (n = 4 groups of mice). Five ORs (Olfr1440, Olfr1433, Olfr235, Olfr1431, and Olfr1437) show significant differences (FDR < 0.10) after muscone exposure (green triangles) compared with vehicle exposure. Blue circles reflect the GFP+/GFP− ratio coordinates of these five ORs in other experiments that did not use muscone. C, Mean GFP+/GFP− ratios and corresponding FDR probabilities for the 13 ORs with FDR probabilities <0.5. D, A portion of the mouse OR phylogenetic tree depicting the relatedness of the members of the MOR214 and MOR215 mouse OR families that gave significant responses to muscone (green).
- Figure 5.
Confirmation of muscone responses from two mouse ORs by heterologous expression. A, Olfr1440. B, Olfr235. C, Rho-pCI, empty plasmid vector control. IPM used as vehicle.
- Figure 6.
Activation of muscone-responsive mouse and human ORs by certain macrocyclic musk odorants in heterologous expression assays. A, OR5AN1, a human OR, responded to the macrocyclic ketone musks muscone and Exaltone and to the macrocyclic lactone musk Exaltolide. B, The OR5AN1 response to muscone is stronger than responses of mouse ORs. C, Olfr1440 and Olfr235 responses to muscone. D, E, Polycyclic musk odors fail to evoke responses different from the plasmid vector negative control, Rho-pCI, in cells transfected with OR5AN1 or the mouse ORs responsive to muscone. Data for some ORs cannot be seen because of overlap of the curves. F, Astrotone, a macrocyclic diester musk odorant, also fails to evoke responses different from the plasmid vector negative control, Rho-pCI, in cells transfected with OR5AN1 or the mouse ORs responsive to muscone. Data for some ORs cannot be seen because of overlap of the curves.
- Figure 7.
Distribution of the 50 lowest FDR probabilities from the in vivo eugenol and muscone experiments illustrate the ability of the assay to separate odorant-responsive receptors from nonresponders. Receptors with FDR probabilities <0.001 were truncated at 0.001.
- Figure 8.
Heterologous expression assay data for additional ORs responsive to eugenol or muscone. A–E, Eugenol concentration–response relationships for five eugenol-responsive ORs that were previously orphan receptors. F, G, Muscone concentration–response relationships for two muscone-responsive ORs that were previously orphan receptors. Rho–pCI, Expression plasmid used as a negative control. IPM used as vehicle.
Tables
Eugenol Muscone Olfr MOR FDR Olfr MOR FDR Olfr961* MOR224-5 0.0002 Olfr1433 MOR214-4 0.0000 Olfr958* MOR224-9 0.0188 Olfr1440* MOR215-1 0.0000 Olfr960* MOR188-5 0.0728 Olfr235* MOR214-3 0.0000 Olfr1101 MOR179-3 0.3015 Olfr1437 MOR214-6 0.0024 Olfr1085 MOR191-1 0.3283 Olfr1431 MOR214-5 0.0074 Olfr1501 MOR212-3 0.3516 Olfr1020 MOR201-2 0.4447 Olfr97 MOR156-2 0.4372 Olfr769 MOR114-4 0.4480 Olfr1160 MOR173-1 0.9179 Olfr867 MOR143-2 0.4587 Olfr610* MOR9-2 0.9197 Olfr250 MOR170-14 0.4630 Olfr1052 MOR172-1 0.9245 Olfr1257 MOR232-1 0.4773 Olfr1234* MOR231-2 0.9832 Olfr1496 MOR127-1 0.4837 Olfr73* MOR174-9 1.0000 Olfr74* MOR174-4 0.5204 Olfr151 MOR171-2 1.0000 Olfr488 MOR204-15 0.5354 Olfr1031 MOR200-1 1.0000 Olfr816* MOR113-1 0.6397 Olfr178* MOR184-6 1.0000 Olfr394 MOR135-8 0.8547 Olfr922 MOR161-3 1.0000 Olfr1213 MOR233-7 1.0000 Olfr1274 MOR228-4 1.0000 Olfr821 MOR109-1 1.0000 Olfr811 MOR110-6 1.0000 Olfr1090 MOR188-4 1.0000 Olfr1325 MOR102-1 1.0000 Olfr1496 MOR127-1 1.0000 Olfr904 MOR167-3 1.0000 Olfr591 MOR24-1 1.0000 Olfr215 MOR119-2 1.0000 Olfr432* MOR123-2 1.0000 Olfr1054 MOR188-2 1.0000 Olfr1014 MOR213-5 1.0000 Olfr360 MOR159-1 1.0000 Olfr913* MOR165-10 1.0000 ↵*Positive responses.
