Table 1.
Olfactory receptor expression levels in olfactory marker protein-positive non-olfactory tissues using RT-qPCR analyses.
Fig 1.
Protein expression profiles of OMP in various mouse tissues.
Double immunoassay for olfactory marker protein (OMP). (A) In all tissues except for kidney (KD), the results of double immunoassays demonstrate the presence of OMP. Total mouse tissue extract (100 μg for olfactory epithelium [OE] and 20 mg for KD, skeletal muscle [SM], and thymus [TM]) was immunoprecipitated (IP) with goat anti-OMP antibodies and immunoblotted with rabbit anti-OMP antibody (double immunoassay). The apparent molecular mass of OMP (arrow) is 19 kDa in OE, SM, and TM [OMP (-)], and this band is completely blocked by preincubating the OMP antibody with purified recombinant OMP [OMP (+)]. (B) Mouse tissue survey for OMP expression. Total tissue extract (100 μg for OE as a positive control, 3 mg for thyroid (TR), and 20 mg for all other tissues) was used for IP. * indicates immunoglobulin. LV, liver; BL, bladder; PC, pancreas; ST, stomach; DD, duodenum; TT, testis; SP, spleen; HT, heart; LG, lung.
Fig 2.
Immunohistochemical detection of OMP in mouse non-olfactory tissues.
(A) Olfactory marker protein (OMP) staining in olfactory epithelium (OE) was used as a positive control. In the thyroid (TR), OMP (+) cells are observed between follicles (FL) and are similar to a parafollicular cell-like population. OMP (+) cells of the bladder (BL) are assumed to be in the submucosal layer. In the testis (TT), OMP signals are displayed in Leydig cell-like populations belonging to interstitial tissue (IT), and in the thymus (TM), OMP is detected in the medullary area. The asterisks indicate OMP (+) cells. (B) Each tissue represents samples stained in the absence of primary antibody. MU, mucosa; SML, submucosal layer; ML, muscle layer; ST, seminiferous tubule. Scale bar: 10 μm.
Fig 3.
Co-expression of OMP with either ACIII or Golf in non-olfactory tissues using immunohistochemical analysis.
The distribution of each olfactory signaling-associated molecule, namely, olfactory marker protein (OMP), adenylate cyclase III (ACIII), and olfactory G-protein (Golf), in non-olfactory tissues shows different expression patterns. In bladder (BL) and thymus (TM), OMP (+) cells are co-expressed with ACIII and Golf. OMP signals expressed in the thyroid (TR) are colocalized with ACIII, but not with Golf. In the heart (HT) and testis (TT), OMP signals are displayed alone, which is not coincident with Golf or ACIII.
Fig 4.
Identification of OMP (+) cells in non-olfactory tissues such as bladder and thyroid.
Olfactory marker protein (OMP) expression (red in b and f) is superimposed with each marker using double-labeling immunohistochemical techniques. A representative image is shown for each antibody combination. The left panels show each marker protein with Dylight 488-conjugated anti-rabbit IgG green fluorescence (a, e), the middle panels show OMP with Cy3-conjugated anti-goat IgG red fluorescence (b, f), and the right panels are the merged images of the two individual images in the corresponding rows (c, g). C-kit (green in a), a marker for the bladder (BL in A), stains interstitial cells of Cajal (ICC). Calcitonin (green in e) is a marker for parafollicular cells (also known as C cells) of the thyroid (TR in B). OMP is expressed in each cell type of mouse tissue as evidenced by colocalization with c-kit (yellow in c) and calcitonin (yellow in g). C-kit/OMP (d) and calcitonin/OMP (h) colocalization was supported by quantitative analysis. ***, P < 0.0001 using an unpaired t-test; ##, P = 0.0004, and ###, P = 0.0001 using a Mann—Whitney U test OMP + c-kit, or calcitonin versus marker or OMP alone. Results are the means ± SEM of three or four independent areas (n = 3–5 mice). Scale bar: 10 μm.
Fig 5.
Identification of OMP (+) cells in thymus.
Olfactory marker protein (OMP) expression was superimposed on each marker using double-labeling immunohistochemical assays. A representative image is shown for each antibody combination. The left panels show each marker protein (CD8 for T cells; CD45R for B cells; Iba-1 for macrophages; and keratin 14 [K14] for medullary epithelial cells). The middle panels show OMP, and the right panels are the merged images of the two individual images in the corresponding rows. OMP is co-labeled the subpopulation of K14-positive cells, but not other cell types, indicating that OMP is within thymic medullary epithelial cells (mTEC).
Fig 6.
Validation of commercial antibodies against the olfactory receptor (OR).
A heterologous OR expression system and the endogenous OR expression in olfactory epithelium (OE) was used to validate commercially available OR antibodies against specific ORs. HEK 293 cells were transiently transfected with cDNAs coding for several associated proteins (Golf, Ric8b, and RTP1S) and ORs (olfr544, olfr1386, olfr558, olfr1496, or OR51E1 [a human olfr558 ortholog]), and immunolabeled using antibodies against both the N-terminal epitope-Rho tag (A, E, I, M, and P) and each OR-specific antibody (B, F, J, N, and Q). The olfr1386 (B) and olfr544 (F) antibody detect Lucy-Flag-Rho-olfr1386- and Rho-olfr544-constructs in the transfected cells, respectively. These results are confirmed using the anti-Rho antibody (A and E; C and G for overlay). As a positive control, both antibodies recognize unique olfactory receptor neuron (ORN) (D and H) in OE. OR51E1 and anti-Rho antibody detect Rho-OR51E1 (J and I) and Rho-olfr558 (N and M) constructs in the transfected cells and ORN (L), which is expected since they share 94% amino acid identity as orthologs. The olfr1496 is detected by anti-Rho antibody (P), but not by commercial olfr1496 antibody (Q; R for overlay), which nonspecifically recognizes all ORN (S) in OE. Scale bar = 10 μm.
Fig 7.
Olfactory receptors (ORs) are expressed in OMP (+) cells.
Each OR is co-expressed with the olfactory marker protein (OMP) in non-olfactory tissues using co-labeling assays. A representative image is shown for each antibody combination. The left panels show each OR with Dylight 488-conjugated anti-rabbit IgG green fluorescence (A, E, I, and M). The middle panels show OMP with Cy3-conjugated anti-goat IgG red fluorescence (B, F, J, and N), and the right panels are merged images of the two individual images in the corresponding rows (C, G, K, and O). OR51E1 (mouse olfr558 ortholog; green in A) or olfr544 (green in E) is expressed with OMP (red in B and F) in the interstitial cells of Cajal (ICC) of the bladder (BL). The olfr544 (green in I) or olfr1386 (green in M) is expressed with OMP (red in J and N) in the parafollicular cells of thyroid (TR). OR (+) cells may be OMP (+) cells in each cell type of mouse tissue as evidenced by the nearly complete overlap of OR and OMP expression. Nuclei are counterstained with DAPI (blue fluorescence), except the image for olfr1386 in thyroid. OR51E1 (olfr558)/OMP (D), olfr544/OMP (H), olfr544/OMP (L), and olfr1386/OMP (P) colocalization is supported by quantitative analysis. ***, P < 0.0001 using unpaired t-test; ###, P = 0.0001, and ####, P < 0.0001 using the Mann—Whitney U test OMP + OR51E1 (olfr558), olfr544, or olfr1386 versus each OR or OMP alone. Results are the means ± SEM of three or four independent areas (n = 3–5 mice). Scale bar: 10 μm.