Fig. 7: TAAR enhancer 1 and enhancer 2 possess TAAR OSN-specific enhancer activity.
From: Coordination of two enhancers drives expression of olfactory trace amine-associated receptors
a Schematic illustration of the design strategy for TAAR enhancer 1-GFP (left) and TAAR enhancer 2-tdTomato (right) transgene constructs. The boxes in dark blue represent Hsp68 minimal promoter. The numbers of founders having GFP-positive and tdTomato-positive cells in the MOE were also indicated. We kept one founder for each enhancer transgenic line and used them for further experiments. b Top, confocal images of two-color in situ hybridization using probes for GFP (green) and all of the Taar genes (magenta) in a TAAR enhancer 1-GFP transgenic mouse. Bottom, confocal images of in situ hybridization of mixed probes of OR genes (magenta) and the following immunohistochemistry by the GFP antibody (green) in a TAAR enhancer 1-GFP transgenic mouse. Scale bar = 25 μm (n ≥ 3). c Top, bar plots showing the percentages of GFP-positive cells that were co-labeled with mixed Taar probes, mixed OR probes, mixed class I OR probes, or OR degenerate probe (1083 out of 1284 cells for mixed Taar probes, 25 out of 616 cells for mixed OR probes, 2 out of 471 cells for mixed class I OR probes, 1 out of 351 cells for OR degenerate probe). Bottom, bar plots showing the percentages of TAAR or OR OSNs that co-expressed GFP using mixed Taar probes, mixed OR probes, mixed class I OR probes, or OR degenerate probe (564 out of 3920 cells for mixed Taar probes, 17 out of 898 cells for mixed OR probes, 2 out of 1153 cells for mixed class I OR probes, 1 out of 203 cells for OR degenerate probe). d Confocal images of in situ hybridization of mixed probes of Taar genes or OR genes (green) and the following immunohistochemistry by the tdTomato antibody (magenta) in a TAAR enhancer 2-tdTomato transgenic mouse. Scale bar = 25 μm (n ≥ 3). e Top, bar plots showing the percentages of tdTomato-positive cells that were co-labeled with mixed Taar probes, mixed OR probes, mixed class I OR probes, or OR degenerate probe (817 out of 1116 cells for mixed Taar probes, 9 out of 896 cells for mixed OR probes, 5 out of 349 cells for mixed class I OR probes, 2 out of 657 cells for OR degenerate probe). Bottom, bar plots showing the percentages of TAAR or OR OSNs that co-expressed tdTomato using mixed Taar probes, mixed OR probes, mixed class I OR probes, or OR degenerate probe (817 out of 5863 cells for mixed Taar probes, 9 out of 4726 cells for mixed OR probes, 5 out of 748 cells for mixed class I OR probes, 2 out of 2713 cells for OR degenerate probe). f Representative confocal images showing cells with GFP and tdTomato signals in TAAR enhancer 1-GFP; TAAR enhancer 2-tdTomato transgenic mice. Right, the numbers of cells expressing each reporter alone (GFP, tdTomato) or together were counted. Scale bar = 25 μm (n ≥ 3). g Whole-mount fluorescent images of the dorsal view of olfactory bulb in TAAR enhancer 1-GFP; TAAR enhancer 2-tdTomato transgenic mice. The area in the black dashed box is enlarged on the bottom. The white arrows indicate GFP-specific glomeruli, while the white arrow heads indicate tdTomato-specific glomeruli. Scale bar = 200 μm (n ≥ 3). In c, e, ***p < 0.001 by Fisher’s exact test (two-sided).
