Fig 1.
Effect of Skn-1a deficiency on the functional differentiation of Trpm5-expressing brush cells in trachea.
A: Skn-1a-expressing cells were characterized using immunohistochemistry with anti-Skn-1a and anti-villin antibodies. Villin-positive brush cells were divided into two types, Skn-1a-positive (arrowhead) and Skn-1a-negative brush cells (arrow). B: Skn-1a-expressing cells were characterized by two-color in situ hybridization with RNA probes for Skn-1a and Trpm5. Skn-1a-positive brush cells were co-labeled with Trpm5 riboprobe (arrowheads). Scale bars, 20 μm. C: The impact of Skn-1a deficiency on the functional differentiation of Trpm5/Skn1a-positive brush cells in the tracheal epithelium was examined by in situ hybridization using probes for taste signaling molecules of Tas1r3, Tas2rs (Tas2r105, Tas2r108, Tas2r131), Gnat3, Plcb2 and Trpm5. The mRNA signals of taste signaling molecules observed in wild-type mice were completely absent in the Skn-1a-/- mice, indicating that Skn-1a is required for the functional differentiation of Trpm5-positive brush cells. Scale bar, 100 μm. D: The expression of taste signaling molecules (Tas1r3, Tas2r105, Tas2r108, Tas2r131, Gnat3, Plcb2, and Trpm5) in wild-type (WT) and Skn-1a-/- (KO) trachea was examined by RT-PCR. The expression of taste signaling molecule genes was not detected in Skn-1a-/- trachea. A housekeeping gene, GAPDH was used as a positive control.
Fig 2.
Skn-1a is required for the functional differentiation of Trpm5-positive tracheal brush cells.
A: Immunostaining of Trpm5 and ChAT on coronal sections of the trachea of wild-type and Skn-1a-/- mice. Trpm5-positive brush cells were ChAT positive in the wild-type trachea (arrows), whereas no immunoreactive signals for Trpm5 and ChAT was observed in the Skn-1a-/- trachea. B: Immunostaining of Trpm5 and villin on coronal sections of the trachea of wild-type and Skn-1a-/- mice. In wild-type mice, both Trpm5 and villin-double positive (arrowhead) and villin-single positive (arrow) brush cells were observed. In Skn-1a-/- mice, Trpm5-positive brush cells were absent and only villin-single positive brush cells (arrows) were observed. Scale bars, 20 μm. C: Quantification of the number of immunosignals for Trpm5 and villin in the wild-type and Skn-1a-/- tracheal epithelium. The signals of Trpm5 were completely absent in the Skn-1a-/- tracheal epithelium, and the number of villin-single positive cells was significantly decreased in Skn-1a-/- mice. Each symbol represents an individual mouse. The error bars represent the mean ± SEM (n = 3, *P < 0.05, Student’s t-test).
Fig 3.
Impact of Skn-1a deficiency on Trpm5-positive tuft cells in digestive tracts.
A: Two-color in situ hybridization of Skn-1a (green) and Trpm5 (magenta) on sections of digestive tracts of stomach, small intestine, and large intestine of wild-type adult mice. The mRNA signals of Skn-1a were co-labeled with Trpm5 signals (arrowheads) in all tissues examined. Scale bar, 20 μm. B: Co-immunostaining using antibodies against Skn-1a (green) and villin (magenta) on sections of stomach, small intestine, and large intestine of wild-type adult mice. Skn-1a-positive cells were overlapped with villin-positive cells (arrowheads). Scale bar, 20 μm. C: The impact of Skn-1a deficiency on Trpm5-positive tuft cells was examined by double-label immunohistochemistry of Trpm5 and villin using sections of stomach, small intestine, and large intestine of wild-type (top) and Skn-1a-/- mice. Trpm5-positive cells were co-labeled with anti-villin antibody (arrowheads) in wild-type mice, whereas the expression of Trpm5 was abolished in all tested tissues in Skn-1a-/- mice. Scale bars, 20 μm. The immunoreactive signals for villin detected in wild-type mice (arrows) were not observed in Skn-1a-/- mice. D: The signals of intestinal tuft cells marker gene, Dclk1 mRNA were observed in wild-type digestive tracts, whereas no signals of Dclk1 mRNA were observed in Skn-1a-/-. Scale bars, 100 μm.
Fig 4.
Loss of the Trpm5-positive chemosensory cells in multiple tissues in Skn-1a-/- mice.
A: Two-color in situ hybridization of Skn-1a (green) and Trpm5 (magenta) in various tissues of auditory tube, urethra, thymus, and pancreatic duct of wild-type adult mice. The mRNA signals of Skn-1a were co-labeled with Trpm5 signals (arrowheads) in all tissues examined. Scale bar, 20 μm. B: Co-immunostaining using antibodies against Skn-1a (green) and villin (magenta) on cryosections of auditory tube, urethra, thymus, and pancreatic duct of wild-type adult mice. Skn-1a-positive cells were overlapped with villin-positive cells (arrowheads). The arrows indicate Skn-1a negative and villin-single positive cells. Scale bar, 20 μm. C: Double-label immunohistochemistry of Trpm5 and villin on sections of auditory tube, urethra, thymus, and pancreatic duct of wild-type (top) and Skn-1a-/- mice (bottom) was carried out to examine the impact of Skn-1a deficiency on Trpm5-positive chemosensory cells. Trpm5-positive cells were co-labeled with anti-villin antibody (arrowheads) in wild-type mice, whereas the expression of Trpm5 was abolished in all tested tissues in Skn-1a-/- mice. The immunoreactive signals for villin were detected in Skn-1a-/- urethral epithelium and thymic medulla (arrows), but not in auditory tube and pancreatic duct. Scale bar, 20 μm. D: The expression of taste signaling molecules (Tas1r3, Tas2r105, Tas2r108, Tas2r131, Gnat3, Plcb2, and Trpm5) in auditory tube, urethra, thymus, and pancreatic duct was examined by RT-PCR in wild-type (WT) and Skn-1a-/- (KO) mice. The expression of taste signaling molecules observed in wilt-type mice was not detected in Skn-1a-/- mice. A housekeeping gene, GAPDH was used as a positive control.