Genetic Lineage Tracing in Taste Tissues Using Sox2-CreERT2 Strain

Abstract

Taste cells in taste buds are epithelial sensory cells. Old taste bud cells die and are replaced by new ones generated from taste stem cells. Identifying and characterizing adult taste stem cells is therefore important to understand how peripheral taste tissues are maintained. SOX2 is expressed in oral epithelium including gustatory papillae and has been proposed to be a marker of adult taste stem/progenitor cells. Nevertheless, this hypothesis has never been directly tested. Here, by single-color genetic lineage tracing using Sox2-CreERT2 strain, we reveal that all types of taste bud cells distributed throughout the oral epithelium are derived from stem cells that express SOX2. Short-term tracing shows that SOX2-positive taste stem cells actively supply taste bud cells. At the base of epithelium outside taste buds are distributed proliferation marker- and SOX2-positive cells. Consistently, taste stem cells identified by Lgr5 expression in the circumvallate papillae also express SOX2. Together, taste stem cells distributed in oral epithelia express SOX2.

Keywords: lineage tracing; oral epithelium; stem cell; taste bud cell.

Figure 1.

Long-term lineage tracing of Sox2⁺ cells in the gustatory areas of oral epithelium. (A) Fluorescence of tdTomato in the oral epithelium of Sox2^CreERT2/+; Rosa26^lsl-Tom/+ mice at 21 months after tamoxifen injections for 5 consecutive days: fluorescent labeling of pan-taste-bud-cell marker KCNQ1 (green, bottom) and nuclei (stained with DAPI, blue, bottom) in the soft palate (left), fungiform papillae (FuP, middle), and circumvallate papillae (CvP, right) with tdTomato fluorescence (red). All taste bud cells are labeled with tdTomato at 21 months after tamoxifen injection. Taste bud cells marked by arrowheads are the representative cells exhibiting lower tdTomato fluorescence than other taste bud cells. (B) Fluorescent labeling of SOX2 (green, left), TRPM5 (green, middle), and DDC (green, right) and tdTomato (red) in the CvP of Sox2^CreERT2/+; Rosa26^lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. (C) Fluorescent labeling of the combination of TRPM5 and DDC (green) and KCNQ1 (blue) with tdTomato (red) in the taste buds of CvP of Sox2^CreERT2/+; Rosa26^lsl-Tom/+ mice at 6 months after tamoxifen injections for 5 consecutive days. Taste bud cells marked by asterisks (*) are the cells positive for KCNQ1 but negative for TRPM5 and DDC and labeled with tdTomato. Scale bar: 50 µm.

Figure 2.

Short-term lineage tracing of Sox2⁺ cells in the circumvallate papillae. (A) Fluorescent labeling of KCNQ1 (green, bottom) with tdTomato fluorescence (red) in CvP of Sox2^CreERT2/+; Rosa26^lsl-Tom/+ mice. Most KCNQ1⁺ cells are tdTomato-positive at 24 days after tamoxifen injections for 5 consecutive days (right), although some KCNQ1⁺ cells are tdTomato-negative at 3 days after one tamoxifen injection for 1 day (left). (B) Fluorescence of tdTomato (red) and merged images labeled with SOX2 (green, bottom) in the CvP of Sox2^CreERT2/+; Rosa26^lsl-Tom/+ mice without tamoxifen treatment at the age of 2 months (left) and 12 months (right). Some SOX2⁺ cells and a subset of taste bud cells that do not express SOX2 are tdTomato⁺. Spontaneous labeling with tdTomato is observed in taste bud cells and nongustatory epithelial cells, showing no strict correlation between spontaneous expression of tdTomato and SOX2 expression. Scale bar: 50 µm.

Figure 3.

Identification of actively proliferating cells in the gustatory areas of oral epithelium. (A) Triple fluorescent labeling of SOX2 (red), PCNA (green), and KCNQ1 (blue) in the CvP (top and second-top), FuP (second-bottom), and the soft palate (bottom). Actively proliferating PCNA⁺ cells are observed in the basal epithelial cells outside taste buds, which are colabeled with SOX2. (B) Nucleoside analog (EdU) incorporation in actively proliferating cells in CvP (top and second-top), FuP (second-bottom), and soft palate (bottom): triple fluorescent labeling of EdU (green), SOX2 (red), and KCNQ1 (blue) at 4 h after EdU administration. Cells positive for both SOX2 and EdU are observed in the basal epithelium outside KCNQ1⁺ cells. Areas of magnified fluorescent images in row 2 (second-top) are indicated by white squares in row 1 (top). Scale bar: 50 µm.

Figure 4.

Relationship between Lgr5⁺ taste stem cells and SOX2⁺ cells in the circumvallate papillae. Triple fluorescence labeling of SOX2 (red), green fluorescent protein (GFP; green), and KCNQ1 (blue) in the CvP of Lgr5^{EGFP-ires-CreERT2/+} mice. All GFP⁺ cells are observed outside taste buds and colabeled with SOX2. Areas of magnified fluorescent images (bottom) are indicated by white squares. Scale bar: 50 µm.