SOX2 regulates homeostasis of taste bud cells and lingual epithelial cells in posterior tongue

Abstract

Taste bud cells arise from local epithelial stem cells in the oral cavity and are continuously replaced by newborn cells throughout an animal's life. However, little is known about the molecular and cellular mechanisms of taste cell turnover. Recently, it has been demonstrated that SOX2, a transcription factor expressed in epithelial stem/progenitor cells of the oral cavity, regulates turnover of anterior tongue epithelium including gustatory and non-gustatory papillae. Yet, the role of SOX2 in regulating taste cell turnover in the posterior tongue is unclear. Prompted by the fact that there are regional differences in the cellular and molecular composition of taste buds and stem/progenitor cells in the anterior and posterior portions of tongue, which are derived from distinct embryonic origins, we set out to determine the role of SOX2 in epithelial tissue homeostasis in the posterior tongue. Here we report the differential requirement of SOX2 in the stem/progenitor cells for the normal turnover of lingual epithelial cells in the posterior tongue. Sox2 deletion in the stem/progenitor cells neither induced active caspase 3-mediated apoptotic cell death nor altered stem/progenitor cell population in the posterior tongue. Nevertheless, morphology and molecular feature of non-gustatory epithelial cells were impaired in the circumvallate papilla but not in the filiform papillae. Remarkably, taste buds became thinner, collapsed, and undetectable over time. Lineage tracing of Sox2-deleted stem/progenitor cells demonstrated an almost complete lack of newly generated basal precursor cells in the taste buds, suggesting mechanistically that Sox2 is involved in determining stem/progenitor cells to differentiate to gustatory lineage cells. Together, these results demonstrate that SOX2 plays key roles in regulating epithelial tissue homeostasis in the posterior tongue, similar but not identical to its function in the anterior tongue.

Fig 1. Sox2 deletion in epithelial stem/progenitor cells results in the disappearance of taste buds.

Immunohistochemistry to marker proteins of taste bud cells in the circumvallate papillae (CvP) of Krt5^CreERT2/+; Sox2^flox/flox mice with and without tamoxifen injection (–Tam, control): SOX2 (top) and KCNQ1 (bottom) as a pan-taste bud cell marker. The broken lines show the boundary of epithelium and connective tissue. Number of analyzed mice was 3 at each point. Scale bar, 50 μm.

Fig 2. Taste bud cell populations are decreased after Sox2 deletion in epithelial stem/progenitor cells.

A, C, E: Immunohistochemical detection of taste bud cell marker SOX2 (non-sensory taste bud cells, A), POU2F3 (sweet, umami, and bitter taste cells, C), and DDC (sour taste cells, E) in the CvP of Krt5^CreERT2/+; Sox2^flox/flox mice with and without tamoxifen injection (–Tam, control). Double fluorescent labeling of SOX2 (red) and KCNQ1 (green) was done to identify SOX2 immunoreactive signals inside taste buds. B, D, F: Quantitative analyses of SOX2⁺ (B), POU2F3⁺ (D), and DDC⁺ (F) cells in taste buds in the CvP. Immunoreactive signals were counted, and the ratios of signals after the induction of Sox2 deletion to those in control (–Tam) per trench were statistically analyzed using Welch’s ANOVA to evaluate significant change over time (n = 3 at each time point). The data are expressed as the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars, 50 μm.

Fig 3. Unaltered apoptotic cell death after Sox2 deletion in epithelial stem/progenitor cells.

A: Double fluorescence immunohistochemical labeling of active CASP3 (red) and KCNQ1 (green) in CvP of Krt5^CreERT2/+; Sox2^flox/flox mice with and without tamoxifen injection (–Tam, control). Scale bar, 50 μm. B: Quantitative analyses of active CASP3⁺ cells in (blue) and outside taste buds (orange). Numbers of active CASP3⁺ cells per trench wall were statistically analyzed using Welch’s ANOVA to evaluate significant change over time (n = 3 at each time point). The data are expressed as the mean ± s.e.m.

Fig 4. Sox2-deleted stem/progenitor cells are incapable of supplying new cells in taste buds.

A: Lineage tracing of epithelial stem/progenitor cells in CvP of wild type (top) and Sox2-deleted mice (bottom). Sox2 deletion was induced concominantly with the induction of tdTomato expression by tamoxifen into Krt5^CreERT2/+; Sox2^flox/flox; Rosa26^lsl-Tom/+ mice. Taste buds were identified by KCNQ1 immunoreactivity (green). Sox2 wild type mice,–Tam (n = 1), 3 days (n = 4), and 7 days (n = 2); Sox2-deleted mice,–Tam (control: no tamoxifen injection, n = 2), 3 days (n = 4), and 7 days (n = 2). B: Quantitative analyses of newly generated basal taste bud cells from wild type (Krt5^CreERT2/+; Sox2^+/+; Rosa26^lsl-Tom/+) and Sox2-deleted (Krt5^CreERT2/+; Sox2^LoxP/LoxP (cKO); Rosa26^lsl-Tom/+) epithelial stem/progenitor cells. tdTomato⁺ basal cells in taste buds per trench were analyzed using Welch’s t-test to evaluate if the difference is statistically significant (n = 4). C: Immunohistochemical detection of Ki67 (top) and PCNA (bottom) in CvP of Krt5^CreERT2/+; Sox2^flox/flox mice with and without tamoxifen injection (–Tam, control). D: Quantitative analyses of immunoreactive signals to Ki67 (left) and PCNA (right) in CvP trench of Krt5^CreERT2/+; Sox2^flox/flox mice 3 days after tamoxifen injection and without tamoxifen injection (–Tam, control). Numbers of immunoreactive singals to proliferative cell markers per trench were statistically analyzed using Welch’s t-test (n = 3). Scale bars, 50 μm.

Fig 5. Impact of Sox2 deletion in epithelial stem/progenitor cells on gene expression in stem/progenitor cells and non-gustatory epithelial cells.

A, C: Qualitative histochemical analyses by in situ hybridization of stem/progenitor cell (A) and non-gustatory epithelial cell (C) genes in the CvP of Krt5^CreERT2/+; Sox2^flox/flox mice (n≥3 at each point) before and after tamoxifen injection. B, D: Quantitative analyses of mRNA expression by PCR of Lgr5 (B, left), Krt5 (B, right), and Sprr2a2 (D) in the CvP of Krt5^CreERT2/+; Sox2^flox/flox mice before and after tamoxifen injection. Relative gene expression levels were normalized using Gapdh and statistically evaluated by Welch’s t-test (n = 4). Scale bars, 50 μm.

Fig 6. Long-term impact of Sox2 deletion in the CvP.

A: Lineage tracing of Sox2-deleted stem/progenitor cells together with immunohistochemical staining of KCNQ1 in the CvP of Krt5^CreERT2/+; Sox2^flox/flox; Rosa26^lsl-Tom/+ mice (n = 2) 3 months after tamoxifen injection. B: Immunohistochemical staining of Ki67 in the CvP of Krt5^CreERT2/+; Sox2^flox/flox (n = 1) and Krt5^CreERT2/+; Sox2^flox/flox; Rosa26^lsl-Tom/+ mice (n = 2) 3 months after tamoxifen injection. C-E: Expression of Krt5 (C), Lgr5 (D), and Sprr2a2 (E) in CvP of Krt5^CreERT2/+; Sox2^flox/flox; Rosa26^lsl-Tom/+ mice (n = 3) after 3 months of tamoxifen injection. Mice without tamoxifen injection (–Tam) were used as controls. Dotted lines show the boundary of epithelium and connective tissue. Scale bars, 100 μm (B, C) and 50 μm (A, D, E).

Fig 7. Sox2 is dispensable for the normal turnover of non-gustatory papillary epithelial cells.

A: Immunohistochemical staining of SOX2 (top) and Ki67 (bottom) in filiform papillae (FiP) in the intermolar eminence with and without tamoxifen injection (–Tam, control). B: Lineage tracing by tdTomato induced concurrently with Sox2 deletion in stem/progenitor cells. Immunoreactive signal to PCNA (green) and tdTomato epifluorescence (red) are overlaid. C: Expression of marker genes and protein expressed in epithelial cells at distinct differentiation stages.Mice used are Krt5^CreERT2/+; Sox2^flox/flox (n = 2 for–Tam, 3 days, and 7 days) and Krt5^CreERT2/+; Sox2^flox/flox; Rosa26^lsl-Tom/+ mice (n = 1 for–Tam, 3 days, and 7 days; n = 3 for 90 days). Scale bars, 50 μm. D: Quantitative PCR analyses to evaluate epithelial cell marker gene expression in FiP in the intermolar eminence. Relative gene expression levels were normalized using Gapdh and statistically evaluated by Welch’s t-test (n = 4 each, Krt5^CreERT2/+; Sox2^flox/flox mice before and 3 days after tamoxifen injection).