Sox2 is required for development of taste bud sensory cells

Abstract

Sox2 is expressed in basal epithelial cells of the tongue, with high levels in taste bud placodes, fungiform papillae, and mature taste cells, and low levels in filiform papillae. High Sox2 expression appears to lie downstream from canonical Wnt signaling. In hypomorphic Sox2(EGFP/LP) embryos, placodes form but no mature taste buds develop. In contrast, transgenic overexpression of Sox2 in the basal cells inhibits differentiation of filiform keratinocytes. Together, our loss-of-function and gain-of-function studies suggest that Sox2 functions in a dose-dependent manner to regulate the differentiation of endodermal progenitor cells of the tongue into taste bud sensory cells versus keratinocytes.

FIGURE 1.

Sox2 expression during TB development. Sox2^EGFP expression in whole tongues at E12.5 (A), E13.5 (B), and E17.5 (E), and in posterior palate at E17.5 (F). (C) Cryosection of E13.5 Sox2^{EGFP/ +} tongue shows fluorescence in columnar placode cells. (D) Whole-mount immunostaining for Sox2 in E15.5 tongue. Sox2^EGFP/+ tissues at P5 (G–I) and P60 (J–L) show expression in the intact anterior tongue (G,J), and in cryosections of fungiform (H,K), and vallate and foliate papillae (I,L). The superficial fluorescence in K is nonspecific. Bars: C,H, 50 μm; K,L, 100 μm.

FIGURE 2.

Phenotype of Sox2 hypomorphic mutant tongue. All samples are P0 except K and L. EGFP expression in Sox2^EGFP/+ (A) and Sox2^EGFP/LP (B) tongues. Histological sections of Sox2^+/+ (C,E) and Sox2^EGFP/LP (D,F) tongues stained with hematoxylin and eosin (H&E). Note the well-formed fungiform papillae in wild type (arrowheads) and their absence in mutant. (G) Immunostaining of wild-type tongues shows high Sox2 expression in TBs and lower levels in keratinocytes. (H) In Sox2^EGFP/LP tongues, only very low levels are seen in the keratinocytes. Antibodies to K8 reveal positive cells in wild-type TBs (I), but no staining in Sox2^EGFP/LP tongues (J). BrdU staining of Sox2^+/+ (K) and Sox2^EGFP/LP (L) embryos at E18.5 after a 1-h pulse fails to detect a significant change in cell proliferation. Innervation of Sox2 ^+/+ (M) and Sox2^EGFP/LP (N) tongues as revealed by staining for PGP9.5. Arrowheads show fungiform papillae. (O) To isolate total RNA from epithelial cells free of mesoderm, tongues were treated with dispase for 30 min at 37°C. (P) RT–PCR analysis of Sox2, Adh7, Bdnf, and Ntf3 in epithelial cell RNA. Actin is internal control. Bars, 50 μm.

FIGURE 3.

Phenotype of K5-Sox2 transgenic tongues. (A) Schematic of Sox2 expression vector. (B–I) Sections of E18.5 tongues with fungiform papillae marked by closed arrowheads. (B,C) Transgenic expression of Sox2 throughout the basal layer confirmed by immunohistochemistry. Histology shows multilayered, keratinized filiform papillae expressing AE15 in wild type (D,F) but absence in mutant (E,G). Instead, mutant tongues have many small, domed papilla-like structures (E, open arrowheads). BrdU-labeled cells are confined to basal layer and top of fungiform papillae in wild type (H), but a less ordered distribution in transgenic (I). Bars, 50 μm.

FIGURE 4.

(A–I) Canonical Wnt signaling and TB development. LacZ expression in BAT-gal reporter embryos. (A) Tongue at E12.5. (B) Section of E13.5 tongue. (C) Tongues at E14.5. (D) Tongue at E17.5. (E) Tongue at P5. (F) Section of P5 fungiform. (G) Section of P5 vallate papilla. (H) Whole palate at P5. (I) Section of fungiform papilla at P60. (J) Expression of β-catenin in tongue at E13.5. Immunostaining for β-catenin in E16.5 tongue (K), and P0 fungiform papilla (L). Nuclei are counterstained with DAPI. Expression of Wnt6 in E13.5 tongue by whole-mount in situ hybridization (M) and after sectioning (M′). Organ culture of E13.5 Sox2^{EGFP /+} tongue. EGFP expression after 2 d treatment with 40 mM NaCl (N,O,P) or LiCl (Q,R,S). Note significant up-regulation of EGFP expression after LiCl treatment. Dorsal (N,Q) and frontal (O,R) view of the cultured tongues. (P,S) Expression of β-catenin. Nuclei were counterstained with DAPI. Bars, 30 μm.

FIGURE 5.

A model for the roles of Sox2 in TB development. (Left panels) Normal development. (Right panels) Defective development in hypomorpic mutants. During placode formation stage (E12–E14), localized clusters of tongue epithelial cells express high Sox2 activated by canonical Wnt signaling. During papilla induction stage (E14–E16), the high Sox2-expressing placode cells subsequently form fungiform papillae where Wnt signaling is still active. Concomitantly there is migration of mesenchymal cells and nerves into the center of the papilla. During the TB formation stage (E17 to P0) high Sox2-expressing cells finally differentiate into TB cells, and sensory cells become innervated. In contrast, low Sox2-expressing cells normally differentiate into the keratinocytes of filiform papillae. In Sox2 hypomorphic mutants, tongue epithelial cells have lower expression of Sox2 and cannot differentiate into TB cells.