Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium

Abstract

Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation.

Keywords: Cell lineage; Mouse molecular genetics; Regeneration; Taste.

Fig. 1.

Ectopic K8⁺ clusters form in SHH-YFPcKI⁺ non-taste epithelium. (A) Lingual taste buds occur in three types of taste papillae: FF, fungiform; CV, circumvallate; and FL, foliate. IE, intermolar eminence. (A′) The anterior 1.5 mm of the tongue, with fungiform papillae (pink) distributed in non-taste epithelium (gray), was analyzed in this study. (A″) Taste epithelium of the anterior tongue comprises fungiform taste buds and papilla epithelium (dark and light pink, respectively); the remainder of the tongue surface is non-taste epithelium (gray) with dense mechanosensory filiform papillae (asterisks). (B) Clusters of K8⁺ cells (red) form within SHH-YFPcKI⁺ domains (green), often near filiform papillae (asterisk). (C-E) Low-magnification images of ectopic K8⁺ cells (red, arrowheads) within SHH-YFPcKI⁺ patches (green) of non-taste lingual epithelium of three different mice show the distribution of K8⁺ ectopic clusters; not all SHH-YFPcKI⁺ patches contain ectopic taste bud-like structures (arrows). Note that the green signal in the lingual muscle is non-specific background. Nuclei are counterstained with Draq5 (blue). White dashed lines delimit the basement membrane; asterisks mark the mesenchymal core of filiform papillae. All images are confocal z-stacks. Scale bars: 10 μm in B; 50 μm in C-E.

Fig. 2.

The ectopic K8⁺ clusters that form in SHH-YFPcKI⁺ non-taste epithelium have taste bud-like characteristics and comprise all three differentiated taste cell types. (A-F) Like endogenous taste buds (A-C), ectopic K8⁺ cells express claudin 4 (D-F, cyan, arrowheads), which marks taste receptor cells and the cornified epithelial layer in an endogenous fungiform papilla (B,C, arrows) and non-taste epithelium (E,F, arrows). (G-I) Ectopic K8⁺ cells (red) within SHH-YFPcKI⁺ domains (green) co-express KCNQ1 (cyan). (J-L) Ectopic K8⁺ cells (red, arrowhead) adjacent to a K8⁺ endogenous taste bud (red, arrow) do not express K13 (green). (M-O) Groups of ectopic type I (M, NTPDase2⁺, red) and II (N, gustducin, red) taste cells reside in SHH-YFPcKI⁺ lingual epithelium (green); type III cells (O, SNAP25, red) generally occur singly. (P-S) All differentiated taste cell types (I, NTPDase2⁺, cyan; II, TRPM5, green; and III, CAR4, red) are present within an ectopic taste bud. Nuclei are counterstained with Draq5 (blue); white dashed lines delimit the basement membrane; white solid lines delimit the epithelial surface; asterisks mark the mesenchymal core of filiform papillae. All images are confocal z-stacks. Scale bars: 10 μm.

Fig. 3.

Ectopic taste buds increase in number and are located progressively more superficially in the lingual epithelium. (A) Ectopic K8⁺ taste buds are evident at 7 days post-tamoxifen and increase over time. (B-D) Over time, more ectopic taste buds containing type I (B), II (C) and III (D) cells are detected. (E) Progressively more ectopic K8⁺ clusters are found in the surface layers of the epithelium at later times post-induction. Three mice per time point; error bars indicate s.e.m. *P<0.05, **P<0.01; one-way ANOVA with Tukey–Kramer post-hoc test.

Fig. 4.

Ectopic taste buds recapitulate the differentiation program of endogenous taste buds. (A-C) An ectopic K8⁺ taste bud (red) lacks K14 expression (cyan), whereas adjacent progenitors are K14⁺. (D-F) Gli1-lacZ⁺ cells, revealed with anti-β-gal (cyan, nicked arrowheads), surround an ectopic K8⁺ taste bud (red, arrowheads), which is SHH-YFPcKI⁺ (green) in the non-taste epithelium of a K14CreER;SHH-YFPcKI;Gli1-lacZ mouse. K8⁺ cells are dimly β-gal⁺. (G-I) SOX2 (cyan) is highly expressed by ectopic K8⁺ cells (red, arrowheads) and by epithelial cells immediately adjacent (cyan, nicked arrowheads). SOX2 expression is low (cyan, arrows) in epithelial cells distant from SHH-YFPcKI⁺ cells (green). (J-L) SKN-1A (cyan) is expressed solely within K8⁺ ectopic taste buds (red). White dashed lines delimit the basement membrane; white solid lines delimit the epithelial surface (in A-C,G-I the surface is up, and out of the field of view). All images are confocal z-stacks except D-F, which show a single optical section (0.76 μm). Scale bars: 10 μm.

Fig. 5.

Fungiform taste buds are innervated by P2X2⁺ and PGP9.5⁺ nerve fibers, whereas ectopic taste buds form in lingual epithelium innervated only by PGP9.5⁺ fibers. (A) A fungiform taste bud is innervated by P2X2⁺ taste fibers, whereas ectopic taste buds (B) lack P2X2⁺ fibers (P2X2⁺, cyan; K8⁺, red; SHH-YFPcKI⁺, green). (C) Diagram of intragemmal versus perigemmal innervation of an endogenous fungiform taste bud. The taste bud (dark pink) is innervated by P2X2⁺ and PGP9.5⁺ intragemmal fibers (black); the papilla epithelium (light pink) is innervated by PGP9.5⁺ perigemmal fibers only (blue). (D) PGP9.5⁺ neurites innervate endogenous taste buds (PGP9.5⁺, cyan; K8⁺, red). (E,F) PGP9.5⁺ neurites are found near an ectopic taste bud with SHH-YFPcKI⁺ expression (arrow in E), while another ectopic bud lies in SHH-YFPcKI⁺ epithelium remote from sparse PGP9.5⁺ fibers (F, arrowheads) (PGP9.5⁺, cyan; K8⁺, red; SHH-YFPcKI⁺, green). White dashed lines delimit the basement membrane; white solid lines delimit the epithelial surface. All images are confocal z-stacks. Scale bars: 10 μm.

Fig. 6.

Ectopic taste buds are independent of intragemmal innervation. Significantly fewer PGP9.5⁺ neurites are found in ectopic taste buds (gray symbols) than in endogenous taste buds (black symbols) at all time points examined. Two-tailed Mann–Whitney U-test: (A) 7 days, U=5.0; (B) 14 days, U=43.0; (C) 21 days, U=54.0; (D) 28 days, U=139.0; 22-45 ectopic and 23-30 endogenous taste buds per time point, randomly selected from all taste buds in three mice per time point; ****P<0.0001. (E) At 14 days post-tamoxifen, endogenous taste bud size and PGP9.5⁺ innervation are strongly correlated (black; 30 endogenous taste buds from three mice), whereas ectopic taste bud size and PGP9.5⁺ innervation are not (gray; 45 ectopic taste buds from three mice).