Wnt signaling interacts with Shh to regulate taste papilla development

Abstract

Wnt and Shh signaling pathways are critical for the development and maturation of many epithelial tissues. Both pathways have roles in stem cell maintenance, tissue development, and tumorigenesis. However, linkage between these pathways in mammalian systems had not been well established. Here, we report that Shh expression in fungiform papillae and formation of normal mature fungiform papillae depend on signaling through Wnt and beta-catenin. We observed that during fungiform papilla formation in mice, Shh and components of the Wnt/beta-catenin signaling pathway are expressed together in the developing placode. The elimination of Wnt/beta-catenin signaling in either Lef1 or Wnt10b knockout mice resulted in down-regulation of Shh expression. In addition, the size and number of fungiform papillae were greatly reduced in Lef1 knockout mice. By examining embryonic mouse tongues in culture we determined that activation of Wnt/beta-catenin signaling up-regulates Shh expression. We observed that blocking Shh signaling in cultured tongue explants enhanced papillae formation and was accompanied by an up-regulation of Wnt/beta-catenin signaling, indicating that Shh inhibits the Wnt/beta-catenin pathway. Exogenously added Shh suppressed expression of endogenous Shh and inhibited Wnt/beta-catenin signaling (assessed in TOPGAL mice), further implicating Shh as an inhibitor of the Wnt/beta-catenin pathway. Our observations indicate that Wnt/beta-catenin signaling and interactions between the Wnt and Shh pathways play essential roles in the development of fungiform papillae.

Fig. 1.

Wnt/β-catenin signaling elements are expressed in the developing tongue. Expression in the developing tongue of β-galactosidase (β-gal) (A–J, V, and Y), Lef1 (K–O and X), Wnt10b (P–T), and β-catenin (U) was detected by X-Gal staining in TOPGAL mice (A–E), by in situ hybridization (K–T), or by immunofluorescence (U–Z) in wild-type mice during developmental stages E11.5–E18.5. At E11.5, β-gal activity is absent from the lateral swelling (within dotted line in A and the white arrow in F). β-gal activity first appears in the placode of the dorsal tongue at E12.5 (B and G, arrows). Arrowheads in B and Insets in C–E indicate CV papillae. β-gal activity is absent from CV papillae until E14.5 (C). Lef1 expression is detected at E11.5 in both the mesenchyme and epithelium of the tongue (K), then increases in the fungiform papilla placode at E12.5 (L). At later stages, Lef1 expression continues in the fungiform papillae but at E16.5, and thereafter Lef1 also is highly expressed in the developing filiform papillae (N and O), whereas its expression still remains in fungiform papillae at later stages (O Inset) in accordance with β-gal expression (compare with J Inset). Wnt10b expression is first seen at E11.5–E12.5 in the developing CV papillae (P and Q, arrowheads), then at E12.5 in the developing placode (Q), and continues to be expressed through E14.5 (R), but its expression declines by E16.5 (S). Fluorescent micrographs of E12.5 tongue placode sections from TOPGAL mice show that β-gal expression (V and Y) associates with β-catenin (U) and Lef1 (X). W and Z are merged images of U and V and X and Y, respectively. Asterisks in F, K, and P indicate the border between the mandible and developing tongue. (Scale bars: A–E, 500 μm; F–T, 200 μm; U–Z, 100 μm.)

Fig. 2.

Shh and Wnt/β-catenin signaling elements are expressed similarly in the developing tongue. Expression in the developing tongue of Shh (A–I), Shh plus Lef1 (J–L), and Shh plus β-gal (M and N) was detected by whole-mount immunostaining (A–D), in situ hybridization (E–I), or double immunofluorescence (J–N) in wild-type (A–L) or TOPGAL (M and N) mice during developmental stages E11.5–E18.5. The dotted line in A indicates the border between the lateral swelling and the mandible. Arrows in B and F indicate the placode; arrows in C and D indicate fungiform papillae closest to the median sulcus. The arrowhead in B indicates the CV papilla. The Inset of I is a higher magnification view of a fungiform papilla. The pattern of expression of Shh is similar to that of β-gal during fungiform papillae formation (compare B–D with Fig. 1 B–D; also compare F–H with Fig. 1 G–I). The asterisk in E indicates the border between the mandible and developing tongue. Merged fluorescent micrographs of tongue placode sections from E12.0 (J), E12.5 (K and M), and E13.5 (L) stage mice show coexpression of Shh (red; J–N) with Lef1 (green; J–L), or β-gal (green; M). The CV papilla at E12.5 (N) expresses Shh (red) but not β-gal (green). The component single fluorescence images corresponding to merged images K–M are shown in supporting information (SI) Fig. 6. (Scale bars: A–I, 500 μm; J–N, 50 μm.)

Fig. 3.

Ablating the Wnt/β-catenin pathway disrupts fungiform papillae development and reduces Shh expression. Fungiform papillae in the developing tongue were examined by SEM of E14 (A, B, M, and N) or postnatal day 0 (C and D) stage mice. The fungiform papillae at E14 of either Lef1 or Wnt10b null mice are reduced in size (B and N) in comparison to those of Lef1+/− heterozygous (A) or wild-type (M) mice. Lef1 null mice at postnatal day 0 show a marked decrease in the size and number of fungiform papillae (D; asterisks indicate missing or atrophied papillae, and arrows indicate remaining papillae), in comparison to those of the wild-type mice at postnatal day 0 (C; arrows mark fungiform papillae). Shh expression in developing tongues from wild-type and null mice was monitored by immunohistochemistry in whole mounts of tongues and mandibles (E–H and O–R) and in sections (I–L). Reduced Shh expression was observed in the fungiform papillae of developing tongues from Lef1 null mice (G, H, and L) and Wnt10b null mice (Q and R) compared with wild-type mice (E, F, J, O, and P). Normal Shh expression was observed in the CV papillae of Lef1 null (G and K, arrowheads), Wnt10b null (Q, arrowhead), and wild-type (E, I, and O, arrowheads) mice. (A–L) C57B/6 mice. (M–R) FVB mice. (Scale bars: A–D, M, and N, 200 μm; E–H and O–R, 500 μm; I–L, 100 μm.)

Fig. 4.

Wnt and Shh interact in the developing tongue. Organ culture of E12.5 tongues from wild-type (Left) or TOPGAL (Right) mice treated for 2 days in culture with 5 mM LiCl (A), 50 μg/ml anti-Shh antibody (5E1) (C), or 2.5 μg/ml Shh-N (E) and examined for expression of Shh and β-gal in fungiform papillae. Control tongues (A, C, and E) were cultured for 2 days under standard culture conditions (STAND). Treatment with LiCl (A) or the anti-Shh antibody (C) up-regulated expression of both Shh and β-gal. Treatment with Shh-N (E) nearly abolished expression of Shh and down-regulated expression of β-gal. (B, D, and F) Histograms of the percent increase/decrease of the number of the Shh-immunoreactive fungiform papillae in treated vs. standard culture conditions, corresponding to images in A, C, and E, respectively (n = 6–7; ∗, P < 0.001; means ± SD). (Scale bar: 0.5 mm.)

Fig. 5.

Schematic diagram of Wnt–Shh regulatory loop during fungiform papillae formation.