Morphogenesis of fungiform papillae in developing miniature pigs

Abstract

Objective: Fungiform papillae contain taste buds and play a critical role in mastication and the gustatory system. In this study, we report a series of sequential observations of organogenesis of fungiform papillae in miniature pigs, as well as changes in the expression of BMP2, BMP4, Wnt5a, Sox2, and Notch1 signaling pathway components.

Design: In this study, we investigated the spatiotemporal expression patterns of BMP, Wnt, Sox2 and Notch in the fungiform papillae of miniature pigs at the bud stage (E40), cap stage (E50) and bell stage (E60). Pregnant miniature pigs were obtained, and the samples were processed for histological staining. Immunohistochemistry and real-time PCR were used to detect the mRNA and protein expression levels of BMP2, BMP4, Wnt5a, Sox2, and Notch1.

Results: At E40, fungiform papillae were present on the anterior two-thirds of the tongue in a specific array and pattern. The fungiform papillae were enlarged and basically developed at E50 and were largest at the earlier stage (E60). Most of the BMP2 was concentrated in the epithelial layer and the connective tissue core of the fungal papilloma and gradually accumulated from E40-E60. BMP-4 was weakly expressed in the fungiform papillae epithelia, but BMP-4-positive cells were also observed in the developing tongue muscle at E50 and E60. Wnt5a-positive cells were observed in the fungiform papillae epithelia and developing tongue muscle at all three time points. Sox2-positive cells were observed only in fungiform papillae epithelial cells, and Notch1-positive cells could not be detected.

Conclusions: This study provides primary data regarding the morphogenesis and expression of developmental signals in the fungiform papillae of miniature pigs, establishing a foundation for further research in both this model and humans.

Keywords: Development; Fungiform papilla; Immunohistochemistry; Miniature pig.

Fig. 1

SEM image of fungiform papillae during morphogenesis. At E40, E50, and E60, fungiform papilla morphology (arrows) was observed on the surface of the tongue dorsum. At different time points, the fungiform papilla morphology was basically consistent, with granular protrusions on the surface. The papilla gradually decreased in number per unit area (A–I). From E40 to E60, the fungiform papilla gradually increased, the area of a single papilla at E60 significantly increased, and there were significant differences at different time points, with units of square millimeters (J). From E40 to E60, the number of fungiform papillae per unit area (0.3 × 0.3 mm) gradually decreased, and there was a significant difference at different time points (K). Scale bar (A, D and G) 300 μm; (B, E and H) 40 μm; (C, F and I) 20 μm.

Fig. 2

Histology of fungiform papillae during morphogenesis. (A, D, G) The epithelium became concave, and the mesenchymal cells were condensed at E40. (B, E, H) Archlike structures arose, and the underlying mesenchymal cells continued to migrate upward at E50. (C, F, I) The epithelium continued to thicken, and squamous epithelial cells were detected on the surface of fungiform papillae at E60. The epithelium (EP), mesenchyme (MZ), lamina propria mucosae (M), apical papilla epithelium (APE), light superficial cells of the epithelium (LSC), and connective tissue core of the primordium of the fungiform papilla (star) were used. Scale bars: 100 μm (A, B, C), 50 μm (D, E, F) and 20 μm (G, H, I).

Fig. 3

Location of BMP-2, BMP-4, Wnt5a, Sox2, and Notch1. (A-C, P) BMP-2 expression was observed in the epithelial cells of fungiform papillae and the developing tongue muscle. (D-F, Q) BMP-4 was weakly expressed in fungiform papillae epithelia, and BMP-4-positive cells were observed in the developing tongue muscle at E50 and E60. (G-I, R) Wnt5a-positive cells were observed in fungiform papillae epithelia and the developing tongue muscle at all three timepoints. (J-L, S) Sox2-positive cells were observed throughout the lingual epithelium and fungiform papillae. (M − O, T) Notch1-positive cells could not be detected. Fungiform papillae are marked with arrowheads. Mesenchyme (MZ), lamina propria mucosae (M), apical papilla epithelium (APE), light superficial cells of the epithelium (LSC), and connective tissue core of the primordium of the fungiform papilla (star). Scale bar, 30 μm.

Fig. 4

Quantitative analysis of BMP-2, BMP-4, Wnt5a, Sox2, and Notch1 mRNA expression levels in the developmental stages of the fungiform papillae in miniature pigs. (A) Relative BMP2 mRNA expression in the fungiform papillae at E40-E60 determined by RT‒PCR. (B) Relative BMP4 mRNA expression in the fungiform papillae at E40-E60 determined by RT‒PCR. (C) Relative Wnt5a mRNA expression at E40-E60 determined by RT‒PCR in fungiform papillae. (D) Relative Sox2 mRNA expression in the fungiform papillae at E40-E60 determined by RT‒PCR. (E) Relative Notch1 mRNA expression in fungiform papillae at E40-E60 determined by RT‒PCR. *p < 0.05, **p < 0.01, ***p < 0.001.