Fig 4. Early loss of SHH signaling in the tongue impinges upon growth and morphogenesis but is conducive to taste bud differentiation.
(A-N) Tongues and parasagittal tongue sections from control and ShhCreERT2/Shhf mutant embryos first exposed to tamoxifen at E10.5. (A,B) Anti-Sonic hedgehog-stained (SHH; dark purple) sections of E15.5 control (A) and mutant (B) tongues showing focal epithelial hyperplasia (arrow in B) and severely decreased SHH immunostaining in the mutant. (C,D) Gli1 in situ hybridization (brown) in sections from E13.5 control (C) and mutant (D) tongues showing severe downregulation of Gli1 expression in the mutant. (E,F) E15 control (E) and mutant (F) tongues after in situ hybridization with a riboprobe targeting both deleted (exon2) and non-deleted (exon1) Shh-coding sequences (dark purple). Abnormally small mutant tongue with a bifid tip, and exhibiting oversized Shh+ spots (arrowheads in F). (G-L) Sections of E15.5 (G-I) and E17.5 (J-L) control (G,K) and mutant (H-J,L) tongues immunostained (dark purple) for keratin 8 (K8; G-I, K,L) and Rab3c (J). The insets in (K) and (L) are enlarged images of the boxed areas in (K) and (L), respectively. The control and mutant tongues show K8+ taste buds (TB) in fungiform papillae (FuP), and the mutant tongues exhibit K8+ and Rab3c+ ectopic Merkel cells (MC). (M,N) Tongue sections from E18 control (M) and mutant (N) embryos after K8 (green) and P2X2 (red) double staining showing innervated TBs. (O) RT-qPCR analysis for Ptch1 relative to Actb (β-actin) in tongues from E13.5 controls (n = 6) and mutants (n = 6) first exposed to tamoxifen (TAM) at E10.5. Severely decreased Ptch1 levels in the mutant tongues as compared to the controls (P = 0.0000; mean values ± SD). PD, periderm; CvP, circumvallate papilla; Ic, incisor; RP, rugae palatinae; T, tongue. Scale bars: 500 μm (E,F,K,L), 200 μm (A-D), 100 μm (G,H), 50 μm (I,J), and 25 μm (M,N).